Milton Bernard Adesnik, Ph.D.

David Sabatini--a lifelong fascination with organelles
Adesnik, Milton
2002 Jul;12(7):347-349, Trends in cell biology
As a pioneer molecular cell biologist, highly skilled in both morphological and biochemical approaches, David Sabatini was a key figure in laying the foundation for the field of intracellular protein trafficking with his seminal studies on cotranslational translocation of nascent polypeptides in the endoplasmic reticulum and the intracellular sorting of plasma membrane proteins in polarized epithelial cells
— id: 39412, year: 2002, vol: 12, page: 347, stat: Journal Article,

GLUT4 retention in adipocytes requires two intracellular insulin-regulated transport steps
Zeigerer, Anja; Lampson, Michael A; Karylowski, Ola; Sabatini, David D; Adesnik, Milton; Ren, Mindong; McGraw, Timothy E
2002 Jul;13(7):2421-2435, Molecular biology of the cell
Insulin regulates glucose uptake into fat and muscle by modulating the distribution of the GLUT4 glucose transporter between the surface and interior of cells. The GLUT4 trafficking pathway overlaps with the general endocytic recycling pathway, but the degree and functional significance of the overlap are not known. In this study of intact adipocytes, we demonstrate, by using a compartment-specific fluorescence-quenching assay, that GLUT4 is equally distributed between two intracellular pools: the transferrin receptor-containing endosomes and a specialized compartment that excludes the transferrin receptor. These pools of GLUT4 are in dynamic communication with one another and with the cell surface. Insulin-induced redistribution of GLUT4 to the surface requires mobilization of both pools. These data establish a role for the general endosomal system in the specialized, insulin-regulated trafficking of GLUT4. Trafficking through the general endosomal system is regulated by rab11. Herein, we show that rab11 is required for the transport of GLUT4 from endosomes to the specialized compartment and for the insulin-induced translocation to the cell surface, emphasizing the importance of the general endosomal pathway in the specialized trafficking of GLUT4. Based on these findings we propose a two-step model for GLUT4 trafficking in which the general endosomal recycling compartment plays a specialized role in the insulin-regulated traffic of GLUT4. This compartment-based model provides the framework for understanding insulin-regulated trafficking at a molecular level
— id: 55808, year: 2002, vol: 13, page: 2421, stat: Journal Article,

A role of a rab11BP-AP1 adaptor interaction in vesicle budding from recycling endosomes
Zhou, L; Li, J; Ma, JP; Sabatini, DD; Adesnik, M; Ren, MD
2001 NOV ;12(1):344A-344A, Molecular biology of the cell
— id: 55363, year: 2001, vol: 12, page: 344A, stat: Journal Article,

In vitro generation from the trans-Golgi network of coatomer-coated vesicles containing sialylated vesicular stomatitis virus-G protein
Simon JP; Ivanov IE; Adesnik M; Sabatini DD
2000 Apr;20(4):437-454, Methods
We describe an in vitro system in which post-Golgi vesicles containing metabolically labeled, sialylated, vesicular stomatitis virus (VSV) G protein molecules (VSV-G) are produced from the trans-Golgi network (TGN) of an isolated Golgi membrane fraction. This fraction is prepared from VSV-infected Madin-Darby canine kidney (MDCK) cells in which the (35)S-labeled viral envelope glycoprotein was allowed to accumulate in the trans-Golgi network during a prolonged incubation at 20 degrees C. The vesicles produced in this system are separated from the remnant Golgi membranes by differential centrifugation or by velocity sedimentation in a sucrose gradient. Vesicle production, quantified as the percentage of labeled VSV-G released from the Golgi membranes, is optimal at 37 degrees C and does not occur below 20 degrees C. It requires GTP and the small GTP-binding protein Arf (ADP-ribosylation factor), as well as coat protein type I (COPI) coat components (coatomer) and vesicle scission factors-one of which corresponds to the phosphatidylinositol transfer protein (PITP). Formation of the vesicles does not require GTP hydrolysis which, however, is necessary for their uncoating. Thus, vesicles generated in the presence of the nonhydrolyzable GTP analogs, GTPgammaS or GMP-PNP, retain a coatomer coat visible in the electron microscope, sediment more rapidly in sucrose density gradients than those generated with ATP or GTP, and can be captured with anticoatomerantibodies. The process of coatomer-coated vesicle formation from the TGN can be dissected into two distinct sequential phases, corresponding to coat assembly/bud formation and vesicle scission. The first phase is completed when Golgi fractions are incubated with cytosolic proteins and nonhydrolyzable GTP analogs at 20 degrees C. The scission phase, which leads to vesicle release, takes place when coated Golgi membranes, recovered after phase I, are incubated at higher temperatures in the presence of cytosolic proteins. The scission phase does not take place if protein kinase C inhibitors are added during the first phase, even though these inhibitors do not prevent membrane coating and bud formation. The phosphorylating activity of a protein kinase C, however, plays no role in vesicle formation, since this process does not require ATP.
— id: 11801, year: 2000, vol: 20, page: 437, stat: Journal Article,

