Gert Kreibich

Involvement of vps33a in the fusion of uroplakin-degrading multivesicular bodies with lysosomes
Guo, Xuemei; Tu, Liyu; Gumper, Iwona; Plesken, Heide; Novak, Edward K; Chintala, Sreenivasulu; Swank, Richard T; Pastores, Gregory; Torres, Paola; Izumi, Tetsuro; Sun, Tung-Tien; Sabatini, David D; Kreibich, Gert
2009 Sep;10(9):1350-1361, Traffic
The apical surface of the terminally differentiated mouse bladder urothelium is largely covered by urothelial plaques, consisting of hexagonally packed 16-nm uroplakin particles. These plaques are delivered to the cell surface by fusiform vesicles (FVs) that are the most abundant cytoplasmic organelles. We have analyzed the functional involvement of several proteins in the apical delivery and endocytic degradation of uroplakin proteins. Although FVs have an acidified lumen and Rab27b, which localizes to these organelles, is known to be involved in the targeting of lysosome-related organelles (LROs), FVs are CD63 negative and are therefore not typical LROs. Vps33a is a Sec1-related protein that plays a role in vesicular transport to the lysosomal compartment. A point mutation in mouse Vps33a (Buff mouse) causes albinism and bleeding (Hermansky-Pudlak syndrome) because of abnormalities in the trafficking of melanosomes and platelets. These Buff mice showed a novel phenotype observed in urothelial umbrella cells, where the uroplakin-delivering FVs were almost completely replaced by Rab27b-negative multivesicular bodies (MVBs) involved in uroplakin degradation. MVB accumulation leads to an increase in the amounts of uroplakins, Lysosomal-associated membrane protein (LAMP)-1/2, and the activities of beta-hexosaminidase and beta-glucocerebrosidase. These results suggest that FVs can be regarded as specialized secretory granules that deliver crystalline arrays of uroplakins to the cell surface, and that the Vps33a mutation interferes with the fusion of MVBs with mature lysosomes thus blocking uroplakin degradation
— id: 101636, year: 2009, vol: 10, page: 1350, stat: Journal Article,

Uroplakins in urothelial biology, function, and disease
Wu, Xue-Ru; Kong, Xiang-Peng; Pellicer, Angel; Kreibich, Gert; Sun, Tung-Tien
2009 Jun;75(11):1153-1165, Kidney international
Urothelium covers the inner surfaces of the renal pelvis, ureter, bladder, and prostatic urethra. Although morphologically similar, the urothelia in these anatomic locations differ in their embryonic origin and lineages of cellular differentiation, as reflected in their different uroplakin content, expandability during micturition, and susceptibility to chemical carcinogens. Previously thought to be an inert tissue forming a passive barrier between the urine and blood, urothelia have recently been shown to have a secretory activity that actively modifies urine composition. Urothelial cells express a number of ion channels, receptors, and ligands, enabling them to receive and send signals and communicate with adjoining cells and their broader environment. The urothelial surface bears specific receptors that not only allow uropathogenic E. coli to attach to and invade the bladder mucosa, but also provide a route by which the bacteria ascend through the ureters to the kidney to cause pyelonephritis. Genetic ablation of one or more uroplakin genes in mice causes severe retrograde vesicoureteral reflux, hydronephrosis, and renal failure, conditions that mirror certain human congenital diseases. Clearly, abnormalities of the lower urinary tract can impact the upper tract, and vice versa, through the urothelial connection. In this review, we highlight recent advances in the field of urothelial biology by focusing on the uroplakins, a group of urothelium-specific and differentiation-dependent integral membrane proteins. We discuss these proteins' biochemistry, structure, assembly, intracellular trafficking, and their emerging roles in urothelial biology, function, and pathological processes. We also call attention to important areas where greater investigative efforts are warranted.Kidney International (2009) 75, 1153-1165; doi:10.1038/ki.2009.73; published online 1 April 2009
— id: 98907, year: 2009, vol: 75, page: 1153, stat: Journal Article,

Assembly of a membrane receptor complex: roles of the uroplakin II prosequence in regulating uroplakin bacterial receptor oligomerization
Hu, Chih-Chi Andrew; Bachmann, Thomas; Zhou, Ge; Liang, Feng-Xia; Ghiso, Jorge; Kreibich, Gert; Sun, Tung-Tien
2008 Sep 1;414(2):195-203, Biochemical journal
The apical surface of the mammalian urothelium is almost completely covered by two-dimensional protein crystals (known as urothelial plaques) of hexagonally packed 16 nm particles consisting of two UP (uroplakin) heterodimers, i.e. UPs Ia/II and Ib/III pairs. UPs are functionally important as they contribute to the urothelial permeability barrier function, and UPIa may serve as the receptor for the uropathogenic Escherichia coli that causes over 90% of urinary tract infections. We study here how the UP proteins are assembled and targeted to the urothelial apical surface, paying special attention to the roles of the prosequence of UPII in UP oligomerization. We show that (i) the formation of the UPIa/UPII heterodimer, necessary for ER (endoplasmic reticulum) exit, requires disulfide formation in the prosequence domain of proUPII (the immature form of UPII still containing its prosequence); (ii) differentiation-dependent N-glycosylation of the prosequence leads to UP stabilization; (iii) a failure to form tetramers in cultured urothelial cells, in part due to altered glycosylation of the prosequence, may block two-dimensional crystal formation; and (iv) the prosequence of UPII remains attached to the mature protein complex on the urothelial apical surface even after it has been cleaved by the trans-Golgi-network-associated furin. Our results indicate that proper secondary modifications of the prosequence of UPII play important roles in regulating the oligomerization and function of the UP protein complex
— id: 81088, year: 2008, vol: 414, page: 195, stat: Journal Article,

Climp-63-mediated binding of microtubules to the ER affects the lateral mobility of translocon complexes
Nikonov, Andrei V; Hauri, Hans-Peter; Lauring, Brett; Kreibich, Gert
2007 Jul 1;120(Pt 13):2248-2258, Journal of cell science
Microtubules are frequently seen in close proximity to membranes of the endoplasmic reticulum (ER), and the membrane protein CLIMP-63 is thought to mediate specific interaction between these two structures. It was, therefore, of interest to investigate whether these microtubules are in fact responsible for the highly restricted lateral mobility of the translocon complexes in M3/18 cells as described before. As determined by fluorescence recovery after photobleaching, the breakdown of microtubules caused by drug treatment or by overexpression of the microtubule-severing protein spastin, resulted in an increased lateral mobility of the translocons that are assembled into polysomes. Also, the expression of a CLIMP-63 mutant lacking the microtubule-binding domain resulted in a significant increase of the lateral mobility of the translocon complexes. The most striking increase in the diffusion rate of the translocon complexes was observed in M3/18 cells transfected with a siRNA that effectively knocked down the expression of the endogenous CLIMP-63. It appears, therefore, that interaction of microtubules with the ER results in the immobilization of translocon complexes that are part of membrane-bound polysomes, and may play a role in the mechanism that segregates the rough and smooth domains of the ER.
— id: 72855, year: 2007, vol: 120, page: 2248, stat: Journal Article,

Cotranslocational degradation protects the stressed endoplasmic reticulum from protein overload
Oyadomari, Seiichi; Yun, Chi; Fisher, Edward A; Kreglinger, Nicola; Kreibich, Gert; Oyadomari, Miho; Harding, Heather P; Goodman, Alan G; Harant, Hanna; Garrison, Jennifer L; Taunton, Jack; Katze, Michael G; Ron, David
2006 Aug 25;126(4):727-739, Cell
The ER's capacity to process proteins is limited, and stress caused by accumulation of unfolded and misfolded proteins (ER stress) contributes to human disease. ER stress elicits the unfolded protein response (UPR), whose components attenuate protein synthesis, increase folding capacity, and enhance misfolded protein degradation. Here, we report that P58(IPK)/DNAJC3, a UPR-responsive gene previously implicated in translational control, encodes a cytosolic cochaperone that associates with the ER protein translocation channel Sec61. P58(IPK) recruits HSP70 chaperones to the cytosolic face of Sec61 and can be crosslinked to proteins entering the ER that are delayed at the translocon. Proteasome-mediated cytosolic degradation of translocating proteins delayed at Sec61 is cochaperone dependent. In P58(IPK-/-) mice, cells with a high secretory burden are markedly compromised in their ability to cope with ER stress. Thus, P58(IPK) is a key mediator of cotranslocational ER protein degradation, and this process likely contributes to ER homeostasis in stressed cells
— id: 69025, year: 2006, vol: 126, page: 727, stat: Journal Article,

Integrity of all four transmembrane domains of the tetraspanin uroplakin Ib is required for its exit from the ER
Tu, Liyu; Kong, Xiang-Peng; Sun, Tung-Tien; Kreibich, Gert
2006 Dec 15;119(Pt 24):5077-5086, Journal of cell science
The surface of the mammalian urinary bladder is covered by a crystalline, asymmetric unit membrane (AUM) structure that contains the four major uroplakins (UPs): Ia, Ib, II and IIIa. UPIa and UPIb belong to the family of tetraspanins. Although UPIa and UPIb are structurally conserved, only UPIb could exit from the endoplasmic reticulum (ER) and reach the cell surface when expressed alone in 293T cells. Modifications of the large extracellular loop of UPIb, such as mutation of the N-glycosylation site or the cysteines involved in the formation of three disulfide bridges, or exchanging the large luminal loop of UPIb with that of UPIa did not affect the ability of UPIb to reach the cell surface. However, modifications of any of the four transmembrane domains of UPIb led to ER retention, suggesting that the proper formation of helical bundles consisting of the tetraspanin transmembrane domains is a prerequisite for UPIb to exit from the ER. Results of sedimentation analysis suggested that aggregate formation is a mechanism for ER retention
— id: 71581, year: 2006, vol: 119, page: 5077, stat: Journal Article,

Cholesterol and steroid synthesizing smooth endoplasmic reticulum of adrenocortical cells contains high levels of proteins associated with the translocation channel
Black, Virginia H; Sanjay, Archana; van Leyen, Klaus; Lauring, Brett; Kreibich, Gert
2005 Oct;146(10):4234-4249, Endocrinology
Steroid-secreting cells are characterized by abundant smooth endoplasmic reticulum whose membranes contain many enzymes involved in sterol and steroid synthesis. Yet they have relatively little morphologically identifiable rough endoplasmic reticulum, presumably required for synthesis and maintenance of the smooth membranes. In this study, we demonstrate that adrenal smooth microsomal subfractions enriched in smooth endoplasmic reticulum membranes contain high levels of translocation apparatus and oligosaccharyltransferase complex proteins, previously thought confined to rough endoplasmic reticulum. We further demonstrate that these smooth microsomal subfractions are capable of effecting cotranslational translocation, signal peptide cleavage, and N-glycosylation of newly synthesized polypeptides. This shifts the paradigm for distinction between smooth and rough endoplasmic reticulum. Confocal microscopy revealed the proteins to be distributed throughout the abundant tubular endoplasmic reticulum in these cells, which is predominantly smooth surfaced. We hypothesize that the broadly distributed translocon and oligosaccharyltransferase proteins participate in local synthesis and/or quality control of membrane proteins involved in cholesterol and steroid metabolism in a sterol-dependent and hormonally regulated manner
— id: 58659, year: 2005, vol: 146, page: 4234, stat: Journal Article,

Interaction of microtubule-associated protein-2 and p63: a new link between microtubules and rough endoplasmic reticulum membranes in neurons
Farah, Carole Abi; Liazoghli, Dalinda; Perreault, Sebastien; Desjardins, Mylene; Guimont, Alain; Anton, Angela; Lauzon, Michel; Kreibich, Gert; Paiement, Jacques; Leclerc, Nicole
2005 Mar 11;280(10):9439-9449, Journal of biological chemistry
Neurons are polarized cells presenting two distinct compartments, dendrites and an axon. Dendrites can be distinguished from the axon by the presence of rough endoplasmic reticulum (RER). The mechanism by which the structure and distribution of the RER is maintained in these cells is poorly understood. In the present study, we investigated the role of the dendritic microtubule-associated protein-2 (MAP2) in the RER membrane positioning by comparing their distribution in brain subcellular fractions and in primary hippocampal cells and by examining the MAP2-microtubule interaction with RER membranes in vitro. Subcellular fractionation of rat brain revealed a high MAP2 content in a subfraction enriched with the endoplasmic reticulum markers ribophorin and p63. Electron microscope morphometry confirmed the enrichment of this subfraction with RER membranes. In cultured hippocampal neurons, MAP2 and p63 were found to concomitantly compartmentalize to the dendritic processes during neuronal differentiation. Protein blot overlays using purified MAP2c protein revealed its interaction with p63, and immunoprecipitation experiments performed in HeLa cells showed that this interaction involves the projection domain of MAP2. In an in vitro reconstitution assay, MAP2-containing microtubules were observed to bind to RER membranes in contrast to microtubules containing tau, the axonal MAP. This binding of MAP2c microtubules was reduced when an anti-p63 antibody was added to the assay. The present results suggest that MAP2 is involved in the association of RER membranes with microtubules and thereby could participate in the differential distribution of RER membranes within a neuron
— id: 95764, year: 2005, vol: 280, page: 9439, stat: Journal Article,

Rab27b is associated with fusiform vesicles and may be involved in targeting uroplakins to urothelial apical membranes
Chen, Yanru; Guo, Xuemei; Deng, Fang-Ming; Liang, Feng-Xia; Sun, Wenyu; Ren, Mindong; Izumi, Tetsuro; Sabatini, David D; Sun, Tung-Tien; Kreibich, Gert
2003 Nov 25;100(24):14012-14017, Proceedings of the National Academy of Sciences of the United States of America
The terminally differentiated umbrella cells of bladder epithelium contain unique cytoplasmic organelles, the fusiform vesicles, which deliver preassembled crystalline arrays of uroplakin proteins to the apical cell surface of urothelial umbrella cells. We have investigated the possible role of Rab proteins in this delivery process, and found Rab27b to be expressed at an extraordinary high level (0.1% of total protein) in urothelium, whereas Rab27b levels were greatly reduced (to <5% of normal urothelium) in cultured urothelial cells, which synthesized only small amounts of uroplakins and failed to form fusiform vesicles. Immuno-electron microscopy showed that Rab27b was associated with the cytoplasmic face of the fusiform vesicles, but not with that of the apical plasma membrane. The association of Rab27b with fusiform vesicles and its differentiation-dependent expression suggest that this Rab protein plays a role in regulating the delivery of fusiform vesicles to the apical plasma membrane of umbrella cells
— id: 42018, year: 2003, vol: 100, page: 14012, stat: Journal Article,

Organization of translocon complexes in ER membranes
Nikonov, A V; Kreibich, G
2003 Dec;31(Pt 6):1253-1256, Transactions (Biochemical Society (Great Britain))
Protein translocation in the ER (endoplasmic reticulum) and N-glycosylation are fundamental processes essential for the normal functioning of eukaryotic cells. They are the initial steps in the intracellular pathway that are followed by secretory proteins and membrane proteins of the endomembrane system and the plasma membrane. The translocation and concurrent N-glycosylation of these proteins take place on a large molecular machine, the TC (translocon complex), which is associated with membrane-bound polysomes. Segregation of TCs into a differentiated domain of the ER, the rough ER, may increase the efficiency of protein synthesis on membrane-bound polysomes. Our research is concerned with the assembly, functional organization and dynamics of the TCs in the ER, and their contribution to the functioning and the morphological appearance of this organelle. We hypothesize that the TCs form higher-order structures defining the rough domain of the ER. These structures, which are immobilized or diffuse slowly in the plain of the ER membrane, may be formed and stabilized by mRNAs interconnecting the TCs, by cytoskeletal elements and/or by hypothetical proteins that form links between the TCs. We have established the M3/18 cell line, which expresses the GFP (green fluorescent protein)-Dad1 fusion protein quantitatively and functionally incorporated into the OST (oligosaccharyltransferase). GFP-Dad1 can be used as a reporter molecule for the lateral mobility of the TCs since the OST is tightly associated with the complex. As determined by FRAP (fluorescence recovery after photobleaching), the lateral mobility of GFP-Dad1-tagged TCs was much more restricted than expected from the estimated size of the TC and can be affected by the functional state of the TCs. Currently, we are studying the possible involvement of cytoskeletal elements in the organization of the TCs. Our data suggest that microtubules also play a role in the immobilization of the TCs
— id: 44807, year: 2003, vol: 31, page: 1253, stat: Journal Article,

Cholesterol and steroid synthesizing smooth endoplasmic reticulum of adrenocortical cells contains high levels of translocation apparatus proteins
Black, V H; Sanjay, A; van Leyen, K; Moeller, I; Lauring, B; Kreibich, G
2002 Nov;28(4):425-430, Endocrine research
Steroid-secreting cells possess abundant smooth endoplasmic reticulum whose membranes contain many enzymes involved in sterol and steroid synthesis. In this study we demonstrate that adrenal smooth microsomal subfractions enriched in these membranes also possess high levels of proteins belonging to the translocation apparatus, proteins previously assumed to be confined to morphologically identifiable rough endoplasmic reticulum (RER). We further demonstrate that these smooth microsomal subfractions are capable of effecting the functions of these protein complexes: co-translational translocation, signal peptide cleavage and N-glycosylation of newly synthesized polypeptides. We hypothesize that these elements participate in regulating the levels of ER-targeted membrane proteins involved in cholesterol and steroid metabolism in a sterol-dependent and hormonally-regulated manner
— id: 34612, year: 2002, vol: 28, page: 425, stat: Journal Article,

Cholesterol and steroid synthesizing smooth endoplasmic reticulum of adrenocortical cells contains high levels of translocation apparatus and oligosaccharyltransferase complex proteins
Black, VH; Sanjay, A; Van Leyen, K; Moeller, I; Lauring, B; Kreibich, G
2002 NOV ;13(4):263A-263A, Molecular biology of the cell
— id: 37188, year: 2002, vol: 13, page: 263A, stat: Journal Article,

Rab27b association with melanosomes: dominant negative mutants disrupt melanosomal movement
Chen, Yanru; Samaraweera, Preminda; Sun, Tung-Tien; Kreibich, Gert; Orlow, Seth J
2002 Jun;118(6):933-940, Journal of investigative dermatology
The movement of melanosomes from post-Golgi compartments to the periphery of melanocytes is known to be regulated by factors including myosin Va and at least one Rab protein, Rab27a. Mutations in the genes encoding either protein in the mouse result in a hypopigmented phenotype mimicking the human disease Griscelli syndrome. Rab27b and Rab27a share 72% identity and they belong to the same melanocyte/platelet subfamily of Rab proteins. Rab27a orchestrates the transport of melanosomes by recruitment of the actin motor, myosin Va, onto melanosomes. By contrast, the function of Rab27b has remained elusive. In this study, we found that Rab27b mRNA is present in melanocytes and demonstrated the intrinsic GTPase activity of Rab27b protein. We explored the function of Rab27b by overexpression of two dominant negative mutants as well as the wild-type Rab27b in melan-a melanocytes. Green-fluorescent-protein-tagged Rab27b colocalizes with the melanosome marker tyrosinase-related protein 1 and with myosin Va at the cell periphery, whereas Rab27b mutants do not decorate melanosomes, and melanosomes in these mutant transfected cells redistribute from cell periphery to the perinuclear region. Furthermore, transient overexpression of the dominant negative forms of Rab27b caused diminution in both numbers and length of dendrites of melan-a cells. Our results suggest that Rab27b may regulate the outward movement of melanosomes and the formation or maintenance of dendritic extensions in melanocytes
— id: 32487, year: 2002, vol: 118, page: 933, stat: Journal Article,

Uroplakin IIIb, a urothelial differentiation marker, dimerizes with uroplakin Ib as an early step of urothelial plaque assembly
Deng, Fang-Ming; Liang, Feng-Xia; Tu, Liyu; Resing, Katheryn A; Hu, Ping; Supino, Mark; Hu, Chih-Chi Andrew; Zhou, Ge; Ding, Mingxiao; Kreibich, Gert; Sun, Tung-Tien
2002 Nov 25;159(4):685-694, Journal of cell biology
Urothelial plaques consist of four major uroplakins (Ia, Ib, II, and III) that form two-dimensional crystals covering the apical surface of urothelium, and provide unique opportunities for studying membrane protein assembly. Here, we describe a novel 35-kD urothelial plaque-associated glycoprotein that is closely related to uroplakin III: they have a similar overall type 1 transmembrane topology; their amino acid sequences are 34% identical; they share an extracellular juxtamembrane stretch of 19 amino acids; their exit from the ER requires their forming a heterodimer with uroplakin Ib, but not with any other uroplakins; and UPIII-knockout leads to p35 up-regulation, possibly as a compensatory mechanism. Interestingly, p35 contains a stretch of 80 amino acid residues homologous to a hypothetical human DNA mismatch repair enzyme-related protein. Human p35 gene is mapped to chromosome 7q11.23 near the telomeric duplicated region of Williams-Beuren syndrome, a developmental disorder affecting multiple organs including the urinary tract. These results indicate that p35 (uroplakin IIIb) is a urothelial differentiation product structurally and functionally related to uroplakin III, and that p35-UPIb interaction in the ER is an important early step in urothelial plaque assembly
— id: 33060, year: 2002, vol: 159, page: 685, stat: Journal Article,

Active translocon complexes labeled with GFP-Dad1 diffuse slowly as large polysome arrays in the endoplasmic reticulum
Nikonov, Andrei V; Snapp, Erik; Lippincott-Schwartz, Jennifer; Kreibich, Gert
2002 Aug 5;158(3):497-506, Journal of cell biology
In the ER, the translocon complex (TC) functions in the translocation and cotranslational modification of proteins made on membrane-bound ribosomes. The oligosaccharyltransferase (OST) complex is associated with the TC, and performs the cotranslational N-glycosylation of nascent polypeptide chains. Here we use a GFP-tagged subunit of the OST complex (GFP-Dad1) that rescues the temperature-sensitive (ts) phenotype of tsBN7 cells, where Dad1 is degraded and N-glycosylation is inhibited, to study the lateral mobility of the TC by FRAP. GFP-Dad1 that is functionally incorporated into TCs diffuses extremely slow, exhibiting an effective diffusion constant (Deff) about seven times lower than that of GFP-tagged ER membrane proteins unhindered in their lateral mobility. Termination of protein synthesis significantly increases the lateral mobility of GFP-Dad1 in the ER membranes, but to a level that is still lower than that of free GFP-Dad1. This suggests that GFP-Dad1 as part of the OST remains associated with inactive TCs. Our findings that TCs assembled into membrane-bound polysomes diffuse slowly within the ER have mechanistic implications for the segregation of the ER into smooth and rough domains
— id: 34614, year: 2002, vol: 158, page: 497, stat: Journal Article,

Specific heterodimer formation is a prerequisite for uroplakins to exit from the endoplasmic reticulum
Tu, Liyu; Sun, Tung-Tien; Kreibich, Gert
2002 Dec;13(12):4221-4230, Molecular biology of the cell
Much of the lower urinary tract, including the bladder, is lined by a stratified urothelium forming a highly differentiated, superficial umbrella cell layer. The apical plasma membrane as well as abundant cytoplasmic fusiform vesicles of the umbrella cells is covered by two-dimensional crystals that are formed by four membrane proteins named uroplakins (UPs) Ia, Ib, II, and III. UPs are synthesized on membrane-bound polysomes, and after several co- and posttranslational modifications they assemble into planar crystals in a post-Golgi vesicular compartment. Distension of the bladder may cause fusiform vesicles to fuse with the apical plasma membrane. We have investigated the early stages of uroplakin assembly by expressing the four uroplakins in 293T cells. Transfection experiments showed that, when expressed individually, only UPIb can exit from the endoplasmic reticulum (ER) and move to the plasma membrane, whereas UPII and UPIII reach the plasma membrane only when they form heterodimeric complexes with UPIa and UPIb, respectively. Heterodimer formation in the ER was confirmed by pulse-chase experiment followed by coimmunoprecipitation. Our results indicate that the initial building blocks for the assembly of crystalline uroplakin plaques are heterodimeric uroplakin complexes that form in the ER
— id: 34613, year: 2002, vol: 13, page: 4221, stat: Journal Article,

Retention of subunits of the oligosaccharyltransferase complex in the endoplasmic reticulum
Fu J; Kreibich G
2000 Feb 11;275(6):3984-3990, Journal of biological chemistry
Membrane proteins of the endoplasmic reticulum (ER) may be localized to this organelle by mechanisms that involve retention, retrieval, or a combination of both. For luminal ER proteins, which contain a KDEL domain, and for type I transmembrane proteins carrying a dilysine motif, specific retrieval mechanisms have been identified. However, most ER membrane proteins do not contain easily identifiable retrieval motifs. ER localization information has been found in cytoplasmic, transmembrane, or luminal domains. In this study, we have identified ER localization domains within the three type I transmembrane proteins, ribophorin I (RI), ribophorin II (RII), and OST48. Together with DAD1, these membrane proteins form an oligomeric complex that has oligosaccharyltransferase (OST) activity. We have previously shown that ER retention information is independently contained within the transmembrane and the cytoplasmic domain of RII, and in the case of RI, a truncated form consisting of the luminal domain was retained in the ER. To determine whether other domains of RI carry additional retention information, we have generated chimeras by exchanging individual domains of the Tac antigen with the corresponding ones of RI. We demonstrate here that only the luminal domain of RI contains ER retention information. We also show that the dilysine motif in OST48 functions as an ER localization motif because OST48 in which the two lysine residues are replaced by serine (OST48ss) is no longer retained in the ER and is found instead also at the plasma membrane. OST48ss is, however, retained in the ER when coexpressed with RI, RII, or chimeras, which by themselves do not exit from the ER, indicating that they may form partial oligomeric complexes by interacting with the luminal domain of OST48. In the case of the Tac chimera containing only the luminal domain of RII, which by itself exits from the ER and is rapidly degraded, it is retained in the ER and becomes stabilized when coexpressed with OST48
— id: 8554, year: 2000, vol: 275, page: 3984, stat: Journal Article,

Localization of ribophorin II to the endoplasmic reticulum involves both its transmembrane and cytoplasmic domains
Fu J; Pirozzi G; Sanjay A; Levy R; Chen Y; De Lemos-Chiarandini C; Sabatini D; Kreibich G
2000 Apr;79(4):219-228, European journal of cell biology
Proteins that are concentrated in specific compartments of the endomembrane system in order to exert their organelle-specific function must possess specific localization signals that prevent their transport to distal regions of the exocytic pathway. Some resident proteins of the endoplasmic reticulum (ER) that are known to escape with low efficiency from this organelle to a post ER compartment are recognized by a recycling receptor and brought back to their site of residence. Other ER proteins, however, appear to be retained in the ER by mechanisms that operate in the organelle itself. The mammalian oligosaccharyltransferase (OST) is a protein complex that effects the cotranslational N-glycosylation of newly synthesized polypeptides, and is composed of at least four rough ER-specific membrane proteins: ribophorins I and II (RI and RII), OST48, and Dadl. The mechanism(s) by which the subunits of this complex are retained in the ER are not well understood. In an effort to identify the domains within RII responsible for its ER localization we have studied the fate of chimeric proteins in which one or more RII domains were replaced by the corresponding ones of the Tac antigen, the latter being a well characterized plasma membrane protein that lacks intrinsic ER retention signals and serves to provide a neutral framework for the identification of retention signals in other proteins. We found that the luminal domain of RII by itself does not contain retention information, while the cytoplasmic and transmembrane domains contain independent ER localization signals. We also show that the retention function of the transmembrane domain is strengthened by the presence of a flanking luminal region consisting of 15 amino acids
— id: 11683, year: 2000, vol: 79, page: 219, stat: Journal Article,

Degradation of a short-lived glycoprotein from the lumen of the endoplasmic reticulum: the role of N-linked glycans and the unfolded protein response
de Virgilio M; Kitzmuller C; Schwaiger E; Klein M; Kreibich G; Ivessa NE
1999 Dec;10(12):4059-4073, Molecular biology of the cell
We are studying endoplasmic reticulum-associated degradation (ERAD) with the use of a truncated variant of the type I ER transmembrane glycoprotein ribophorin I (RI). The mutant protein, RI(332), containing only the N-terminal 332 amino acids of the luminal domain of RI, has been shown to interact with calnexin and to be a substrate for the ubiquitin-proteasome pathway. When RI(332) was expressed in HeLa cells, it was degraded with biphasic kinetics; an initial, slow phase of approximately 45 min was followed by a second phase of threefold accelerated degradation. On the other hand, the kinetics of degradation of a form of RI(332) in which the single used N-glycosylation consensus site had been removed (RI(332)-Thr) was monophasic and rapid, implying a role of the N-linked glycan in the first proteolytic phase. RI(332) degradation was enhanced when the binding of glycoproteins to calnexin was prevented. Moreover, the truncated glycoprotein interacted with calnexin preferentially during the first proteolytic phase, which strongly suggests that binding of RI(332) to the lectin-like protein may result in the slow, initial phase of degradation. Additionally, mannose trimming appears to be required for efficient proteolysis of RI(332). After treatment of cells with the inhibitor of N-glycosylation, tunicamycin, destruction of the truncated RI variants was severely inhibited; likewise, in cells preincubated with the calcium ionophore A23187, both RI(332) and RI(332)-Thr were stabilized, despite the presence or absence of the N-linked glycan. On the other hand, both drugs are known to trigger the unfolded protein response (UPR), resulting in the induction of BiP and other ER-resident proteins. Indeed, only in drug-treated cells could an interaction between BiP and RI(332) and RI(332)-Thr be detected. Induction of BiP was also evident after overexpression of murine Ire1, an ER transmembrane kinase known to play a central role in the UPR pathway; at the same time, stabilization of RI(332) was observed. Together, these results suggest that binding of the substrate proteins to UPR-induced chaperones affects their half lives
— id: 18409, year: 1999, vol: 10, page: 4059, stat: Journal Article,

The bladder as a bioreactor: urothelium production and secretion of growth hormone into urine [see comments]
Kerr DE; Liang F; Bondioli KR; Zhao H; Kreibich G; Wall RJ; Sun TT
1998 Jan;16(1):75-79, Nature biotechnology
Uroplakin genes are expressed in a bladder-specific and differentiation-dependent fashion. Using a 3.6-kb promoter of mouse uroplakin II gene, we have generated transgenic mice that express human growth hormone (hGH) in their bladder epithelium, resulting in its secretion into the urine at 100-500 ng/ml. The levels of urine hGH concentration remain constant for longer than 8 months. hGH is present as aggregates mostly in the uroplakin-delivering cytoplasmic vesicles that are targeted to fuse with the apical surface. Using the bladder as a bioreactor offers unique advantages, including the utility of all animals throughout their lives. Using urine, which contains little protein and lipid, as a starting material facilitates recombinant protein purification
— id: 8368, year: 1998, vol: 16, page: 75, stat: Journal Article,

Unregulated exposure of the ribosomal M-site caused by NAC depletion results in delivery of non-secretory polypeptides to the Sec61 complex
Moller I; Beatrix B; Kreibich G; Sakai H; Lauring B; Wiedmann M
1998 Dec 11;441(1):1-5, FEBS letters
Nascent polypeptide associated complex (NAC) interacts with nascent polypeptides emerging from ribosomes. Both signal recognition particle (SRP) and NAC work together to ensure specificity in co-translational targeting by competing for binding to the ribosomal membrane attachment site. While SRP selects signal-containing ribosomes for targeting, NAC prevents targeting of signal peptide-less nascent chains to the endoplasmic reticulum membrane. Here we show that the ribosome binding that occurs in NAC's absence delivers signalless nascent chains to the Sec61 complex, underscoring the danger of unregulated exposure of the ribosomal M-site. Recently, the idea that NAC prevents ribosome binding has been challenged. By carefully examining the physiologic NAC concentration in a variety of tissues from different species we here demonstrate that the discrepancy resulted from subphysiologic NAC concentrations
— id: 18410, year: 1998, vol: 441, page: 1, stat: Journal Article,

A general mechanism for regulation of access to the translocon: competition for a membrane attachment site on ribosomes
Moller I; Jung M; Beatrix B; Levy R; Kreibich G; Zimmermann R; Wiedmann M; Lauring B
1998 Nov 10;95(23):13425-13430, Proceedings of the National Academy of Sciences of the United States of America
For proteins to enter the secretory pathway, the membrane attachment site (M-site) on ribosomes must bind cotranslationally to the Sec61 complex present in the endoplasmic reticulum membrane. The signal recognition particle (SRP) and its receptor (SR) are required for targeting, and the nascent polypeptide associated complex (NAC) prevents inappropriate targeting of nonsecretory nascent chains. In the absence of NAC, any ribosome, regardless of the polypeptide being synthesized, binds to the endoplasmic reticulum membrane, and even nonsecretory proteins are translocated across the endoplasmic reticulum membrane. By occupying the M-site, NAC prevents all ribosome binding unless a signal peptide and SRP are present. The mechanism by which SRP overcomes the NAC block is unknown. We show that signal peptide-bound SRP occupies the M-site and therefore keeps it free of NAC. To expose the M-site and permit ribosome binding, SR can pull SRP away from the M-site without prior release of SRP from the signal peptide
— id: 18411, year: 1998, vol: 95, page: 13425, stat: Journal Article,

DAD1 is required for the function and the structural integrity of the oligosaccharyltransferase complex
Sanjay A; Fu J; Kreibich G
1998 Oct 2;273(40):26094-26099, Journal of biological chemistry
Asparagine-linked glycosylation is a highly conserved protein modification reaction that occurs in all eukaryotic organisms. The oligosaccharyltransferase (OST), which has its active site exposed on the luminal face of the endoplasmic reticulum (ER), catalyzes the transfer of preassembled high mannose oligosaccharides onto certain asparagine residues of nascent polypeptides. The mammalian OST complex was initially thought to be composed of three transmembrane proteins, ribophorin I (RI), ribophorin II (RII), and OST48. Most recently, a small integral membrane protein of 12 kDa called DAD1 has been identified as an additional member of the mammalian OST complex. A point mutation in the DAD1 gene is responsible for the temperature-sensitive phenotype of a baby hamster kidney-derived cell line (tsBN7) that undergoes apoptosis at the non-permissive temperature. Furthermore, the mutant protein DAD1 is not detectable in tsBN7 cells 6 h after shifting the cells to the non-permissive temperature. This temperature-sensitive cell line offered unique opportunities to study the effects caused by the loss of one OST subunit on the other three subunits and also on N-linked glycosylation. Western blot analysis of cell lysates showed that after 6 h at the non-permissive temperature, steady-state levels of the ribophorins were reduced by about 50%, and OST48 was barely detectable. On the other hand, steady-state levels of other components of the rough ER, such as the alpha-subunits of the TRAP (translocon-associated membrane protein) and the Sec61 complex, which are components of the translocation apparatus, are not affected by the instability of the OST subunits. Furthermore, N-glycosylation of the ribophorins was seriously affected 6 h after shifting the cells to the non-permissive temperature, and after 12 h they were synthesized only in the non-glycosylated form. As may be expected, this defect in the OST complex at the non-permissive temperature caused also the underglycosylation of a secretory glycoprotein. We concluded that degradation of DAD1 at the non-permissive temperature not only affects the stability of OST48 and the ribophorins but also results in the functional inactivation of the OST complex
— id: 12069, year: 1998, vol: 273, page: 26094, stat: Journal Article,