Rab11 stimulates fluid phase and mannose receptor-mediated endocytosis by regulating endosome fusion
Aballay, A; Damiani, MT; Ren, MD; Adesnik, M; Mayorga, LS; Sabatini, DD; Stahl, PD; Colombo, MI
1999 ;10(4):1284-1284, Molecular biology of the cell
— id: 104641, year: 1999, vol: 10, page: 1284, stat: Journal Article,

Identification of a putative effector protein for rab11 that participates in transferrin recycling
Zeng J; Ren M; Gravotta D; De Lemos-Chiarandini C; Lui M; Erdjument-Bromage H; Tempst P; Xu G; Shen TH; Morimoto T; Adesnik M; Sabatini DD
1999 Mar 16;96(6):2840-2845, Proceedings of the National Academy of Sciences of the United States of America
We have identified and cloned the cDNA for a 912-aa protein, rab11BP, that interacts with the GTP-containing active form of rab11, a GTP-binding protein that plays a critical role in receptor recycling. Although rab11BP is primarily cytosolic, a significant fraction colocalizes with rab11 in endosomal membranes of both the sorting and recycling subcompartments. In vitro binding of rab11 to native rab11BP requires partial denaturation of the latter to expose an internal binding site located between residues 334 and 504 that is apparently masked by the C-terminal portion of the protein, which includes six repeats known as WD40 domains. Within the cell, rab11BP must undergo a conformational change in which the rab11-binding site becomes exposed, because when coexpressed with rab11 in transfected cells the two proteins formed abundant complexes in association with membranes. Furthermore, although overexpression of rab11BP did not affect transferrin recycling, overexpression of a truncated form of the protein, rab11BP(1-504), that includes the rab11-binding site but lacks the WD40 domains inhibited recycling as strongly as does a dominant negative rab11 mutant protein that does not bind GTP. Strikingly, the inhibition caused by the truncated rab11BP was prevented completely when the cells also expressed a C-terminally deleted, nonprenylatable form of rab11 that, by itself, has no effect on recycling. We propose that rab11BP is an effector for rab11, whose association with this GTP-binding protein is dependent on the action of another membrane-associated factor that promotes the unmasking of the rab11-binding site in rab11BP
— id: 56409, year: 1999, vol: 96, page: 2840, stat: Journal Article,

Hydrolysis of GTP on rab11 is required for the direct delivery of transferrin from the pericentriolar recycling compartment to the cell surface but not from sorting endosomes
Ren M; Xu G; Zeng J; De Lemos-Chiarandini C; Adesnik M; Sabatini DD
1998 May 26;95(11):6187-6192, Proceedings of the National Academy of Sciences of the United States of America
Rab11 is a small GTP-binding protein that in cultured mammalian cells has been shown to be concentrated in the pericentriolar endosomal recycling compartment and to play a key role in passage of the recycling transferrin receptor through that compartment [Ullrich, O., Reinsch, S., Urbe, S., Zerial, M. & Parton, R. G. (1996) J. Cell Biol. 135, 913-924]. To obtain insights into the site(s) of action of rab11 within the recycling pathway, we have now compared the effects on recycling at 37 degreesC of overexpression of wild-type rab11 and various mutant forms of this protein in cells that had been loaded with transferrin at either 37 degreesC or 16 degreesC. We show that incubation at 16 degreesC blocks passage of endocytosed transferrin into the recycling compartment and that, whereas the rab11 dominant negative mutant form (S25N) inhibits transferrin recycling after interiorization at either temperature, the wild-type rab11 and constitutively active mutant (Q70L) have no inhibitory effect on the recycling of molecules that were interiorized at 16 degreesC. This differential inhibitory effect shows that two distinct pathways for recycling are followed by the bulk of the transferrin molecules interiorized at the two different temperatures. The incapacity of the constitutively active form of rab11 (Q70L) to inhibit recycling of molecules interiorized at 16 degreesC is consistent with their recycling taking place directly from sorting endosomes, in a process that does not require hydrolysis of GTP on rab11. The fact that the dominant negative (S25N) form of rab11 inhibits recycling of molecules interiorized at both temperatures indicates that activation of rab11 by GTP is required for exit of transferrin from sorting endosomes, regardless of whether this exit is toward the recycling compartment or directly to the plasma membrane
— id: 57335, year: 1998, vol: 95, page: 6187, stat: Journal Article,

Rab11PB, a novel Rab11 effector, regulates transferrin recycling
Ren, M; Zeng, J; Gravotta, D; Xu, G; De Lemos-Chiarandini, C; Shen, T; Morimoto, T; Adesnik, M; Sabatini, DD
1998 ;9(11):2693-2693, Molecular biology of the cell
— id: 104642, year: 1998, vol: 9, page: 2693, stat: Journal Article,

An essential role for the phosphatidylinositol transfer protein in the scission of coatomer-coated vesicles from the trans-Golgi network
Simon JP; Morimoto T; Bankaitis VA; Gottlieb TA; Ivanov IE; Adesnik M; Sabatini DD
1998 Sep 15;95(19):11181-11186, Proceedings of the National Academy of Sciences of the United States of America
We identified the phosphatidylinositol transfer protein (PITP) as being responsible for a powerful latent, nucleotide-independent, Golgi-vesiculating activity that is present in the cytosol but is only manifested as an uncontrolled activity in a cytosolic protein subfraction, in which it is separated from regulatory components that appear to normally limit its action to the scission of COPI-coated buds from trans-Golgi network membranes. A specific anti-PITP antibody that recognizes the two mammalian PITP isoforms fully inhibited the capacity of the cytosol to support normal vesicle generation as well as the uncontrolled vesiculating activity manifested by the cytosolic protein subfraction. The phosphatidylinositol- (PI) loaded form of the yeast PITP, Sec14p, but not the phosphatidylcholine- (PC) loaded form of the protein, was capable of substituting for the cytosolic subfraction in promoting the scission of coated buds from the trans-Golgi network. At higher concentration, however, Sec14p, when loaded with PI, but not with PC or phosphatidylglycerol, caused by itself an indiscriminate vesiculation of uncoated Golgi membranes that could be suppressed by PC-Sec14p, which also suppresses the uncontrolled vesiculation caused by the cytosolic subfraction. We propose that, by delivering PI to specific sites in the Golgi membrane near the necks of coated buds, PITP induces local changes in the organization of the lipid bilayer, possibly involving PI metabolites, that triggers the fusion of the ectoplasmic faces of the Golgi membrane necessary for the scission of COPI-coated vesicles
— id: 57548, year: 1998, vol: 95, page: 11181, stat: Journal Article,

Coatomer, but not P200/myosin II, is required for the in vitro formation of trans-Golgi network-derived vesicles containing the envelope glycoprotein of vesicular stomatitis virus
Simon JP; Shen TH; Ivanov IE; Gravotta D; Morimoto T; Adesnik M; Sabatini DD
1998 Feb 3;95(3):1073-1078, Proceedings of the National Academy of Sciences of the United States of America
Using a cytosol and nucleotide dependent assay that we previously developed, we have investigated the requirement for coat proteins in the in vitro production of trans-Golgi network (TGN)-derived vesicles from a Madin-Darby canine kidney (MDCK) cell Golgi fraction that contains the 35S-labeled, terminally glycosylated, envelope glycoprotein of vesicular stomatitis virus (VSV-G) accumulated in the TGN. We found that the TGN-derived vesicles, like those involved in intra-Golgi transport and in retrograde transport to the endoplasmic reticulum, contain a coatomer coat and that coatomer is required for their formation. Thus, after they are produced with GTPgammaS, the coated vesicles could be captured on beads containing anticoatomer antibody. Moreover, a cytosolic protein fraction depleted of coatomer could not support vesicle formation but it did so after purified coatomer was added. We also determined that P200/myosin II does not play an essential role in the in vitro generation of TGN-derived vesicles. Thus, removal of this protein from the cytosol, by differential salt precipitation or binding to phalloidin-induced actin filaments, had no effect on vesicle generation. Nevertheless, immunodepletion of cytosol using the anti-P200/myosin II AD7 antibody abolished vesicle generation and that antibody was capable of effectively immunocapturing coated vesicles, even when these were generated in the absence of P200/myosin II. These effects, however, are explained by the unexpected finding that the AD7 antibody interacts with undenatured coatomer
— id: 57547, year: 1998, vol: 95, page: 1073, stat: Journal Article,