Interactions among subunits of the oligosaccharyltransferase complex
Fu J; Ren M; Kreibich G
1997 Nov 21;272(47):29687-29692, Journal of biological chemistry
The mammalian oligosaccharyltransferase (OST) is an oligomeric complex composed of three membrane proteins of the endoplasmic reticulum: ribophorin I (RI), ribophorin II (RII), and OST48. In addition, sequence homology between the Ost2 subunit of the yeast OST complex and Dad1 (defender against apoptotic death) suggests that Dad1 may represent a fourth subunit of the mammalian OST complex. In attempts to elucidate the structural organization of this complex, we have studied the interactions among its subunits. Using the yeast two-hybrid system, we have shown that the luminal domains of RI and RII (RIL and RIIL, respectively) interacted with the luminal domain of OST48 (OST48L), but no direct interaction was observed between RIL and RIIL. These results were confirmed by biochemical assays. Deletion analyses using the yeast two-hybrid system showed that subdomain of RIL or RIIL adjacent to the respective transmembrane domains interacted with OST48L. Of the three equal length subdomains of OST48L, the one at the N terminus and the one next to the transmembrane domain interacted with RIL. None of these three subdomains of OST48L interacted with RIIL. The yeast two-hybrid assay also revealed affinity between the cytoplasmically located N-terminal region of Dad1 and the short cytoplasmic tail of OST48, thus placing Dad1 firmly into the OST complex. In addition, we found a homotypic interaction between the cytoplasmic domains of RI, which may play a role in the formation of the oligomeric array formed by components of the translocation machinery
— id: 8251, year: 1997, vol: 272, page: 29687, stat: Journal Article,

Functional protein prenylation is required for the brefeldin A-dependent retrograde transport from the Golgi apparatus to the endoplasmic reticulum
Ivessa NE; Gravotta D; De Lemos-Chiarandini C; Kreibich G
1997 Aug 15;272(33):20828-20834, Journal of biological chemistry
In cells exposed to brefeldin A (BFA), enzymes of the Golgi apparatus are redistributed to the endoplasmic reticulum (ER) by retrograde membrane flow, where they may cause modifications on resident ER proteins. We have used a truncated form of the rough ER-specific type I transmembrane glycoprotein ribophorin I as a probe to detect Golgi glycosyltransferases relocated to the ER in a BFA-dependent fashion. This polypeptide (RI332) comprises the 332 amino-terminal amino acids of ribophorin I and behaves like a luminal ER protein when expressed in HeLa cells. Upon treatment of the cells with BFA, RI332 becomes quantitatively O-glycosylated by Golgi glycosyltransferases that are transported back to the ER. Here we demonstrate that pretreatment of the cells with lovastatin, an inhibitor of HMG-CoA reductase, abrogates this modification and that mevalonate, the product formed in the step inhibited by the drug, is able to counteract the effect of lovastatin. We also show by immunofluorescence using mannosidase II as a Golgi marker that the BFA-induced retrograde transport of Golgi enzymes is blocked by lovastatin, although electron microscopy indicates that BFA causes disassembly of the Golgi apparatus into swollen vesicles and tubules. Our observations support the role of a prenylated protein, such as the geranylgeranylated small G protein Rab6, in the retrograde transport from the Golgi apparatus to the ER, since lovastatin acts by inhibiting its prenylation
— id: 8250, year: 1997, vol: 272, page: 20828, stat: Journal Article,

Incomplete endoplasmic reticulum (ER) retention in immature thymocytes as revealed by surface expression of "ER-resident" molecular chaperones
Wiest DL; Bhandoola A; Punt J; Kreibich G; McKean D; Singer A
1997 Mar 4;94(5):1884-1889, Proceedings of the National Academy of Sciences of the United States of America
The folding and assembly of nascent proteins in the endoplasmic reticulum (ER) is assisted by molecular chaperones that are themselves retained within the ER. We now report that a number of different ER proteins, including molecular chaperones, are selectively expressed on the surface of immature thymocytes, but their surface expression is extinguished upon further differentiation. Escape from the ER is only possible for newly synthesized ER proteins before they become permanently retained. Thus, the cellular process of ER retention is incomplete in immature thymocytes and provides an explanation for surface expression of partial receptor complexes that transduce differentiative signals during thymic development
— id: 18412, year: 1997, vol: 94, page: 1884, stat: Journal Article,

Signals from the stressed endoplasmic reticulum induce C/EBP-homologous protein (CHOP/GADD153)
Wang XZ; Lawson B; Brewer JW; Zinszner H; Sanjay A; Mi LJ; Boorstein R; Kreibich G; Hendershot LM; Ron D
1996 Aug;16(8):4273-4280, Molecular & cellular biology
The gene encoding C/EBP-homologous protein (CHOP), also known as growth arrest and DNA-damage-inducible gene 153 (GADD153), is activated by agents that adversely affect the function of the endoplasmic reticulum (ER). Because of the pleiotropic effects of such agents on other cellular processes, the role of ER stress in inducing CHOP gene expression has remained unclear. We find that cells with conditional (temperature-sensitive) defects in protein glycosylation (CHO K12 and BHK tsBN7) induce CHOP when cultured at the nonpermissive temperature. In addition, cells that are defective in initiating the ER stress response, because of overexpression of an exogenous ER chaperone, BiP/GRP78, exhibit attenuated inducibility of CHOP. Surprisingly, attenuated induction of CHOP was also noted in BiP-overexpressing cells treated with methyl methanesulfonate, an agent thought to activate CHOP by causing DNA damage. The roles of DNA damage and growth arrest in the induction of CHOP were therefore reexamined. Induction of growth arrest by culture to confluence or treatment with the enzymatic inhibitor N-(phosphonacetyl)-L-aspartate did not induce CHOP. Furthermore, both a DNA-damage-causing nucleoside analog (5-hydroxymethyl-2'-deoxyuridine) and UV light alone did not induce CHOP. These results suggest that CHOP is more responsive to ER stress than to growth arrest or DNA damage and indicate a potential role for CHOP in linking stress in the ER to alterations in gene expression
— id: 12576, year: 1996, vol: 16, page: 4273, stat: Journal Article,

The Brefeldin A-induced retrograde transport from the Golgi apparatus to the endoplasmic reticulum depends on calcium sequestered to intracellular stores
Ivessa NE; De Lemos-Chiarandini C; Gravotta D; Sabatini DD; Kreibich G
1995 Oct 27;270(43):25960-25967, Journal of biological chemistry
Ribophorin I is a type I transmembrane glycoprotein specific to the rough endoplasmic reticulum. We have previously shown that, when expressed in transfected HeLa cells, a carboxyl-terminally truncated form of ribophorin I that contains most of the luminal domain (RI332) is, like the native protein, retained in the endoplasmic reticulum (ER). Brefeldin A (BFA) treatment of these HeLa cells leads to O-glycosylation of RI332 by glycosyltransferases that are redistributed from the Golgi apparatus to the ER (Ivessa, N. E., De Lemos-Chiarandini, C., Tsao, Y.-S., Takatsuki, A., Adesnik, M., Sabatini, D. D., and Kreibich, G. (1992) J. Cell Biol. 117, 949-958). Using the state of glycosylation of RI332 as a measure for the BFA-induced backflow of enzymes of the Golgi apparatus to the ER, we now demonstrate that the retrograde transport is inhibited when cells are treated with various agents that affect intracellular Ca2+ concentrations, such as the dipeptide benzyloxycarbonyl (Cbz)-Gly-Phe-amide, the Ca2+ ionophore A23187, and thapsigargin, an inhibitor of the Ca(2+)-transporting ATPase of the ER. These treatments prevent the BFA-induced O-glycosylation of RI332. Immunofluorescence localization of the Golgi markers, MG-160 and galactosyltransferase, shows that when BFA is applied in the presence of Ca2+ modulating agents, the markers remain confined to the Golgi apparatus and are not redistributed to the ER, as is the case when BFA alone is used. Cbz-Gly-Phe-amide does not, however, interfere with the BFA-induced release of beta-COP from the Golgi apparatus. We conclude that the maintenance of a Ca2+ gradient between the cytoplasm and the lumen of the ER and the Golgi apparatus is required for the BFA-induced retrograde transport from the Golgi apparatus to the ER to occur
— id: 12718, year: 1995, vol: 270, page: 25960, stat: Journal Article,

The intrinsic ability of ribosomes to bind to endoplasmic reticulum membranes is regulated by signal recognition particle and nascent-polypeptide-associated complex [see comments]
Lauring B; Kreibich G; Weidmann M
1995 Oct 10;92(21):9435-9439, Proceedings of the National Academy of Sciences of the United States of America
Signal peptides direct the cotranslational targeting of nascent polypeptides to the endoplasmic reticulum (ER). It is currently believed that the signal recognition particle (SRP) mediates this targeting by first binding to signal peptides and then by directing the ribosome/nascent chain/SRP complex to the SRP receptor at the ER. We show that ribosomes can mediate targeting by directly binding to translocation sites. When purified away from cytosolic factors, including SRP and nascent-polypeptide-associated complex (NAC), in vitro assembled translation intermediates representing ribosome/nascent-chain complexes efficiently bound to microsomal membranes, and their nascent polypeptides could subsequently be efficiently translocated. Because removal of cytosolic factors from the ribosome/nascent-chain complexes also resulted in mistargeting of signalless nascent polypeptides, we previously investigated whether readdition of cytosolic factors, such as NAC and SRP, could restore fidelity to targeting. Without SRP, NAC prevented all nascent-chain-containing ribosomes from binding to the ER membrane. Furthermore, SRP prevented NAC from blocking ribosome-membrane association only when the nascent polypeptide contained a signal. Thus, NAC is a global ribosome-binding prevention factor regulated in activity by signal-peptide-directed SRP binding. A model presents ribosomes as the targeting vectors for delivering nascent polypeptides to translocation sites. In conjunction with signal peptides, SRP and NAC contribute to this specificity of ribosomal function by regulating exposure of a ribosomal membrane attachment site that binds to receptors in the ER membrane
— id: 12720, year: 1995, vol: 92, page: 9435, stat: Journal Article,

Nascent polypeptide-associated complex protein prevents mistargeting of nascent chains to the endoplasmic reticulum [published erratum appears in Proc Natl Acad Sci U S A 1995 Aug 15;92(17):8088]
Lauring B; Sakai H; Kreibich G; Wiedmann M
1995 Jun 6;92(12):5411-5415, Proceedings of the National Academy of Sciences of the United States of America
We show that, after removal of the nascent polypeptide-associated complex (NAC) from ribosome-associated nascent chains, ribosomes synthesizing proteins lacking signal peptides are efficiently targeted to the endoplasmic reticulum (ER) membrane. After this mistargeting, translocation across the ER membrane occurs, albeit less efficiently than for a nascent secretory polypeptide, perhaps because the signal peptide is needed to catalyze the opening of the translocation pore. The mistargeting was prevented by the addition of purified NAC and was shown not to be mediated by the signal recognition particle and its receptor. Instead, it appears to be a consequence of the intrinsic affinity of ribosomes for membrane binding sites, since it can be blocked by competing ribosomes that lack associated nascent polypeptides. We propose that, when bound to a signalless ribosome-associated nascent polypeptide, NAC sterically blocks the site in the ribosome for membrane binding
— id: 12763, year: 1995, vol: 92, page: 5411, stat: Journal Article,

Nascent-polypeptide-associated complex: a bridge between ribosome and cytosol
Lauring B; Wang S; Sakai H; Davis TA; Wiedmann B; Kreibich G; Wiedmann M
1995 ;60:47-56, Cold Spring Harbor symposia on quantitative biology
— id: 12819, year: 1995, vol: 60, page: 47, stat: Journal Article,

Functional characterization of the cis-regulatory elements of the rat ribophorin I gene
Rajasekaran AK; Zhou Z; Prakash K; Das G; Kreibich G
1995 Feb 11;23(3):313-319, Nucleic acids research
The ribophorin I gene encodes a rough endoplasmic reticulum (RER) specific membrane protein which is a subunit of the oligosaccharyltransferase. To establish the functional activity of its promoter region we have performed transient gene transcription experiments employing plasmid constructs that contain 5' flanking regions of the ribophorin I gene cloned upstream of the CAT reporter gene. Among the restriction fragments obtained from the 1.3-kilobase 5' flanking region, a proximal fragment (-42 to +24) containing two GC-rich elements was required for basic promoter activity, while a fragment (-364 to +24) encoding an additional GC-box and an octamer like motif at -233 conferred the maximal promoter activity. In order to investigate the functionality of an octomer-like sequence co-transfection experiments were performed with Oct-2 cDNA and the CAT reporter gene containing the ribophorin I fragment (-364 to +24). A 3-4-fold increase in the transcriptional activity was observed with this construct. In addition, gel shift experiments showed Oct-2 binding to this construct. These results indicate that Oct-2 is most likely involved in the regulation of the ribophorin I gene transcription. We suggest that the GC-rich elements are necessary for constitutive ribophorin I expression while octamer motif binding proteins function synergistically with the GC-rich element binding proteins to increase the expression of the ribophorin I gene during the proliferation of RER
— id: 6721, year: 1995, vol: 23, page: 313, stat: Journal Article,

Precursor sequence, processing, and urothelium-specific expression of a major 15-kDa protein subunit of asymmetric unit membrane
Lin JH; Wu XR; Kreibich G; Sun TT
1994 Jan 21;269(3):1775-1784, Journal of biological chemistry
The asymmetric unit membrane (AUM) is a highly specialized biomembrane elaborated by terminally differentiated urothelial cells. It contains quasi-crystalline arrays of 12-nm protein particles each of which is composed of six dumbbell-shaped subdomains. In this paper we describe the precursor sequence, processing and in vitro membrane insertion properties of bovine uroplakin II (UPII), a 15-kDa major protein component of AUM. The cDNA-deduced amino acid sequence revealed that UPII is synthesized as a precursor protein containing a cleavable signal peptide of approximately 26 amino acids, a long pro-sequence of approximately 59 residues harboring three potential N-glycosylation sites, and the mature polypeptide of 100 residues. In vitro translation of UPII mRNA demonstrated that UPII is indeed first synthesized as a 19-kDa precursor, which loses its signal peptide upon insertion into added microsomes; this process is accompanied by the acquisition of high mannose-type oligosaccharides giving rise to a 28-kDa precursor which is completely protected from the digestion by exogenous proteases. These results, together with the presence of a stretch of 25 hydrophobic amino acids at the C terminus, suggest that UPII protein is anchored to the lipid bilayer via its C-terminal membrane-spanning domain with its major N-terminal domain exposed luminally. The formation of the 15-kDa mature UPII requires the removal of the pro-sequence by a furin-like endoprotease. Since only mature UPII devoid of this pro-sequence can interact with 27-kDa uroplakin I, the proteolytic processing of UPII precursor may play an important role in regulating the assembly of AUM. Finally, we showed that genomic sequences cross-hybridizing with bovine UPII cDNA are present in many mammals suggesting that UPII performs a highly conserved function in the terminally differentiated cells of mammalian urinary bladder epithelium
— id: 8249, year: 1994, vol: 269, page: 1775, stat: Journal Article,

Structural reorganization of the rough endoplasmic reticulum without size expansion accounts for dexamethasone-induced secretory activity in AR42J cells
Rajasekaran AK; Morimoto T; Hanzel DK; Rodriguez-Boulan E; Kreibich G
1993 Jun;105 ( Pt 2)(3):333-345, Journal of cell science
A striking reorganization of the rough endoplasmic reticulum (RER) from a tubulo-vesicular (TV-RER) to a stacked cisternal (SC-RER) configuration was observed when the secretory activity of AR42J cells, a cell line derived from a rat pancreatic acinar carcinoma, was induced by dexamethasone. Treatment with 10 nM dexamethasone resulted in a 6.6-fold increase in the intracellular and a 4.6-fold increase in the secreted amylase activity, respectively. On the basis of the morphometric analysis of thin-section electron micrographs it has been previously reported that this increase in secretory activity is accompanied by a 2.4-fold or 30-fold increase in the size of the RER. We have developed a new biochemical method to determine the size of the RER by quantifying the membrane-bound ribosomes. Using this procedure we did not detect any change in the size of the RER after induction of an active secretory state in AR42J cells. Electron microscopic observation showed the predominance of SC-RER in dexamethasone-treated cells compared to the abundance of TV-RER in control cells. Laser scanning confocal microscopy showed a patchy distribution of ER staining in dexamethasone-treated cells compared to more basal localization in control cells. On the basis of our observations we conclude that in AR42J cells the increase in secretory activity induced by dexamethasone is accompanied by a reorganization of the RER rather than by an increase in ER surface area, as reported by others. Our results suggest that SC-RER is a biosynthetically more efficient form of the RER, which is found predominantly in actively secreting cells
— id: 18413, year: 1993, vol: 105 ( Pt 2), page: 333, stat: Journal Article,

Genomic organization of the rat ribophorin II gene: Common cis-regulatory elements are found in the 5' flanking regions of the rat ribophorin I and ribophorin II genes
Zhou, Z.; Prakash, K.; Rajasekaran, A. K.; Adesnik, M.; Sabatini, D. D.; Kreibich, G.
1993 ;4(SUPPL.):423A-423A, Molecular biology of the cell
— id: 104647, year: 1993, vol: 4, page: 423A, stat: Journal Article,

A Golgi-related structure remains after the brefeldin A-induced formation of an ER-Golgi hybrid compartment
De Lemos-Chiarandini C; Ivessa NE; Black VH; Tsao YS; Gumper I; Kreibich G
1992 Aug;58(2):187-201, European journal of cell biology
Brefeldin A (BFA) has previously been shown to block protein transport from the endoplasmic reticulum (ER), to cause the redistribution of Golgi components to the ER, and to change profoundly the morphology of the Golgi apparatus. In order to quantitate the effects of this drug on the morphology of the ER and the Golgi apparatus in HeLa cells, the numerical, surface and volume densities of these organelles were determined by stereological means. We found that in cells treated with BFA (5 micrograms/ml) clusters of vesicles and tubules, often located near transitional elements of the ER, replaced the Golgi apparatus. The numerical density of these clusters in cells treated with BFA for 30 min or 4.5 h is similar to that of Golgi complexes and Golgi-related clusters in control cells. The surface density of the vesicles and tubules contained in these clusters is about 50% of that represented by Golgi elements in control cells. Concomitantly, a corresponding increase in the surface density of the ER-Golgi hybrid compartment was observed. This hybrid compartment contained Golgi-specific enzymes effecting modifications of N-linked oligosaccharides and the transfer of O-linked sugars. Antibodies recognizing different subcompartments of the Golgi apparatus or the intermediate compartment, labeled vesicles and tubules of the Golgi-related clusters. Applying low doses of BFA allowed for the dissection of the disassembly of the Golgi apparatus into at least two phases. At very low doses (10-20 ng/ml) the numerical density of vesicles in the clusters increased up to 4-fold above control, while the surface density did not markedly change, suggesting that vesiculation of the Golgi cisternae had occurred. Fusion of Golgi elements with the ER seemed to occur only at doses of BFA higher than 20 ng/ml. Contrary to observations on other cell types, removal of BFA from HeLa cell cultures resulted in a rather slow reformation (1-2 h) of the Golgi complex, which allowed us to observe several intermediate stages in this process. During this time period an ER was restored which no longer contained Golgi-specific O-glycosylation functions. Our results demonstrate that BFA does not simply cause the disappearance of the Golgi apparatus by fusion with the ER, but instead clusters of vesicles and tubules remain that contain Golgi-specific markers
— id: 13499, year: 1992, vol: 58, page: 187, stat: Journal Article,

O-glycosylation of intact and truncated ribophorins in brefeldin A-treated cells: newly synthesized intact ribophorins are only transiently accessible to the relocated glycosyltransferases
Ivessa NE; De Lemos-Chiarandini C; Tsao YS; Takatsuki A; Adesnik M; Sabatini DD; Kreibich G
1992 Jun;117(5):949-958, Journal of cell biology
Ribophorins I and II are type I transmembrane glycoproteins of the ER that are segregated to the rough domains of this organelle. Both ribophorins appear to be part of the translocation apparatus for nascent polypeptides that is associated with membrane-bound ribosomes and participate in the formation of a proteinaceous network within the ER membrane that also includes other components of the translocation apparatus. The ribophorins are both highly stable proteins that lack O-linked sugars but each contains one high mannose N-linked oligosaccharide that remains endo H sensitive throughout their lifetimes. We have previously shown (Tsao, Y. S., N. E. Ivessa, M. Adesnik, D. D. Sabatini, and G. Kreibich. 1992. J. Cell Biol. 116:57-67) that a COOH-terminally truncated variant of ribophorin I that contains only the first 332 amino acids of the luminal domain (RI332), when synthesized in permanent transformants of HeLa cells, undergoes a rapid degradation with biphasic kinetics in the ER itself and in a second, as yet unidentified nonlysosomal pre-Golgi compartment. We now show that in cells treated with brefeldin A (BFA) RI332 molecules undergo rapid O-glycosylation in a multistep process that involves the sequential addition of N-acetylgalactosamine, galactose, and terminal sialic acid residues. Addition of O-linked sugars affected all newly synthesized RI332 molecules and was completed soon after synthesis with a half time of about 10 min. In the same cells, intact ribophorins I and II also underwent O-linked glycosylation in the presence of BFA, but these molecules were modified only during a short time period immediately after their synthesis was completed, and the modification affected only a fraction of the newly synthesized polypeptides. More important, these molecules synthesized before the addition of BFA were not modified by O-glycosylation. The same is true for ribophorin I when overexpressed in HeLa cells although it is significantly less stable than the native polypeptide in control cells. We, therefore, conclude that soon after their synthesis, ribophorins lose their susceptibility to the relocated Golgi enzymes that effect the O-glycosylation, most likely as a consequence of a conformational change in the ribophorins that occurs during their maturation, although it cannot be excluded that rapid integration of these molecules into a supramolecular complex in the ER membrane leads to their inaccessibility to these enzymes
— id: 13579, year: 1992, vol: 117, page: 949, stat: Journal Article,

Oligosaccharyltransferase activity is associated with a protein complex composed of ribophorins I and II and a 48 kd protein
Kelleher DJ; Kreibich G; Gilmore R
1992 Apr 3;69(1):55-65, Cell
Oligosaccharyltransferase catalyzes the N-linked glycosylation of asparagine residues on nascent polypeptides in the lumen of the rough endoplasmic reticulum (RER). A protein complex composed of 66, 63, and 48 kd subunits copurified with oligosaccharyltransferase from canine pancreas. The 66 and 63 kd subunits were shown by protein immunoblotting to be identical to ribophorin I and II, two previously identified RER glycoproteins that colocalize with membrane-bound ribosomes. The transmembrane segment of ribophorin I was found to be homologous to a recently proposed dolichol recognition consensus sequence. Based on a revision of the consensus sequence, we propose a model for the interaction of dolichol with the glycosyltransferases that catalyze the assembly and transfer of lipid-linked oligosaccharides
— id: 18414, year: 1992, vol: 69, page: 55, stat: Journal Article,

Sticking together for a difficult passage
Kreibich G; Sabatini DD
1992 Feb;2(2):90-92, Current biology. CB
— id: 44806, year: 1992, vol: 2, page: 90, stat: Journal Article,

Carboxy terminally truncated forms of ribophorin I are degraded in pre-Golgi compartments by a calcium-dependent process
Tsao YS; Ivessa NE; Adesnik M; Sabatini DD; Kreibich G
1992 Jan;116(1):57-67, Journal of cell biology
Two COOH terminally truncated variants of ribophorin I (RI), a type I transmembrane glycoprotein of 583 amino acids that is segregated to the rough portions of the ER and is associated with the protein-translocating apparatus of this organelle, were expressed in permanent HeLa cell transformants. Both variants, one membrane anchored but lacking part of the cytoplasmic domain (RL467) and the other consisting of the luminal 332 NH2-terminal amino acids (RI332), were retained intracellularly but, in contrast to the endogenous long lived, full length ribophorin I (t 1/2 = 25 h), were rapidly degraded (t 1/2 less than 50 min) by a nonlysosomal mechanism. The absence of a measurable lag phase in the degradation of both truncated ribophorins indicates that their turnover begins in the ER itself. The degradation of RI467 was monophasic (t 1/2 = 50 min) but the rate of degradation of RI332 molecules increased about threefold approximately 50 min after their synthesis. Several pieces of evidence suggest that the increase in degradative rate is the consequence of the transport of RI332 molecules that are not degraded during the first phase to a second degradative compartment. Thus, when added immediately after labeling, ionophores that inhibit vesicular flow out of the ER, such as carbonyl cyanide m-chlorophenylhydrazone (CCCP) and monensin, suppressed the second phase of degradation of RI332. On the other hand, when CCCP was added after the second phase of degradation of RI332 was initiated, the degradation was unaffected. Moreover, in cells treated with brefeldin A the degradation of RI332 became monophasic, and took place with a half-life intermediate between those of the two normal phases. These results point to the existence of two subcellular compartments where abnormal ER proteins can be degraded. One is the ER itself and the second is a non-lysosomal pre-Golgi compartment to which ER proteins are transported by vesicular flow. A survey of the effects of a variety of other ionophores and protease inhibitors on the turnover of RI332 revealed that metalloproteases are involved in both phases of the turnover and that the maintenance of a high Ca2+ concentration is necessary for the degradation of the luminally truncated ribophorin
— id: 13723, year: 1992, vol: 116, page: 57, stat: Journal Article,

Rat ribophorin II: molecular cloning and chromosomal localization of a highly conserved transmembrane glycoprotein of the rough endoplasmic reticulum
Pirozzi G; Zhou ZM; D'Eustachio P; Sabatini DD; Kreibich G
1991 May 15;176(3):1482-1486, Biochemical & biophysical research communications
We report here the complete nucleotide sequence of rat ribophorin II. The predicted amino acid sequence is highly homologous to the corresponding human protein and consists of 631 amino acid residues, including a 22 amino acid N-terminal cleavable signal sequence, and a single 23 amino acid putative transmembrane domain. Northern blot analysis reveals a single -2.4 kb message expressed in a number of rat cell lines and in adult liver. The gene was mapped to mouse chromosome 2, close to the Src proto-oncogene
— id: 14023, year: 1991, vol: 176, page: 1482, stat: Journal Article,

Structure and chromosomal location of the rat ribophorin I gene
Behal A; Prakash K; D'Eustachio P; Adesnik M; Sabatini DD; Kreibich G
1990 May 15;265(14):8252-8258, Journal of biological chemistry
Ribophorin I is a type I transmembrane glycoprotein characteristic of the rough portions of the endoplasmic reticulum where it is thought to play a role in the cotranslational insertion of nascent polypeptides. A rat ribophorin I cDNA was used to isolate four overlapping genomic clones from a rat EMBL3 genomic library. Restriction mapping, Southern blotting, and DNA sequencing showed that these clones, spanning approximately 21 kilobases of chromosomal DNA, include the entire ribophorin I gene, as well as 15 kilobases (kb) of upstream sequences. Southern blotting analysis of DNA from a panel of mouse-Chinese hamster cell hybrids demonstrated that the ribophorin I gene is located on mouse chromosome six. The ribophorin I gene contains 10 exons, seven of which encode the luminal domain of the polypeptide. Exon 8 encodes the trans-membrane domain and small portions of the flanking luminal and cytoplasmic domains. Exons 9 and 10 encode the remainder of the cytoplasmic domain, and the latter includes the 3'-untranslated portion of the mRNA. Six closely spaced transcription start sites located 3 to 24 base pairs upstream from the initiation codon were identified by primer extension analysis and S1 mapping. The sequence of a 1.3-kb region upstream of the cap sites was determined and found to contain three GC-rich potential Sp1-binding sites beginning at -14, -24, and -91 base pairs (bp), two octamer-like sequences at -233 and -1248 bp, and a CAAT-like box at -41 bp. The possible roles of these elements in regulating expression of the ribophorin gene in all cells and in differentiated cell types characterized by a well developed rough endoplasmic reticulum is discussed
— id: 17245, year: 1990, vol: 265, page: 8252, stat: Journal Article,

Antiribophorin antibodies inhibit the targeting to the ER membrane of ribosomes containing nascent secretory polypeptides
Yu YH; Sabatini DD; Kreibich G
1990 Oct;111(4):1335-1342, Journal of cell biology
Polyclonal antibodies directed against ribophorins I and II, two membrane glycoproteins characteristic of the rough endoplasmic reticulum, inhibit the cotranslational translocation of a secretory protein growth hormone into the lumen of dog pancreas or rat liver microsomes. As expected, site-specific antibodies to epitopes located within the cytoplasmic domain of ribophorin I, but not antibodies to epitopes in the luminal domain of this protein, were effective in inhibiting translocation. Since monovalent Fab fragments were as inhibitory as intact IgG molecules, ribophorins must be closely associated with the translocation site and, therefore, are likely to function at some stage in the translocation process. In all cases, the antibodies that inhibited translocation also caused a significant reduction in total protein synthesis and treatments that neutralized their capacity to inhibit translocation also prevented their inhibitory effect on protein synthesis. This would be expected if the antibodies blocked the membrane-mediated relief of the SRP-induced arrest of polypeptide elongation. The antibodies were effective only when added before translocation was allowed to begin. In this case, they prevented the targeting of active ribosomes containing mRNA and nascent chains to the ER membrane. Thus, ribophorins must either directly participate in targeting or be so close to the targeting site that the antibodies sterically blocked this early phase of the translocation process
— id: 18415, year: 1990, vol: 111, page: 1335, stat: Journal Article,

Endolyn-78, a membrane glycoprotein present in morphologically diverse components of the endosomal and lysosomal compartments: implications for lysosome biogenesis
Croze E; Ivanov IE; Kreibich G; Adesnik M; Sabatini DD; Rosenfeld MG
1989 May;108(5):1597-1613, Journal of cell biology
A monoclonal antibody (2C5) raised against rat liver lysosomal membranes was used to identify a 78-kD glycoprotein that is present in the membranes of both endosomes and lysosomes and, therefore, is designated endolyn-78. In cultures of rat hepatoma (Fu5C8) and kidney cells (NRK), this glycoprotein could not be labeled with [35S]methionine or with [32P]inorganic phosphate but was easily labeled with [35S]cysteine and [3H]mannose. Pulse-chase experiments and determinations of endoglycosidase H (endo H) sensitivity showed that endolyn-78 is derived from a precursor of Mr 58-62 kD that is processed to the mature form with a t1/2 of 15-30 min. The protein has a 22-kD polypeptide backbone that is detected after a brief pulse in tunicamycin-treated cells. During a chase in the presence of the drug, this is converted into an O-glycosylated product of 46 kD that despite the absence of N-linked oligosaccharides is effectively transferred to lysosomes. This demonstrates that the delivery of endolyn-78 to this organelle is not mediated by the mannose-6-phosphate receptor (MPR). Immunocytochemical experiments showed that endolyn-78 is present in the limiting membranes and the interior membranous structures of morphologically identifiable secondary lysosomes that contain the lysosomal hydrolase beta-glucuronidase, lack the MPR, and could not be labeled with alpha-2-macroglobulin at 18.5 degrees C, a temperature which prevents appearance of endocytosed markers in lysosomes. Endolyn-78 was present at low levels in the plasma membrane and in peripheral tubular endosomes, but was prominent in morphologically diverse components of the endosomal compartment (vacuolar endosomes and various types of multivesicular bodies) which acquired alpha-2-macroglobulin at 18.5 degrees C, and frequently contained substantial levels of the MPR and variable levels of beta-glucuronidase. On the other hand, the MPR was very rarely found in endolyn-containing structures that were not labeled with alpha-2-macroglobulin at the low temperature. Thus, the process of lysosomal maturation appears to involve the progressive delivery of lysosomal enzymes to various types of endosomes that may have already received some of the lysosomal membrane proteins. Although endolyn-78 would be one of the proteins added early to endosomes, other lysosomal membrane proteins may be added only to multivesicular endosomes that represent very advanced stages of maturation
— id: 10656, year: 1989, vol: 108, page: 1597, stat: Journal Article,

Reconstitution of translocation-competent membrane vesicles from detergent-solubilized dog pancreas rough microsomes
Yu YH; Zhang YY; Sabatini DD; Kreibich G
1989 Dec;86(24):9931-9935, Proceedings of the National Academy of Sciences of the United States of America
Dog pancreas rough microsomes were solubilized in 1% octyl beta-glucoside, and membrane vesicles were reconstituted by slow 30-fold dilution with a buffer of low ionic strength. Asymmetric assembly of the membranes occurred during reconstitution since the vesicles formed contained ribosomes bound only to the vesicular outer surfaces. The reconstituted vesicles were similar in protein composition to native rough microsomes, although these vesicles were largely devoid of luminal-content proteins. These reconstituted vesicles could translocate and process nascent secretory (human placental lactogen) and membrane proteins (influenza hemagglutinin and rat liver ribophorin I) synthesized in cell-free translation systems programmed with the corresponding mRNAs. Signal cleavage and N-glycosylation only occurred when the reconstituted membranes were present during translation, providing evidence that the translocation apparatus was asymmetrically assembled into the reconstituted membranes. When a supernatant lacking ribosomes and particles greater than 50S from centrifuging the detergent-solubilized microsomes at high speed was used for reconstitution, smooth-surfaced membrane vesicles were obtained that, except for the absence of ribosomal proteins, were similar in protein composition to that of the reconstituted vesicles from total solubilized rough microsomes. The reconstituted smooth-surfaced vesicles, however, were totally inactive in cotranslational processing and translocation of nascent polypeptides. These findings suggest that ribosomes and/or large macromolecular complexes, not dissociated under our solubilization conditions, are essential for in vitro assembly of a functional translocation apparatus
— id: 10409, year: 1989, vol: 86, page: 9931, stat: Journal Article,

STRUCTURE OF THE RAT RIBOPHORIN I GENE
BEHAL A; PRAKASH K; ADESNIK M; SABATINI D D; KREIBICH G
1988 ;107(6 PART 3):762A-762A, Journal of cell biology
— id: 104651, year: 1988, vol: 107, page: 762A, stat: Journal Article,