An essential role for the phosphatidylinositol transfer protein (PITP) in the scission of COPI-coated vesicles from the TGN
Simon, JP; Morimoto, T; Bankaitis, VA; Gottlieb, TA; Ivanov, IE; Adesnik, M; Sabatini, DD
1998 NOV ;9(11):207A-207A, Molecular biology of the cell
— id: 53645, year: 1998, vol: 9, page: 207A, stat: Journal Article,

Rab8 is a specific substrate for the Rab8ip/GC kinase
Ren, M; Lake, R; Xu, G; Adesnik, M; Sabatini, DD
1997 NOV ;8(5):2447-2447, Molecular biology of the cell
— id: 53171, year: 1997, vol: 8, page: 2447, stat: Journal Article,

Rab11-GTP is required early in the endocytic pathway but hydrolysis of its GTP is necessary only for return of receptors from the recycling compartment to the cell surface
Ren, M; Xu, G; DeLemosChiarandini, C; Adesnik, M; Sabatini, DD
1997 NOV ;8(5):2460-2460, Molecular biology of the cell
— id: 53172, year: 1997, vol: 8, page: 2460, stat: Journal Article,

Coatomer is required for the in vitro formation of TGN-derived vesicles containing VSV-G protein
Simon, JP; Shen, TH; Ivanov, I; Gravotta, D; Morimoto, T; Adesnik, M; Sabatini, DD
1997 NOV ;8(5):1129-1129, Molecular biology of the cell
— id: 53165, year: 1997, vol: 8, page: 1129, stat: Journal Article,

Transcriptional regulation of the CYP2B1 and CYP2B2 genes by C/EBP-related proteins
Luc PV; Adesnik M; Ganguly S; Shaw PM
1996 Feb 9;51(3):345-356, Biochemical pharmacology
Cytochrome P450 (CYP) 2B1 and 2B2 are encoded by two closely related genes, CYP2B1 and CYP2B2, that are expressed at low levels in adult rat liver but are induced markedly by the administration of the drug phenobarbital (PB) or other structurally unrelated hydrophobic compounds to animals. Very little is understood about the molecular mechanisms that control both basal and induced transcription of these genes. We have identified two liver specific DNase I hypersensitive sites associated with the CYP2B1 and CYP2B2 (CYP2B) genes. One site, which maps to a region in the 5'-flanking region between -2.2 and -2.3 kb, became more resistant to DNase I cleavage in nuclei from PB-treated rats; the converse was true of the other hypersensitive site, which maps to the proximal promoter region between -0.05 and -0.15 kb. DNase I footprint analysis revealed three prominent and one weak footprinted regions in the promoter region in the vicinity of the proximal hypersensitive site. Using competitor oligonucleotides, we determined that one footprinted region (FT2), between -42 and -66 bp, is likely to represent a binding site for CCAATT enhancer binding protein (C/EBP) family members. Indeed, bacterial expressed recombinant C/EBP alpha bound at this site and formed a footprint pattern identical to the pattern observed with liver nuclear extract. In vitro transcription assays demonstrated that the FT2 site contributed strongly to promoter activity, since its mutation reduced transcription by 80%. Two other sites identified by footprint analysis (FT1 and FT3) are also required to maintain high basal transcription of CYP2B2 promoter constructs in an in vitro transcription assay. Transient transfection experiments confirmed the expectation that C/EBP alpha could activate the 1.4 kb CYP2B promoter constructs, with mutation of the FT2 site impairing both basal transcription and transactivation by exogenous C/EBP alpha
— id: 12644, year: 1996, vol: 51, page: 345, stat: Journal Article,