A molecular map of the chicken major histocompatibility complex: the class II beta genes are closely linked to the class I genes and the nucleolar organizer
Guillemot F; Billault A; Pourquie O; Behar G; Chausse AM; Zoorob R; Kreibich G; Auffray C
1988 Sep;7(9):2775-2785, EMBO journal
We have cloned in a cosmid vector four DNA clusters covering 320 kb of the chicken MHC (B complex), including five class II (B-L) beta genes defining two related isotypic families. Additional B complex genes have been revealed using tissue-specific cDNA probes. A cosmid fragment has been used to isolate a cDNA for a class I (B-F) transcript. This transcript, that is by far the most divergent known member of the class I gene family, hybridized to six B-F genes present in the cosmids. One of the clusters was shown to contain two rRNA transcriptional units from the nucleolar organizer region (NOR), marking the telomeric boundary of the B complex. None of the other B complex genes hybridizes to, or has the transcriptional characteristics of mammalian MHC class II alpha or class III genes. The map we have obtained shows that the B complex does not contain well defined class I and class II regions since B-F and B-L beta genes are closely associated with unrelated genes. Moreover, class II beta genes are very closely linked to class I genes in two clusters, and to the NOR in a third one
— id: 18417, year: 1988, vol: 7, page: 2775, stat: Journal Article,

Signals for the incorporation and orientation of cytochrome P450 in the endoplasmic reticulum membrane
Monier S; Van Luc P; Kreibich G; Sabatini DD; Adesnik M
1988 Aug;107(2):457-470, Journal of cell biology
Cytochrome P450b is an integral membrane protein of the rat hepatocyte endoplasmic reticulum (ER) which is cotranslationally inserted into the membrane but remains largely exposed on its cytoplasmic surface. The extreme hydrophobicity of the amino-terminal portion of P450b suggests that it not only serves to initiate the cotranslational insertion of the nascent polypeptide but that it also halts translocation of downstream portions into the lumen of the ER and anchors the mature protein in the membrane. In an in vitro system, we studied the cotranslational insertion into ER membranes of the normal P450b polypeptide and of various deletion variants and chimeric proteins that contain portion of P450b linked to segments of pregrowth hormone or bovine opsin. The results directly established that the amino-terminal 20 residues of P450b function as a combined insertion-halt-transfer signal. Evidence was also obtained that suggests that during the early stages of insertion, this signal enters the membrane in a loop configuration since, when the amino-terminal hydrophobic segment was placed immediately before a signal peptide cleavage site, cleavage by the luminally located signal peptidase took place. After entering the membrane, the P450b signal, however, appeared to be capable of reorienting within the membrane since a bovine opsin peptide segment linked to the amino terminus of the signal became translocated into the microsomal lumen. It was also found that, in addition to the amino-terminal combined insertion-halt-transfer signal, only one other segment within the P450b polypeptide, located between residues 167 and 185, could serve as a halt-transfer signal and membrane-anchoring domain. This segment was shown to prevent translocation of downstream sequences when the amino-terminal combined signal was replaced by the conventional cleavable insertion signal of a secretory protein
— id: 11017, year: 1988, vol: 107, page: 457, stat: Journal Article,

RAPID INTRACELLULAR DEGRADATION OF A TRUNCATED FORM OF RIBOPHORIN I
TSAO Y-S; IVESSA N E; ADESNIK M; SABATINI D D; KREIBICH G
1988 ;107(6 PART 3):764-764, Journal of cell biology
— id: 104655, year: 1988, vol: 107, page: 764, stat: Journal Article,

Antibodies to liver/kidney microsome1 in chronic active hepatitis recognize specific forms of hepatic cytochrome P-450
Waxman DJ; Lapenson DP; Krishnan M; Bernard O; Kreibich G; Alvarez F
1988 Nov;95(5):1326-1331, Gastroenterology
Anti-liver/kidney microsome1-positive sera from children with chronic active hepatitis were studied in an effort to identify the microsomal antigens selected during induction and progression of this autoimmune disease. Immunoblot analysis of sodium dodecyl sulfate gel-resolved microsomal proteins from human and rat liver using anti-liver/kidney microsome1-positive sera revealed a single polypeptide of 48 kilodaltons (human microsomes) or 50 kilodaltons (rat microsomes). Levels of the 50-kilodalton rat microsomal polypeptide were suppressed in vivo by several drugs known to modulate expression of individual forms (enzymes) of hepatic cytochrome P-450, with the largest decrease effected by phenobarbital. Dot blot analysis using a panel of 10 electrophoretically homogeneous rat liver cytochrome P-450 forms under nondenaturing conditions established that the two methylcholanthrene-inducible forms, P-450 BNF-B and P-450 ISF-G (P-450 gene subfamily IA), are selectively recognized by the anti-liver/kidney microsome1 antibodies. These findings demonstrate that sera associated with autoimmune (anti-liver/kidney microsome1) chronic active hepatitis are specifically reactive with select rat hepatic P-450 forms and suggest that these autoantibodies may be principally directed against one or more constitutive forms of the corresponding human liver cytochromes
— id: 18416, year: 1988, vol: 95, page: 1326, stat: Journal Article,

Anti-liver-kidney microsome antibody is a marker for the rat hepatocyte endoplasmic reticulum
De Lemos-Chiarandini C; Alvarez F; Bernard O; Homberg JC; Kreibich G
1987 May-Jun;7(3):468-475, Hepatology
Human sera, containing anti-liver-kidney microsome antibody as demonstrated by indirect immunofluorescence, were obtained from a subgroup of young patients with autoimmune chronic hepatitis. The anti-liver-kidney microsome antibody-positive sera were used to study the localization of the liver-kidney microsome antigen in hepatocytes. Immunoblot analysis of microsomal subfractions, lysosomal membranes, plasma membranes, mitochondria and purified ribosomes obtained from rat liver demonstrated that this antibody recognizes a protein of 50 kD present only in endoplasmic reticulum membranes. Immunogold labeling of ultrathin frozen sections and immunoperoxidase staining of 11 to 15 micron cryostat sections were used to detect the liver-kidney microsome antigen in rat liver tissue. The anti-liver-kidney microsome antibody binds to antigenic domains on the cytoplasmic face of smooth and rough endoplasmic reticulum membranes of hepatocytes. No labeling was observed of the Golgi apparatus, peroxisomes, mitochondria, lysosomes, nuclei or plasma membranes. Not only was the antigen recognized by the anti-liver-kidney microsome antibody specific for endoplasmic reticulum membranes, but it was also specific for the endoplasmic reticulum of hepatocytes only, since no labeling was observed in any organelle of Kupffer or endothelial cells. Therefore, the anti-liver-kidney microsome antibody can be considered as a marker for endoplasmic reticulum in rat hepatocytes
— id: 18419, year: 1987, vol: 7, page: 468, stat: Journal Article,

Determination of the membrane topology of the phenobarbital-inducible rat liver cytochrome P-450 isoenzyme PB-4 using site-specific antibodies
De Lemos-Chiarandini C; Frey AB; Sabatini DD; Kreibich G
1987 Feb;104(2):209-219, Journal of cell biology
Fifteen peptides, ranging in length from 6 to 31 amino acids and corresponding in sequence to portions of the major phenobarbital-inducible form of rat liver cytochrome P-450 (P-450 PB-4), were previously synthesized chemically and used to prepare site-specific rabbit antibodies (Frey, A. B., D.J. Waxman, and G. Kreibich, 1985, J. Biol. Chem., 260:15253-15265). The antipeptide antibodies were affinity purified using Sepharose resins derivatized with the respective peptides and 14 preparations were obtained that in an ELISA assay showed affinities to immobilized P-450 judged to be adequate for binding studies on intact rat liver microsomes. The binding of these antibodies to rough microsomes from the livers of phenobarbital treated rats was assessed using 125I-labeled IgG and by immunoelectron microscopy employing protein A-gold as a marker. It was found that many of the antibodies bound to the cytoplasmic surface of the membrane but none bound to the luminal face of ruptured or inverted microsomal vesicles or to contaminating membranes of other organelles present in the preparations. These observations eliminate previously proposed models for the transmembrane disposition of P-450 that postulate the existence of multiple transmembrane domains and the exposure of several polar segments of the polypeptide on the luminal side of the membrane. The fact that an antibody raised to the first 31 residues of P-450 bound well to the purified P-450 but very poorly to rough microsomes, whereas an antibody to a peptide comprising residues 24-38 showed relatively strong binding to intact microsomes, is consistent with the proposal that the amino terminal segment of P-450 extending approximately to residue 20 is embedded in the phospholipid bilayer and the immediately following segment is exposed on the cytoplasmic surface of the membrane. All these results favor a model in which the cytochrome P-450 molecule is largely exposed on the cytoplasmic surface of the endoplasmic reticulum membrane to which it is anchored by its short amino terminal hydrophobic segment
— id: 18421, year: 1987, vol: 104, page: 209, stat: Journal Article,

The influenza hemagglutinin insertion signal is not cleaved and does not halt translocation when presented to the endoplasmic reticulum membrane as part of a translocating polypeptide
Finidori J; Rizzolo L; Gonzalez A; Kreibich G; Adesnik M; Sabatini DD
1987 Jun;104(6):1705-1714, Journal of cell biology
The co-translational insertion of polypeptides into endoplasmic reticulum membranes may be initiated by cleavable amino-terminal insertion signals, as well as by permanent insertion signals located at the amino-terminus or in the interior of a polypeptide. To determine whether the location of an insertion signal within a polypeptide affects its function, possibly by affecting its capacity to achieve a loop disposition during its insertion into the membrane, we have investigated the functional properties of relocated insertion signals within chimeric polypeptides. An artificial gene encoding a polypeptide (THA-HA), consisting of the luminal domain of the influenza hemagglutinin preceded by its amino-terminal signal sequence and linked at its carboxy-terminus to an intact prehemagglutinin polypeptide, was constructed and expressed in in vitro translation systems containing microsomal membranes. As expected, the amino-terminal signal initiated co-translational insertion of the hybrid polypeptide into the membranes. The second, identical, interiorized signal, however, was not recognized by the signal peptidase and was translocated across the membrane. The failure of the interiorized signal to be cleaved may be attributed to the fact that it enters the membrane as part of a translocating polypeptide and therefore cannot achieve the loop configuration that is thought to be adopted by signals that initiate insertion. The finding that the interiorized signal did not halt translocation of downstream sequences, even though it contains a hydrophobic region and must enter the membrane in the same configuration as natural stop-transfer signals, indicates that the HA insertion signal lacks essential elements of halt transfer signals that makes the latter effective membrane-anchoring domains. When the amino-terminal insertion signal of the THA-HA chimera was deleted, the interior signal was incapable of mediating insertion, probably because of steric hindrance by the folded preceding portions of the chimera. Several chimeras were constructed in which the interiorized signal was preceded by polypeptide segments of various lengths. A signal preceded by a segment of 111 amino acids was also incapable of initiating insertion, but insertion took place normally when the segment preceding the signal was only 11-amino acids long.(ABSTRACT TRUNCATED AT 400 WORDS)
— id: 18418, year: 1987, vol: 104, page: 1705, stat: Journal Article,

Isolation and characterization of cDNA clones for rat ribophorin I: complete coding sequence and in vitro synthesis and insertion of the encoded product into endoplasmic reticulum membranes
Harnik-Ort V; Prakash K; Marcantonio E; Colman DR; Rosenfeld MG; Adesnik M; Sabatini DD; Kreibich G
1987 Apr;104(4):855-863, Journal of cell biology
Ribophorins I and II are two transmembrane glycoproteins that are characteristic of the rough endoplasmic reticulum and are thought to be part of the apparatus that affects the co-translational translocation of polypeptides synthesized on membrane-bound polysomes. A ribophorin I cDNA clone containing a 0.6-kb insert was isolated from a rat liver lambda gtll cDNA library by immunoscreening with specific antibodies. This cDNA was used to isolate a clone (2.3 kb) from a rat brain lambda gtll cDNA library that contains the entire ribophorin I coding sequence. SP6 RNA transcripts of the insert in this clone directed the in vitro synthesis of a polypeptide of the expected size that was immunoprecipitated with anti-ribophorin I antibodies. When synthesized in the presence of microsomes, this polypeptide, like the translation product of the natural ribophorin I mRNA, underwent membrane insertion, signal cleavage, and co-translational glycosylation. The complete amino acid sequence of the polypeptide encoded in the cDNA insert was derived from the nucleotide sequence and found to contain a segment that corresponds to a partial amino terminal sequence of ribophorin I that was obtained by Edman degradation. This confirmed the identity of the cDNA clone and established that ribophorin I contains 583 amino acids and is synthesized with a cleavable amino terminal insertion signal of 22 residues. Analysis of the amino acid sequence of ribophorin I suggested that the polypeptide has a simple transmembrane disposition with a rather hydrophilic carboxy terminal segment of 150 amino acids exposed on the cytoplasmic face of the membrane, and a luminal domain of 414 amino acids containing three potential N-glycosylation sites. Hybridization measurements using the cloned cDNA as a probe showed that ribophorin I mRNA levels increase fourfold 15 h after partial hepatectomy, in confirmation of measurements made by in vitro translation of liver mRNA. Southern blot analysis of rat genomic DNA suggests that there is a single copy of the ribophorin I gene in the haploid rat genome
— id: 18420, year: 1987, vol: 104, page: 855, stat: Journal Article,

BIOSYNTHESIS OF A CYSTEINE-RICH, HIGHLY GLYCOSYLATED PROTEIN PRESENT IN LYSOSOMAL AND ENDOSOMAL MEMBRANES
CROZE, E; IVANOV, I; SNITKIN, H; KREIBICH, G; SABATINI, DD; ROSENFELD, MG
1986 NOV ;103(5):A355-A355, Journal of cell biology
— id: 41323, year: 1986, vol: 103, page: A355, stat: Journal Article,

3-(Trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine photolabels a substrate-binding site of rat hepatic cytochrome P-450 form PB-4
Frey AB; Kreibich G; Wadhera AB; Clarke L; Waxman DJ
1986 Aug 26;25(17):4797-4803, Biochemistry
Hepatic microsomes isolated from untreated male rats or from rats pretreated with phenobarbital (PB) or 3-methylcholanthrene (3-MC) were labeled with the hydrophobic, photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID). [125I]TID incorporation into 3-MC- and PB-induced liver microsomal protein was enhanced 5- and 8-fold, respectively, relative to the incorporation of [125I]TID into uninduced liver microsomes. The major hepatic microsomal cytochrome P-450 forms inducible by PB and 3-MC, respectively designated P-450s PB-4 and BNF-B, were shown to be the principal polypeptides labeled by [125I]TID in the correspondingly induced microsomes. Trypsin cleavage of [125I]TID-labeled microsomal P-450 PB-4 yielded several radiolabeled fragments, with a single labeled peptide of Mr approximately 4000 resistant to extensive proteolytic digestion. The following experiments suggested that TID binds to the substrate-binding site of P-450 PB-4. [125I]TID incorporation into microsomal P-450 PB-4 was inhibited in a dose-dependent manner by the P-450 PB-4 substrate benzphetamine. In the absence of photoactivation, TID inhibited competitively about 80% of the cytochrome P-450-dependent 7-ethoxycoumarin O-deethylation catalyzed by PB-induced microsomes with a Ki of 10 microM; TID was a markedly less effective inhibitor of the corresponding activity catalyzed by microsomes isolated from uninduced or beta-naphthoflavone-induced livers.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 18423, year: 1986, vol: 25, page: 4797, stat: Journal Article,

SIGNALS FOR INSERTION OF CYTOCHROME-P-450 INTO ENDOPLASMIC- RETICULUM MEMBRANES
Monier, S; Vanluc, P; Kreibich, G; Sabatini, DD; Adesnik, M
1986 Nov;103(5):A290-A290, Journal of cell biology
— id: 31007, year: 1986, vol: 103, page: A290, stat: Journal Article,

Nucleotide sequence of rat preputial gland beta-glucuronidase cDNA and in vitro insertion of its encoded polypeptide into microsomal membranes
Nishimura Y; Rosenfeld MG; Kreibich G; Gubler U; Sabatini DD; Adesnik M; Andy R
1986 Oct;83(19):7292-7296, Proceedings of the National Academy of Sciences of the United States of America
We have selected the rat preputial gland beta-glucuronidase as a model protein to study the sorting of newly synthesized lysosomal hydrolases to the lysosome. The complete coding sequence of beta-glucuronidase messenger RNA was determined from the sequences of a group of overlapping cDNA clones isolated from preputial gland cDNA libraries. The beta-glucuronidase mRNA primary translation product contains 648 amino acids, including an amino-terminal signal sequence of 22 residues. The polypeptide has four potential sites for the addition of asparagine-linked core oligosaccharides. A 376-residue segment of beta-glucuronidase shows extensive homology (23% sequence identity) to a portion of Escherichia coli beta-galactosidase. This homology most likely reflects an evolutionary relationship between the bacterial and eukaryotic enzymes and the conservation of structural features necessary for the glycosidase activity of both proteins. Translation of mRNA synthesized in vitro by transcription of a cDNA containing the entire beta-glucuronidase coding region yielded a polypeptide that was immunoprecipitated with anti-beta-glucuronidase antiserum and had the same electrophoretic mobility as the primary translation product of natural beta-glucuronidase mRNA. In the presence of microsomal membranes, the in vitro-synthesized beta-glucuronidase underwent cotranslational incorporation into the microsomes, as indicated by removal of the signal sequence and the addition of several oligosaccharide chains. The beta-glucuronidase cDNA will provide a useful tool to study the mechanism of mannose phosphorylation and other aspects of the sorting of lysosomal enzymes to lysosomes
— id: 18422, year: 1986, vol: 83, page: 7292, stat: Journal Article,

NUCLEOTIDE-SEQUENCE OF RAT PREPUTIAL GLAND BETA-GLUCURONIDASE CDNA AND INVITRO INSERTION OF ITS ENCODED POLYPE
Nishimura, Y; Rosenfeld, MG; Kreibich, G; Gubler, U; Sabatini, DD; Adesnik, M; Andy, R
1986 Dec;11(4):534-534, Cell structure & function
— id: 31421, year: 1986, vol: 11, page: 534, stat: Journal Article,

3-(TRIFLUOROMETHYL)-3-(META-[I-125]IODOPHENYL)DIAZIRINE ([I-125]TID) LABELS A SUBSTRATE-BINDING SITE OF RAT HEPATIC CYTOCHROME-P-450 (P-450) FORM PB-4
WAXMAN, DJ; FREY, AB; WADHERA, A; CLARKE, L; KREIBICH, G
1986 MAY ;45(6):1506-1506, Federation Proceedings (Federation of American Societies for Experimental Biology)
— id: 41445, year: 1986, vol: 45, page: 1506, stat: Journal Article,

Anti-liver-kidney microsome antibody recognizes a 50,000 molecular weight protein of the endoplasmic reticulum
Alvarez F; Bernard O; Homberg JC; Kreibich G
1985 May 1;161(5):1231-1236, Journal of experimental medicine
Children with autoimmune chronic active hepatitis may have high titers of antibodies detected by immunofluorescence staining of hepatocytes and tubular cells in rat liver and kidney sections, respectively. These antibodies are directed against antigens contained in microsomal fractions prepared from these two organs. We have found that sera from these patients recognized a 50,000 mol wt protein present in higher concentration in smooth microsome subfractions compared with rough microsome subfractions. This protein is an integral membrane protein and is not glycosylated. It is exposed on the cytoplasmic face of the endoplasmic reticulum and is rather resistant to proteolysis with proteinase K. Since patients with liver disease of different etiology and similar severity of cell lysis do not give rise to liver-kidney microsome antibody (LKMA), lysis of hepatocytes is apparently not a sufficient condition for their development
— id: 18425, year: 1985, vol: 161, page: 1231, stat: Journal Article,

The structure of phenobarbital-inducible rat liver cytochrome P-450 isoenzyme PB-4. Production and characterization of site-specific antibodies
Frey AB; Waxman DJ; Kreibich G
1985 Dec 5;260(28):15253-15265, Journal of biological chemistry
Fifteen peptides corresponding in sequence to segments of the major phenobarbital-inducible forms of rat hepatic cytochrome P-450 (termed P-450 PB-4 and P-450 PB-5) were chemically synthesized, conjugated to carrier proteins, and used to prepare site-specific rabbit and/or mouse antipeptide antibodies. Four of the synthetic peptides were recognized by rabbit heterosera raised against purified P-450 PB-4. The titer of these heterosera measured against P-450 PB-4 was only partially reduced upon complete adsorption of antipeptide activity suggesting that these peptides represent minor antigenic determinants. Each of the antipeptide antibodies recognized purified P-450 PB-4 and the highly homologous P-450 PB-5 as demonstrated by a solid-phase enzyme-linked immunosorbent assay. Although each antipeptide immunoprecipitated both purified 125I-labeled P-450 PB-4 and also in vitro-synthesized apo-P-450 PB-4, the yields of immunoprecipitation were low relative to that obtained using anti-P-450 heterosera. Only one of the antipeptide antibodies gave a good signal in an immunoblot analysis of either microsomal or purified P-450s PB-4 and PB-5. Three antipeptide antibodies raised against hydrophilic segments located in the amino-terminal one-third of P-450 PB-4 markedly inhibited the P-450 PB-4-catalyzed O-deethylation of the model substrate 7-ethoxycoumarin. Four of the antipeptide antibodies were found to cross-react with P-450 beta NF-B, the major aromatic hydrocarbon-inducible rat hepatic P-450, suggesting that certain amino acid sequences or regions of secondary structure are conserved between the major phenobarbital-induced and polycyclic-induced rat liver P-450 isoenzymes. These studies demonstrate the utility of antipeptide antibodies for evaluation of antigenic sites exposed in native P-450 PB-4, for identification of specific amino acid sequences important for the interaction of P-450 PB-4 with its substrate and/or with cytochrome P-450 reductase in a reconstituted system and for elucidation of structural and immunochemical homologies between P-450 PB-4 and other P-450 isoenzymes present in rat liver endoplasmic reticulum
— id: 18424, year: 1985, vol: 260, page: 15253, stat: Journal Article,

Segregation of the polypeptide translocation apparatus to regions of the endoplasmic reticulum containing ribophorins and ribosomes. I. Functional tests on rat liver microsomal subfractions
Amar-Costesec A; Todd JA; Kreibich G
1984 Dec;99(6):2247-2253, Journal of cell biology
A preparation of rat liver microsomes containing 70% of the total cellular endoplasmic reticulum (ER) membranes was subfractionated by isopycnic density centrifugation. Twelve subfractions of different ribosome content ranging in density from 1.06 to 1.29 were obtained and analyzed with respect to marker enzymes, RNA, and protein content, as well as the capacity of these membranes to bind 80S ribosomes in vitro. After removal of native polysomes from these microsomal subfractions by puromycin in a buffer of high ionic strength their capacity to rebind 80S ribosomes approached levels found in the corresponding native membranes before ribosome stripping. This indicates that in vitro rebinding of ribosomes occurs to the same sites occupied in the cell by membrane-bound polysomes. Microsomes in the microsomal subfractions were also tested for their capacity to effect the translocation of nascent secretory proteins into the microsomal lumen utilizing a rabbit reticulocyte translation system programmed with mRNA coding for the precursor of human placental lactogen. Membranes from microsomes with the higher isopycnic density and a high ribosome content showed the highest translocation activity, whereas membranes derived from smooth microsomes had only a very low translocation activity. These results indicate the membranes of the rough and smooth portions of the endoplasmic reticulum are functionally differentiated so that sites for ribosome binding and the translocation of nascent polypeptides are segregated to the rough domain of the organelle
— id: 18427, year: 1984, vol: 99, page: 2247, stat: Journal Article,

Induction of cytochrome P-450 isozymes in rat hepatoma-derived cell cultures
Frey AB; Rosenfeld MG; Dolan WJ; Adesnik M; Kreibich G
1984 Aug;120(2):169-180, Journal of cellular physiology
We have investigated the responsiveness of established rat hepatocyte cell cultures to inducers of cytochrome P-450. One Reuber hepatoma-derived line (Fu5-C8), which under normal culture conditions produces no detectable cytochrome P-450(MC) or cytochrome P-450(PB)--the major cytochrome P-450 isozymes induced by 3-methylcholanthrene and phenobarbital, respectively--was tested for the ability to accumulate either cytochrome P-450 isozyme in response to treatment with various xenobiotics. By immune-precipitation from [35S]-methionine-labeled cell extracts, using monospecific anticytochrome P-450(MC) antibody or monoclonal anticytochrome P-450(PB) antibody, it was demonstrated that these cells possess the capability to synthesize cytochrome P-450(MC) in response to 3-methylcholanthrene treatment, while none of the drug treatments caused the synthesis of detectable quantities of cytochrome P-450(PB). RNA extracted from Fu5-C8 cells directed the in vitro synthesis of immune-precipitable cytochrome P-450(MC) only after treatment of the cells with 3-methylcholanthrene. Kinetic analysis of the response of these cells to 3-methylcholanthrene induction revealed detectable levels of immune-precipitable cytochrome P-450(MC) 2 h after drug treatment with maximal induction occurring between 12 and 16 h of exposure. Another cell line (HF 1.5), obtained originally by hybridization of Fao X H5 variants of a Reuber H35 hepatoma, produces cytochrome P-450(MC) and also cytochrome P-450(PB) constitutively, as determined by specific immune-precipitation from labeled cell extracts. Exposure of confluent monolayers to either phenobarbital or 3-methylcholanthrene resulted in an induction of cytochrome P-450(PB) or cytochrome P-450(MC), respectively. Double-labeling immunofluorescence studies indicate that all cells in the culture produce albumin and most of the cells produce cytochrome P-450(MC), but only a subset of cells synthesize cytochrome P-450(PB). Our results demonstrate that some continuously dividing hepatocyte cell cultures retain the capacity to respond to xenobiotics, including phenobarbital, a response which is typically exhibited by fully differentiated liver cells. Such established hepatocyte cell cultures should prove useful for investigating the mechanism of induction of cytochrome P-450(PB)
— id: 18429, year: 1984, vol: 120, page: 169, stat: Journal Article,

QUANTITATIVE-ANALYSIS OF RIBOSOME-BINDING SITES IN RAT-LIVER MICROSOMAL SUBFRACTIONS
KREIBICH, G; SABATINI, DD; AMARCOSTESEC, A
1984 ;92(3):B93-B93, Archives internationales de physiolgoie de biochimie et de biophysique
— id: 40869, year: 1984, vol: 92, page: B93, stat: Journal Article,

Segregation of the polypeptide translocation apparatus to regions of the endoplasmic reticulum containing ribophorins and ribosomes. II. Rat liver microsomal subfractions contain equimolar amounts of ribophorins and ribosomes
Marcantonio EE; Amar-Costesec A; Kreibich G
1984 Dec;99(6):2254-2259, Journal of cell biology
Ribophorins I and II, two transmembrane glycoproteins characteristic of the rough endoplasmic reticulum (ER) are thought to be part of the translocation apparatus for proteins made on membrane bound polysomes. To study the stoichiometry between ribophorins and membrane-bound ribosomes we have determined the RNA and ribophorin content in rat liver microsomes or in microsomal subfractions of different density (i.e., ribosome content). The specificity of antibodies against the ribophorins was demonstrated by Western blot analysis of rat liver rough microsomes separated by 2-dimensional gel electrophoresis. The ribophorin content of microsomal subfractions was determined by indirect immunoprecipitation and for ribophorin I by a radioimmune assay. In the latter assay a molar ratio of ribophorin I/ribosomes approaching one was calculated for total microsomes as well as in the gradient subfractions. We therefore suggest that ribophorins mediate the binding of ribosomes to endoplasmic reticulum membranes or play a role in co-translational process which depend on this binding, such as the insertion of nascent polypeptides into the membrane or their transfer into the cisternal lumen
— id: 18426, year: 1984, vol: 99, page: 2254, stat: Journal Article,

RAT-LIVER ENDOPLASMIC-RETICULUM CONTAINS EQUIMOLAR AMOUNTS OF RIBOPHORINS AND RIBOSOMES
MARCANTONIO, EE; AMARCOSTESEC, A; SABATINI, DD; KREIBICH, G
1984 ;92(3):B95-B95, Archives internationales de physiolgoie de biochimie et de biophysique
— id: 40870, year: 1984, vol: 92, page: B95, stat: Journal Article,

Biosynthesis and processing of ribophorins in the endoplasmic reticulum
Rosenfeld MG; Marcantonio EE; Hakimi J; Ort VM; Atkinson PH; Sabatini D; Kreibich G
1984 Sep;99(3):1076-1082, Journal of cell biology
Ribophorins are two transmembrane glycoproteins characteristic of the rough endoplasmic reticulum, which are thought to be involved in the binding of ribosomes. Their biosynthesis was studied in vivo using lines of cultured rat hepatocytes (clone 9) and pituitary cells (GH 3.1) and in cell-free synthesis experiments. In vitro translation of mRNA extracted from free and bound polysomes of clone 9 cells demonstrated that ribophorins are made exclusively on bound polysomes. The primary translation products of ribophorin messengers obtained from cultured hepatocytes or from regenerating livers co-migrated with the respective mature proteins, but had slightly higher apparent molecular weights (2,000) than the unglycosylated forms immunoprecipitated from cells treated with tunicamycin. This indicates that ribophorins, in contrast to all other endoplasmic reticulum membrane proteins previously studied, contain transient amino-terminal insertion signals which are removed co-translationally. Kinetic and pulse-chase experiments with [35S]methionine and [3H]mannose demonstrated that ribophorins are not subjected to electrophoretically detectable posttranslational modifications, such as proteolytic cleavage or trimming and terminal glycosylation of oligosaccharide side chain(s). Direct analysis of the oligosaccharides of ribophorin l showed that they do not contain the terminal sugars characteristic of complex oligosaccharides and that they range in composition from Man8GlcNAc to Man5GlcNAc. These findings, as well as the observation that the mature proteins are sensitive to endoglycosidase H and insensitive to endoglycosidase D, are consistent with the notion that the biosynthetic pathway of the ribophorins does not require a stage of passage through the Golgi apparatus
— id: 18428, year: 1984, vol: 99, page: 1076, stat: Journal Article,

FUNCTIONAL TRANSLOCATION SITES FOR NEWLY SYNTHESIZED SECRETORY PROTEINS ARE SEGREGATED IN THE ROUGH DOMAIN OF THE RAT-LIVER ENDOPLASMIC-RETICULUM
TODD, JA; KREIBICH, G; SABATINI, DD; AMARCOSTESEC, A
1984 ;92(3):B108-B109, Archives internationales de physiolgoie de biochimie et de biophysique
— id: 40871, year: 1984, vol: 92, page: B108, stat: Journal Article,

Biosynthesis of myelin-specific proteins
Colman DR; Kreibich G; Sabatini DD
1983 ;96(3):378-385, Methods in enzymology
— id: 18435, year: 1983, vol: 96, page: 378, stat: Journal Article,

Studies on the subunit composition of rat liver glutathione S-transferases
Frey AB; Friedberg T; Oesch F; Kreibich G
1983 Sep 25;258(18):11321-11325, Journal of biological chemistry
Native glutathione S-transferases are composed of subunits with apparent molecular weights of 25,000, 23,500, or 22,000 which form either homo- or heterodimers. Glutathione S-transferases A, C, and X which contain two subunits with molecular weights of 23,500 yielded similar but nonidentical proteolytic fragmentation patterns. Fragments unique to the subunits of the homodimers A and X were present in decreased intensities in the patterns of form C. Two-dimensional electrophoresis under denaturing conditions showed single nonoverlapping spots for transferases A and X, while form C yielded two spots corresponding in position to those obtained from forms A and X. Renaturation of dissociated glutathione S-transferase C yielded enzymatically active transferases A, C, and X. These results indicate that form C is a heterodimer composed of one subunit from the homodimeric transferases A and X. This was substantiated by NH2-terminal sequence analysis showing extensive NH2-terminal homology amongst all three forms. However, in the positions where forms A and X yielded different residues, both amino acids were detected in the sequence of form C, indicating that the two subunits of Mr = 23,500 are the products of two different genes. NH2-terminal sequence analysis of the heterodimeric glutathione S-transferase B which is composed of subunits with molecular weights of 22,000 and 25,000 revealed a single unique sequence which bore no resemblance to the sequences of either forms A or X. Despite the identical NH2-terminal sequences, proteolytic fragmentation of the separated subunits showed markedly different fragmentation patterns. This indicates that two different mRNAs code for these two subunits
— id: 18430, year: 1983, vol: 258, page: 11321, stat: Journal Article,

DISTRIBUTION, BIOSYNTHESIS AND PHYSIOLOGICAL-ROLE OF OVINE CORTICOTROPIN RELEASING-FACTOR IN MAN
HOLLANDER, CS; AUDHYA, T; FREY, A; KREIBICH, G; SABATINI, D; NAKANE, T; KAGEYAMA, N; KUWAYAMA, A; GOLBE, L; SCHLESINGER, D
1983 ;31(2):A528-A528, Clinical research
— id: 40552, year: 1983, vol: 31, page: A528, stat: Journal Article,

[Microbiological degradation of glucosinolates in defatted rapeseed meal]
Huber J; Kranz G; Kreibich G; Beining K; Kruger M; Weissbach F
1983 ;27(3):257-263, Nahrung
During the degradation of thioglucosides in defatted rape seed meal (RES) microorganisms were found, whose ability to degrade glucosinolates (GSL) and vinyl thio-oxazolidone (VTO) was not known so far. The isolated microorganisms are two strains of bacteria of the species Bacillus cereus and the yeast Trichosporon cutaneum. The degradation of GSL and VTO in the cultural broths by the Bacillus cereus strains was the more complete the more other Gram-negative bacteria from RES were additionally present. Drinking tests with Wistar rats showed that here is a relation between the watersoluble and mainly bitter toxin substances of rape and their influence on the increase of the animals body weight
— id: 18431, year: 1983, vol: 27, page: 257, stat: Journal Article,

Ribophorins I and II: membrane proteins characteristic of the rough endoplasmic reticulum
Kreibich G; Marcantonio EE; Sabatini DD
1983 ;96(3):520-530, Methods in enzymology
— id: 18434, year: 1983, vol: 96, page: 520, stat: Journal Article,

Biosynthesis of hepatocyte endoplasmic reticulum proteins
Kreibich G; Sabatini DD; Adesnik M
1983 ;96(3):530-542, Methods in enzymology
— id: 18433, year: 1983, vol: 96, page: 530, stat: Journal Article,

INCORPORATION OF PROTEINS INTO CELLULAR MEMBRANES AND ORGANELLES
KREIBICH, G; COLMAN, D; ROSENFELD, MG; SABATINI, DD
1983 ;364(9):1164-1165, Hoppe-Seylers zeitschrift fur physiologische Chemie
— id: 40628, year: 1983, vol: 364, page: 1164, stat: Journal Article,

ISOLATION AND CHARACTERIZATION OF CDNA CLONES FOR THE MYELIN BASIC-PROTEINS
MENTABERRY, A; ADESNIK, M; KREIBICH, G; SABATINI, DD; COLMAN, D
1983 ;97(5):A244-A244, Journal of cell biology
— id: 40497, year: 1983, vol: 97, page: A244, stat: Journal Article,

ISOLATION AND CHARACTERIZATION OF CDNA CLONES FOR BETA-GLUCURONIDASE
NISHIMURA, Y; ROSENFELD, M; KREIBICH, G; ADESNIK, M; SABATINI, DD
1983 ;97(5):A103-A103, Journal of cell biology
— id: 40491, year: 1983, vol: 97, page: A103, stat: Journal Article,

Biosynthesis of lysosomal enzymes
Rosenfeld MG; Kreibich G; Sabatini DD; Kato K
1983 ;96(3):764-777, Methods in enzymology
— id: 18432, year: 1983, vol: 96, page: 764, stat: Journal Article,

IDENTIFICATION OF A PROTEIN CHARACTERISTIC OF THE LYSOSOMAL MEMBRANE USING MONOCLONAL-ANTIBODIES
ROSENFELD, M; DOLAN, W; SABATINI, D; KREIBICH, G
1983 ;97(5):A106-A106, Journal of cell biology
— id: 40492, year: 1983, vol: 97, page: A106, stat: Journal Article,

Synthesis and incorporation of myelin polypeptides into CNS myelin
Colman DR; Kreibich G; Frey AB; Sabatini DD
1982 Nov;95(2 Pt 1):598-608, Journal of cell biology
The distribution of newly synthesized proteolipid protein (PLP, 23 kdaltons) and myelin basic proteins (MBPs, 14-21.5 kdaltons) was determined in microsomal and myelin fractions prepared from the brainstems o1 10-30 d-old rats sacrificed at different times after an intracranial injection of 35S-methionine. Labeled MBPs were found in the myelin fraction 2 min after the injection, whereas PLP appeared first in the rough microsomal fraction and only after a lag of 30 min in the myelin fraction. Cell-free translation experiments using purified mRNAs demonstrated that PLP and MBPs are synthesized in bound and free polysomes, respectively. A mechanism involving the cotranslational insertion into the ER membrane and subsequent passage of the polypeptides through the Golgi apparatus is consistent with the lag observed in the appearance of the in vivo-labeled PLP in the myelin membrane. Newly synthesized PLP and MBPs are not proteolytically processed, because the primary translation products synthesized in vitro had the same electrophoretic mobility and N-terminal amino acid sequence as the mature PLP and MBP polypeptides. It was found that crude myelin fractions are highly enriched in mRNAs coding for the MBPs but not in mRNA coding for PLP. This suggests that whereas the bound polysomes synthesizing PLP are largely confined to the cell body, free polysomes synthesizing MBPs are concentrated in oligodendrocyte processes involved in myelination, which explains the immediate incorporation of MBPs into the developing myelin sheath
— id: 18436, year: 1982, vol: 95, page: 598, stat: Journal Article,

POLARIZED DISTRIBUTION OF SUGAR RESIDUES IN THE GOLGI-APPARATUS
Delemos, C; Kreibich, G; Rodriguezboulan, E; Sabatini, DD
1982 ;95(2):A404-A404, Journal of cell biology
— id: 30516, year: 1982, vol: 95, page: A404, stat: Journal Article,

Recovery of ribophorins and ribosomes in "inverted rough" vesicles derived from rat liver rough microsomes
Kreibich G; Ojakian G; Rodriguez-Boulan E; Sabatini DD
1982 Apr;93(1):111-121, Journal of cell biology
Treatment of rat liver rough microsomes (3.5 mg of protein/ml) with sublytical concentrations (0.08%) of the neutral detergent Triton X-100 caused a lateral displacement of bound ribosomes and the formation of ribosomal aggregates on the microsomal surface. At slightly higher detergent concentrations (0.12-0.16%) membrane areas bearing ribosomal aggregates invaginated into the microsomal lumen and separated from the rest of the membrane. Two distinct classes of vesicles could be isolated by density gradient centrifugation from microsomes treated with 0.16% Triton X-100: one with ribosomes bound to the inner membrane surfaces ('inverted rough' vesicles) and another with no ribosomes attached to the membranes. Analysis of the fractions showed that approximately 30% of the phospholipids and 20-30% of the total membrane protein were released from the membranes by this treatment. Labeling with avidin-ferritin conjugates demonstrated that concanavalin A binding sites, which in native rough microsomes are found in the luminal face of the membranes, were present on the outer surface of the inverted rough vesicles. Freeze-fracture electron microscopy showed that both fracture faces had similar concentrations of intramembrane particles. SDS PAGE analysis of the two vesicle subfractions demonstrated that, of all the integral microsomal membrane proteins, only ribophorins I and II were found exclusively in the inverted rough vesicles bearing ribosomes. These observations are consistent with the proposal that ribophorins are associated with the ribosomal binding sites characteristic of rough microsomal membranes
— id: 18439, year: 1982, vol: 93, page: 111, stat: Journal Article,

Identification of ribophorins in rough microsomal membranes from different organs of several species
Marcantonio EE; Grebenau RC; Sabatini DD; Kreibich G
1982 May;124(1):217-222, European journal of biochemistry
Microsomes prepared from several animal sources were analyzed for the presence of proteins corresponding to the ribophorins (I and II) which have been previously characterized in rat liver rough microsomes and appear to be involved in the binding of polysomes to endoplasmic reticulum membranes. In rough microsomal membranes from rat lacrimal gland, rabbit liver, dog and chicken pancreas, and mouse myeloma, ribophorin-like polypeptides with similar electrophoretic mobilities were detected by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. In all cases the polypeptides remained associated with sedimentable polysomes after solubilization of the microsomal membranes with nonionic detergents. Ribophorin-like polypeptides were absent from smooth microsomes. Antibodies raised against each rat liver ribophorin, purified by preparative sodium dodecyl sulfate/polyacrylamide gel electrophoresis, immunoprecipitated only the corresponding polypeptide, indicating no crossreactivity between ribophorins I and II. These antibodies also immunoprecipitated the homologous ribophorins found in microsomal preparations from other organs and species
— id: 18437, year: 1982, vol: 124, page: 217, stat: Journal Article,

Studies on the biosynthesis of microsomal membrane proteins. Site of synthesis and mode of insertion of cytochrome b5, cytochrome b5 reductase, cytochrome P-450 reductase and epoxide hydrolase
Okada Y; Frey AB; Guenthner TM; Oesch F; Sabatini DD; Kreibich G
1982 Feb;122(2):393-402, European journal of biochemistry
— id: 18440, year: 1982, vol: 122, page: 393, stat: Journal Article,

Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution
Rosenfeld MG; Kreibich G; Popov D; Kato K; Sabatini DD
1982 Apr;93(1):135-143, Journal of cell biology
— id: 18438, year: 1982, vol: 93, page: 135, stat: Journal Article,

Mechanisms for the incorporation of proteins into the plasma membrane
Sabatini D; Colman D; Sabban E; Sherman J; Morimoto T; Kreibich G; Adesnik M
1982 ;46 Pt 2(1):807-818, Cold Spring Harbor symposia on quantitative biology
— id: 18442, year: 1982, vol: 46 Pt 2, page: 807, stat: Journal Article,

Mechanisms for the incorporation of proteins in membranes and organelles
Sabatini DD; Kreibich G; Morimoto T; Adesnik M
1982 Jan;92(1):1-22, Journal of cell biology
— id: 18441, year: 1982, vol: 92, page: 1, stat: Journal Article,

BIOSYNTHESIS OF CNS MYELIN MEMBRANE-PROTEINS
Colman, DR; Kreibich, G; Frey, AB; Sabatini, DD
1981 ;91(2):A404-A404, Journal of cell biology
— id: 30541, year: 1981, vol: 91, page: A404, stat: Journal Article,

Precursor of rat liver alpha 2u-globulin: partial amino acid sequence determination of its signal peptide
Ikehara Y; Oda K; Rosenfeld MG; Bar-Nun S; Kreibich G
1981 Dec;90(6):1833-1836, Journal of biochemistry (Tokyo)
The biosynthesis of alpha 2 u-globulin, the major protein in rat urine, was studied in a cell-free system programmed with messenger RNA isolated from rat liver. Analysis of the primary translation product by SDS-polyacrylamide gel electrophoresis showed a molecular weight of approximatley 22,000 daltons as compared to 20,000 daltons for the mature form. When translation was carried out in the presence of dog pancreas microsomes, alpha 2u-globulin was cotranslationally processed and sequestered in the microsomal lumen. Automated sequencing to mature alpha 2u-globulin and the labeled precursor disclosed the presence of 19 additional amino acids at the amino-terminal end of the primary translation product of the urinary alpha 2u-globulin. The partial amino acid sequence of this extension was determined
— id: 18443, year: 1981, vol: 90, page: 1833, stat: Journal Article,

SYNTHESIS AND COTRANSLATIONAL PROCESSING OF RIBOPHORINS
Rosenfeld, MG; Marcantonio, EE; Harnik, VM; Sabatini, DD; Kreibich, G
1981 ;91(2):A404-A404, Journal of cell biology
— id: 30542, year: 1981, vol: 91, page: A404, stat: Journal Article,

Membrane And Organelle Biogenesis A Brief Synopsis Of Current Concepts
Sabatini DD; Kreibich G; Morimoto T; Adesnik M
Mitochondria and microsomes : in honor of Lars Ernster / Reading, Mass. : Addison-Wesley, 1981,
— id: 5224, year: 1981, vol: , page: 563, stat: Chapter,

Synthesis and insertion of cytochrome P-450 into endoplasmic reticulum membranes
Bar-Nun S; Kreibich G; Adesnik M; Alterman L; Negishi M; Sabatini DD
1980 Feb;77(2):965-969, Proceedings of the National Academy of Sciences of the United States of America
Treatment of rats with phenobarbital leads to a substantial increase in levels of translatable liver cytochrome P-450 mRNA. This mRNA is primarily associated with ribosomes bound to endoplasmic reticulum membranes which in an in vitro system synthesized approximately 10 times more cytochrome P-450 than did free polysomes from the same animals. Cytochrome P-450 synthesized by rough microsomes in vitro appears to be directly inserted into the membranes because it was not released by a treatment with low detergent concentrations that released albumin and other microsomal content proteins. The amino-terminal amino acid sequence of cytochrome P-450 synthesized in an mRNA-dependent system resembles in hydrophobicity the signal segment of presecretory proteins and therefore may serve to insert the polypeptide into the membrane during synthesis. In contrast to the situation with secretory proteins and several other membrane proteins, however, the putative insertion signal of cytochrome P-450 is not removed by a membrane-associated peptidase and remains in the mature polypeptide
— id: 18444, year: 1980, vol: 77, page: 965, stat: Journal Article,

Functional and structural characteristics of endoplasmic reticulum proteins associated with ribosome binding sites
Kreibich G; Czako-Graham M; Grebenau RC; Sabatini DD
1980 ;343(2):17-33, Annals of the New York Academy of Sciences
— id: 18445, year: 1980, vol: 343, page: 17, stat: Journal Article,

FUNCTIONS OF MEMBRANE-BOUND RIBOSOMES IN EUKARYOTIC CELLS
Kreibich, G
1980 ;361(4):497-498, Hoppe-Seylers zeitschrift fur physiologische Chemie
— id: 28128, year: 1980, vol: 361, page: 497, stat: Journal Article,

STRUCTURAL AND FUNCTIONAL-CHARACTERISTICS OF ENDOPLASMIC- RETICULUM MEMBRANE-PROTEINS RELATED TO TRANSLATION ON BOUND POLYSOMES
Kreibich, G; Sabatini, DD
1980 ;22(1):153-153, European journal of cell biology
— id: 28098, year: 1980, vol: 22, page: 153, stat: Journal Article,

Stimulation of choline incorporation in cell cultures by phorbol derivatives and its correlation with their irritant and tumor-promoting activity
Kinzel V; Kreibich G; Hecker E; Suss R
1979 Jul;39(7 Pt 1):2743-2750, Cancer research
— id: 18446, year: 1979, vol: 39, page: 2743, stat: Journal Article,

RECONSTITUTION OF MICROSOMAL VESICLES CONTAINING FUNCTIONAL BINDING-SITES FOR RIBOSOMES
Czakograham, M; Sabatini, DD; Algranati, I; Bard, E; Morimoto, T; Kreibich, G
1978 ;37(6):1568-1568, Federation Proceedings (Federation of American Societies for Experimental Biology)
— id: 29907, year: 1978, vol: 37, page: 1568, stat: Journal Article,

Characterization of the ribosomal binding site in rat liver rough microsomes: ribophorins I and II, two integral membrane proteins related to ribosome binding
Kreibich G; Czako-Graham M; Grebenau R; Mok W; Rodriguez-Boulan E; Sabatini DD
1978 ;8(3):279-302, Journal of supramolecular structure
Rat liver rough endoplasmic reticulum membranes (ER) contain two characteristic transmembrane glycoproteins which have been designated ribophorins I and II and are absent from smooth ER membranes. These proteins (MW 65,000 and 63,000 respectively) are related to the binding sites for ribosomes, as suggested by the following findings: i) The ribophorin content of the rough ER membranes corresponds stoichiometrically to the number of bound ribosomes; ii) ribophorins are quantitatively recovered with the bound polysomes after most other ER membrane proteins are dissolved with the nonionic detergent Kyro EOB; iii) in intact rough microsomes ribophorins can be cross-linked chemically to the ribosomes and therefore are in close proximity to them. Treatment of rough microsomes with a low Triton-X-100 concentration leads to the lateral displacement of ribosomes on the microsomal surface and to the formation of aggregates of bound ribosomes in areas of membranes which frequently invaginate into the microsomal lumen. Subfractionation of Triton-treated microsomes containing invaginations led to the recovery of smooth and 'rough-inverted' vesicles. Ribophorins were present only in the latter fraction, indicating that both proteins are displaced together with the ribosomes when these aggregate without detaching. Measurements of the ribosome-binding capacity of rough and smooth microsomal membranes reconstituted after solubilization with detergents suggest that ribophorins are necessary for in vitro ribosome binding. Ribophorin-like proteins were found in rough microsomes obtained from secretory tissues of several animal species. The two proteins present in rat lacrimal gland microsomes have the same mobility as hepatocyte ribophorins and cross-react with antisera against them
— id: 18452, year: 1978, vol: 8, page: 279, stat: Journal Article,

Proteins of rough microsomal membranes related to ribosome binding. II. Cross-linking of bound ribosomes to specific membrane proteins exposed at the binding sites
Kreibich G; Freienstein CM; Pereyra BN; Ulrich BL; Sabatini DD
1978 May;77(2):488-506, Journal of cell biology
Two proteins (ribophorins I and II), which are integral components of rough microsomal membranes and appear to be related to the bound ribosomes, were shown to be exposed on the surface of rat liver rough microsomes (RM) and to be in close proximity to the bound ribosomes. Both proteins were labeled when intact RM were incubated with a lactoperoxidase iodinating system, but only ribophorin I was digested during mild trypsinization of intact RM. Ribophorin II (63,000 daltons) was only proteolyzed when the luminal face of the microsomal vesicles was made accessible to trypsin by the addition of sublytical detergent concentrations. Only 30--40% of the bound ribosomes were released during trypsinization on intact RM, but ribosome release was almost complete in the presence of low detergent concentrations. Very low glutaraldehyde concentrations (0.005--0.02%) led to the preferential cross-linking of large ribosomal subunits of bound ribosomes to the microsomal membranes. This cross-linking prevented the release of subunits caused by puromycin in media of high ionic strength, but not the incorporation of [3H]puromycin into nascent polypeptide chains. SDS-acrylamide gel electrophoresis of cross-linked samples a preferential reduction in the intensity of the bands representing the ribophorins and the formation of aggregates which did not penetrate into the gels. At low methyl-4-mercaptobutyrimidate (MMB) concentrations (0.26 mg/ml) only 30% of the ribosomes were cross-linked to the microsomal membranes, as shown by the puromycin-KCl test, but membranes could still be solubilized with 1% DOC. This allowed the isolation of the ribophorins together with the sedimentable ribosomes, as was shown by electrophoresis of the sediments after disruption of the cross-links by reduction. Experiments with RM which contained only inactive ribosomes showed that the presence of nascent chains was not necessary for the reversible cross-linking of ribosomes to the membranes. These observations suggest that ribophorins are in close proximity to the bound ribosomes, as may be expected from components of the ribosome-binding sites
— id: 18451, year: 1978, vol: 77, page: 488, stat: Journal Article,

Proteins of rough microsomal membranes related to ribosome binding. I. Identification of ribophorins I and II, membrane proteins characteristics of rough microsomes
Kreibich G; Ulrich BL; Sabatini DD
1978 May;77(2):464-487, Journal of cell biology
Rat liver rough microsomes (RM) contain two integral membrane proteins which are not found in smooth microsomes (SM) and appear to be related to the presence of ribosome-binding sites. These proteins, of molecular weight 65,000 and 63,000, were designated ribophorins I and II, respectively. They were not released from the microsomal membranes by alkali or acid treatment, or when the ribosomes were detached by incubation with puromycin in a high salt medium. The anionic detergent sodium deoxycholate caused solubilization of the ribophorins, but neutral detergents led to their recovery with the sedimentable ribosomes. Ribosomal aggregates containing both ribophorins, but few other membrane proteins, were obtained from RM treated with the nonionic detergent Kyro EOB (2.5 X10(-2) M) in a low ionic strength medium. Sedimentation patterns produced by these aggregates resembled those of large polysomes but were not affected by RNase treatment. The aggregates, however, were dispersed by mild trypsinization (10 microgram trypsin for 30 min at 0 degrees C), incubation with deoxycholate, or in a medium of high salt concentration. These treatments led to a concomitant degradation or release of the ribophorins. It was estimated, from the staining intensity of protein bands in acrylamide gels, that in the Kyro EOB aggregates there were one to two molecules of each ribophorin per ribosome. Sedimentable complexes without ribosomes containing both ribophorins could also be obtained by dissolving RM previously stripped of ribosomes by puromycin-KCl using cholate, a milder detergent than DOC. Electron microscope examination of the residue obtained from RM treated with Kyro EOB showed that the rapidly sedimenting polysome-like aggregates containing the ribophorins consisted of groups of tightly packed ribosomes which were associated with remnants of the microsomal membranes
— id: 18450, year: 1978, vol: 77, page: 464, stat: Journal Article,

RECOVERY OF RIBOPHORINS IN INVERTED ROUGH VESICLES DERIVED FROM RAT-LIVER MICROSOMES
Kreibich, G; Ojakian, G; Rodriguezboulan, E; Feng, T; Sabatini, DD
1978 ;79(2):A231-A231, Journal of cell biology
— id: 29874, year: 1978, vol: 79, page: A231, stat: Journal Article,

Coordinated polypeptide synthesis and insertion of protoheme in cytochrome P-450 during development of endoplasmic reticulum membranes
Negishi M; Kreibich G
1978 Jul 10;253(13):4791-4797, Journal of biological chemistry
— id: 18449, year: 1978, vol: 253, page: 4791, stat: Journal Article,

INVITRO SYNTHESIS OF BETA-GLUCURONIDASE BY RAT-LIVER AND PREPUTIAL GLAND MEMBRANE-BOUND RIBOSOMES
Popov, D; Alterman, L; Sabatini, D; Kreibich, G
1978 ;79(2):A364-A364, Journal of cell biology
— id: 29876, year: 1978, vol: 79, page: A364, stat: Journal Article,

Spatial orientation of glycoproteins in membranes of rat liver rough microsomes. I. Localization of lectin-binding sites in microsomal membranes
Rodriguez Boulan E; Kreibich G; Sabatini DD
1978 Sep;78(3):874-893, Journal of cell biology
Carbohydrate-containing structures in rat liver rough microsomes (RM) were localized and characterized using iodinated lectins of defined specificity. Binding of [125I]Con A increased six- to sevenfold in the presence of low DOC (0.04--0.05%) which opens the vesicles and allows the penetration of the lectins. On the other hand, binding of [125I]WGA and [125I]RCA increased only slightly when the microsomal vesicles were opened by DOC. Sites available in the intact microsomal fraction had an affinity for [125I]Con A 14 times higher than sites for lectin binding which were exposed by the detergent treatment. Lectin-binding sites in RM were also localized electron microscopically with lectins covalently bound to biotin, which, in turn, were visualized after their reaction with ferritin-avidin (F-Av) markers. Using this method, it was demonstrated that in untreated RM samples, binding sites for lectins are not present on the cytoplasmic face of the microsomal vesicles, even after removal of ribosomes by treatment with high salt buffer and puromycin, but are located on smooth membranes which contaminate the rough microsomal fraction. Combining this technique with procedures which render the interior of the microsomal vesicles accessible to lectins and remove luminal proteins, it was found that RM membranes contain binding sites for Con A and for Lens culinaris agglutinin (LCA) located exclusively on the cisternal face of the membrane. No sites for WGA, RCA, soybean (SBA) and Lotus tetragonobulus (LTA) agglutinins were detected on either the cytoplasmic or the luminal faces of the rough microsomes. These observations demonstrate that: (a) sugar moieties of microsomal glycoproteins are exposed only on the luminal surface of the membranes and (b) microsomal membrane glycoproteins have incomplete carbohydrate chains without the characteristic terminal trisaccharides N-acetylglucosamine comes from galactose comes from sialic acid or fucose present in most glycoproteins secreted by the liver. The orientation and composition of the carbohydrate chains in microsomal glycoproteins indicate that the passage of these glycoproteins through the Golgi apparatus, followed by their return to the endoplasmic reticulum, is not required for their biogenesis and insertion into the endoplasmic reticulum (ER) membrane
— id: 18448, year: 1978, vol: 78, page: 874, stat: Journal Article,

Spatial orientation of glycoproteins in membranes of rat liver rough microsomes. II. Transmembrane disposition and characterization of glycoproteins
Rodriguez Boulan E; Sabatini DD; Pereyra BN; Kreibich G
1978 Sep;78(3):894-909, Journal of cell biology
Rat liver microsomal glycoproteins were purified by affinity chromatography on concanavalin A Sepharose columns from membrane and content fractions, separated from rough microsomes (RM) treated with low concentrations of deoxycholate (DOC). All periodic acid-Schiff (PAS)-positive glycoproteins of RM showed affinity for concanavalin A Sepharose; even after sodium dodecyl sulfate (SDS) acrylamide gel electrophoresis, most of the microsomal glycoproteins bound [125I]concanavalin A added to the gels, as detected by autoradiography. Two distinct sets of glycoproteins are present in the membrane and content fractions derived from RM. SDS acrylamide gel electrophoresis showed that RM membranes contain 15--20 glycoproteins (15--22% of the total microsomal protein) which range in apparent mol wt from 23,000 to 240,000 daltons. A smaller set of glycoproteins (five to seven polypeptides), with apparent mol wt between 60,000 and 200,000 daltons, was present in the microsomal content fraction. The disposition of the membrane glycoproteins with respect to the membrane plane was determined by selective iodination with the lactoperoxidase (LPO) technique. Intact RM were labeled on their outer face with 131I and, after opening of the vesicles with 0.05% DOC, in both faces with 125I. An analysis of iodination ratios for individual proteins separated electrophoretically showed that in most membrane glycoproteins, tyrosine residues are predominantly exposed on the luminal face of the vesicles, which is the same face on which the carbohydrate moieties are exposed. Several membrane glycoproteins are also exposed on the cytoplasmic surface and therefore have a transmembrane disposition. In this study, ribophorins I and II, two integral membrane proteins (mol wt 65,000 and 63,000) characteristic of RM, were found to be transmembrane glycoproteins. It is suggested that the transmembrane disposition of the ribophorins may be related to their possible role in ribosome binding and in the vectorial transfer of nascent polypeptides into the microsomal lumen
— id: 18447, year: 1978, vol: 78, page: 894, stat: Journal Article,

2 MEMBRANE PROTEINS OF RAT-LIVER MICROSOMES RELATED TO RIBOSOME BINDING
Kreibich, G; Grebenau, R; Mok, W; Pereyra, B; Rodriguezboulan, E; Ulrich, B; Sabatini, DD
1977 ;36(3):656-656, Federation Proceedings (Federation of American Societies for Experimental Biology)
— id: 29631, year: 1977, vol: 36, page: 656, stat: Journal Article,

CHARACTERIZATION OF 2 MEMBRANE PROTEINS OF RAT-LIVER ROUGH MICROSOMES INVOLVED IN RIBOSOME BINDING
Kreibich, G; Sabatini, DD
1977 ;297(2):166-166, Journal of supramolecular structure
— id: 29577, year: 1977, vol: 297, page: 166, stat: Journal Article,

INVIVO AND INVITRO SYNTHESIS OF CYTOCHROME-P-450
Negishi, M; Sabatini, DD; Kreibich, G
1977 ;75(2):A235-A235, Journal of cell biology
— id: 29567, year: 1977, vol: 75, page: A235, stat: Journal Article,

Mobility of ribosomes bound to microsomal membranes. A freeze-etch and thin-section electron microscope study of the structure and fluidity of the rough endoplasmic reticulum
Ojakian GK; Kreibich G; Sabatini DD
1977 Mar;72(3):530-551, Journal of cell biology
The lateral mobility of ribosomes bound to rough endoplasmic reticulum (RER) membranes was demonstrated under experimental conditions. High-salt-washed rough microsomes were treated with pancreatic ribonuclease (RNase) to cleave the mRNA of bound polyribosomes and allow the movement of individual bound ribosomesmfreeze-etch and thin-section electron microscopy demonstrated that, when rough microsomes were treated with RNase at 4 degrees C and then maintained at this temperature until fixation, the bound ribosomes retained their homogeneous distribution on the microsomal surface. However, when RNase-treated rough microsomes were brought to 24 degrees C, a temperature above the thermotropic phase transition of the microsomal phospholipids, bound ribosomes were no longer distributed homogeneously but, instead, formed large, tightly packed aggregates on the microsomal surface. Bound polyribosomes could also be aggregated by treating rough microsomes with antibodies raised against large ribosomal subunit proteins. In these experiments, extensive cross-linking of ribosomes from adjacent microsomes also occurred, and large ribosome-free membrane areas were produced. Sedimentation analysis in sucrose density gradients demonstrated that the RNase treatment did not release bound ribosomes from the membranes; however, the aggregated ribosomes remain capable of peptide bond synthesis and were released by puromycin. It is proposed that the formation of ribosomal aggregates on the microsomal surface results from the lateral displacement of ribosomes along with their attached binding sites, nascent polypeptide chains, and other associated membrane proteins; The inhibition of ribosome mobility after maintaining rough microsomes at 4 degrees C after RNase, or antibody, treatment suggests that the ribosome binding sites are integral membrane proteins and that their mobility is controlled by the fluidity of the RER membrane. Examination of the hydrophobic interior of microsomal membranes by the freeze-fracture technique revealed the presence of homogeneously distributed 105-A intramembrane particles in control rough microsomes. However, aggregation of ribosomes by RNase, or their removal by treatment with puromycin, led to a redistribution of the particles into large aggregates on the cytoplasmic fracture face, leaving large particle-free regions
— id: 18453, year: 1977, vol: 72, page: 530, stat: Journal Article,

SPECIFICITY AND CONTROL OF BINDING OF RIBOSOMES TO ENDOPLASMIC- RETICULUM MEMBRANES
Mok, W; Freienstein, C; Sabatini, D; Kreibich, G
1976 ;70(2):A393-A393, Journal of cell biology
— id: 29455, year: 1976, vol: 70, page: A393, stat: Journal Article,

LATERAL MOVEMENT OF RIBOSOMES ON MICROSOMAL-MEMBRANES DETECTED BY FREEZE-ETCHING
Ojakian, G; Kreibich, G; Kruppa, J; Mok, W; Sabatini, DD
1975 ;34(3):536-536, Federation Proceedings (Federation of American Societies for Experimental Biology)
— id: 28673, year: 1975, vol: 34, page: 536, stat: Journal Article,

SPATIAL LOCATION OF GLYCOPROTEINS IN MICROSOMAL-MEMBRANES
RODRIQUEZBOULAN, ER; KREIBICH, G; SABATINI, DD
1975 ;34(3):582-582, Federation Proceedings (Federation of American Societies for Experimental Biology)
— id: 98739, year: 1975, vol: 34, page: 582, stat: Journal Article,

Structural and functional aspects of the protein synthesizing apparatus in the rough endoplasmic reticulum
Sabatini DD; Ojakian G; Lande MA; Lewis J; Mok W; Adesnik M; Kreibich G
1975 ;62(3):151-180, Advances in experimental medicine & biology
— id: 18454, year: 1975, vol: 62, page: 151, stat: Journal Article,

Ribosomal-membrane interaction: in vitro binding of ribosomes to microsomal membranes
Borgese N; Mok W; Kreibich G; Sabatini DD
1974 Sep 25;88(3):559-580, Journal of molecular biology
— id: 18455, year: 1974, vol: 88, page: 559, stat: Journal Article,

On the spatial arrangememt of proteins in microsomal membranes from rat liver
Kreibich G; Hubbard AL; Sabatini DD
1974 Mar;60(3):616-627, Journal of cell biology
— id: 18457, year: 1974, vol: 60, page: 616, stat: Journal Article,

Procedure for the selective release of content from microsomal vesicles without membrane disassembly
Kreibich G; Sabatini DD
1974 ;31(Pt A):215-225, Methods in enzymology
— id: 18458, year: 1974, vol: 31, page: 215, stat: Journal Article,

Selective release of content from microsomal vesicles without membrane disassembly. II. Electrophoretic and immunological characterization of microsomal subfractions
Kreibich G; Sabatini DD
1974 Jun;61(3):789-807, Journal of cell biology
— id: 18456, year: 1974, vol: 61, page: 789, stat: Journal Article,

On the biochemical mechanism of tumorigenesis in mouse skin. V. Studies of the metabolism of tumor promoting and non promoting phorbol derivatives in vivo and in vitro
Kreibich G; Suss R; Kinzel V
1974 ;81(2):135-149, Zeitschrift fur krebsforschung & klinische onkologie
— id: 18459, year: 1974, vol: 81, page: 135, stat: Journal Article,

Differential sensitivity of monolayer cells compared with suspension cell cultures to treatment with high concentrations of croton oil factor (TPA) and the surfactants tween 80 and triton X-100
Kinzel V; Schmid E; Suss R; Kreibich G
1973 Dec 1;22(23):3091-3097, Biochemical pharmacology
— id: 18460, year: 1973, vol: 22, page: 3091, stat: Journal Article,

Selective release of content from microsomal vesicles without membrane disassembly. I. Permeability changes induced by low detergent concentrations
Kreibich G; Debey P; Sabatini DD
1973 Aug;58(2):436-462, Journal of cell biology
— id: 18462, year: 1973, vol: 58, page: 436, stat: Journal Article,

Microsomal membranes and the translational apparatus of eukaryotic cells
Kreibich G; Sabatini DD
1973 Nov;32(11):2133-2138, Federation Proceedings (Federation of American Societies for Experimental Biology)
— id: 18461, year: 1973, vol: 32, page: 2133, stat: Journal Article,

SPATIAL ARRANGEMENT OF PROTEINS IN RAT-LIVER MICROSOMAL MEMBRANES
KREIBICH, G; HUBBART, AL; SABATINI, DD
1973 ;32(3):674-&, Federation Proceedings (Federation of American Societies for Experimental Biology)
— id: 39807, year: 1973, vol: 32, page: 674, stat: Journal Article,

RIBOSOME-MEMBRANE ASSOCIATION IN RAT-LIVER ROUGH MICROSOMES
SABATINI, D; KREIBICH, G
1973 ;23(3):231-232, Acta physiologica latinoamerica
— id: 39769, year: 1973, vol: 23, page: 231, stat: Journal Article,

Phorbol esters as a tool in cell research?
Suss R; Kreibich G; Kinzel V
1972 Jun;8(3):299-304, European journal of cancer
— id: 18463, year: 1972, vol: 8, page: 299, stat: Journal Article,

Phorbol ester stimulates choline incorporation
Kreibich, G; Hecker, E; Suss, R; Kinzel, V
1971 Jun;58(6):323-323, Naturwissenschaften
— id: 18464, year: 1971, vol: 58, page: 323, stat: Journal Article,

On the biochemical mechanism of tumorigenesis in mouse skin. IV. Methods for determination of fate and distribution of phorbolester TPA
Kreibich, G; Witte, I; Hecker, E
1971 ;76(2):113-123, Zeitschrift fur krebsforschung & klinische onkologie
— id: 18466, year: 1971, vol: 76, page: 113, stat: Journal Article,

Cocarcinogenic croton oil factor A1 stimulates lipid synthesis in cell cultures
Suss, R; Kinzel, V; Kreibich, G
1971 Jan 15;27(1):46-47, Experientia
— id: 18465, year: 1971, vol: 27, page: 46, stat: Journal Article,

Thymidine incorporation into HeLa cells increased by tumor-producing croton oil factor TPA
Freienstein C; Freienstein S; Kreibich G; Kinzel V; Suss R
1970 Dec;57(12):675-676, Naturwissenschaften
— id: 18467, year: 1970, vol: 57, page: 675, stat: Journal Article,

Active principles of croton oil. X. Preparation of tritium labeled croton oil factor A1 and other tritium labeled phorbol derivatives
Kreibich G; Hecker E
1970 ;74(4):448-456, Zeitschrift fur krebsforschung
— id: 18468, year: 1970, vol: 74, page: 448, stat: Journal Article,

Biochemical mechanism of tumorigenesis in mouse skin. 3. Decrease in tumor yields by poly I-C administered during initiation of skin b y and intragastric dose of 7,12-dimethyl-benz(a)anthracene
Kreibich G; Suss R; Kinzel V; Hecker E
1970 ;74(4):383-389, Zeitschrift fur krebsforschung
— id: 18469, year: 1970, vol: 74, page: 383, stat: Journal Article,

[On the active substances of croton oil. IX. Partial synthesis of active substances in croton oil]
Bresch H; Kreibich G; Kubinyi H; Schairer HU; Thielmann HW; Hecker E
1968 Apr;23(4):538-546, Zeitschrift fur naturforschung. B. Chemie, biochemie, biophysik, biologie & verwandte gebiete
— id: 18472, year: 1968, vol: 23, page: 538, stat: Journal Article,

[On the chemistry of phorbol. V. On some ethers of phorbol]
Kreibich G; Hecker E
1968 Nov;23(11):1444-1452, Zeitschrift fur naturforschung. B. Chemie, biochemie, biophysik, biologie & verwandte gebiete
— id: 18471, year: 1968, vol: 23, page: 1444, stat: Journal Article,

[On the chemistry of phorbol. IV. Polybenzoate and acetate of phorbol and phorbol-3-ol and functional derivatives of the allyl grouping of phorbol]
Schairer HU; Thielmann HW; Gschwendt M; Kreibich G; Schmidt R; Hecker E
1968 Nov;23(11):1430-1443, Zeitschrift fur naturforschung. B. Chemie, biochemie, biophysik, biologie & verwandte gebiete
— id: 18470, year: 1968, vol: 23, page: 1430, stat: Journal Article,