Cell-free reconstitution of the transport of viral glycoproteins from the TGN to the basolateral plasma membrane of MDCK cells
Mayer A; Ivanov IE; Gravotta D; Adesnik M; Sabatini DD
1996 Jul;109(Pt 7):1667-1676, Journal of cell science
An in vitro system to study the transport of plasma membrane proteins from the TGN to the basolateral plasma membrane of polarized MDCK cells has been developed in which purified cell fractions are combined and transport between them is studied under controlled conditions. In this system, a donor Golgi fraction derived from VSV or influenza virus-infected MDCK cells, in which 35S-labeled viral glycoproteins were allowed to accumulate in the TGN during a low temperature block, is incubated with purified immobilized basolateral plasma membranes that have their cytoplasmic face exposed and are obtained by shearing-lysis of MDCK monolayers grown on cytodex beads. Approximately 15-30% of the labeled glycoprotein molecules are transferred from the Golgi fraction to the acceptor plasma membranes and are recovered with the sedimentable (1 g) beads. Transport is temperature, energy and cytosol dependent, and is abolished by alkylation of SH groups and inhibited by the presence of GTP-gamma-S, which implicates GTP-binding proteins and the requirement for GTP hydrolysis in one or more stages of the transport process. Endo H-resistant glycoprotein molecules that had traversed the medial region of the Golgi apparatus are preferentially transported and their luminal domains become accessible to proteases, indicating that membrane fusion with the plasma membrane takes place in the in vitro system. Mild proteolysis of the donor or acceptor membranes abolishes transport, suggesting that protein molecules exposed on the surface of these membranes are involved in the formation and consumption of transport intermediates, possibly as addressing and docking proteins, respectively. Surprisingly, both VSV-G and influenza HA were transported with equal efficiencies to the basolateral acceptor membranes. However, low concentrations of a microtubular protein fraction preferentially inhibited the transport of HA, although this effect was not abolished by microtubule depolymerizing agents. This system shows great promise for elucidating the mechanisms that effect the proper sorting of plasma membrane proteins in the TGN and their subsequent targeting to the appropriate acceptor membrane
— id: 12585, year: 1996, vol: 109, page: 1667, stat: Journal Article,

In its active form, the GTP-binding protein rab8 interacts with a stress-activated protein kinase
Ren M; Zeng J; De Lemos-Chiarandini C; Rosenfeld M; Adesnik M; Sabatini DD
1996 May 14;93(10):5151-5155, Proceedings of the National Academy of Sciences of the United States of America
Rab8 is a small GTP-binding protein that plays a role in vesicular transport from the trans-Golgi network to the basolateral plasma membrane in polarized epithelial cells (MDCK), and to the dendritic surface in hippocampal neurons. As is the case for most other rab proteins, the precise molecular interactions by which rab8 carries out its function remain to be elucidated. Here we report the identification and the complete cDNA-derived amino acid sequence of a murine rab8-interacting protein (rab8ip) that specifically interacts with rab8 in a GTP-dependent manner. Rab8ip displays 93% identity with the GC kinase, a serine/threonine protein kinase recently identified in human lymphoid tissue that is activated in the stress response. Like the GC kinase, rab8ip has protein kinase activity manifested by autophosphorylation and phosphorylation of the classical serine/threonine protein kinase substrates, myelin basic protein and casein. When coexpressed in transfected 293T cells, rab8 and the rab8ip/GC kinase formed a complex that could be recovered by immunoprecipitation with antibodies to rab8. Cell fractionation and immunofluorescence analyses indicate that in MDCK cells endogenous rab8ip is present both in the cytosol and as a peripheral membrane protein concentrated in the Golgi region and basolateral plasma membrane domains, sites where rab8 itself is also located. In light of recent evidence that rab proteins may act by promoting the stabilization of SNARE complexes, the specific GTP-dependent association of rab8 with the rab8ip/GC kinase raises the possibility that rab-regulated protein phosphorylation is important for vesicle targeting or fusion. Moreover, the rab8ip/GC kinase may serve to modulate secretion in response to stress stimuli
— id: 57507, year: 1996, vol: 93, page: 5151, stat: Journal Article,

Mechanism of formation of post Golgi vesicles from TGN membranes: Arf-dependent coat assembly and PKC-regulated vesicle scission
Sabatini DD; Adesnik M; Ivanov IE; Simon JP
1996 Dec;20(3):287-300, Biocell
We have developed an experimental system that utilizes purified Golgi fractions obtained from virus infected infected MDCK cells to reproduce in vitro the process of vesicle generation in the trans Golgi network, an important site for the sorting of proteins addressed to the plasma membrane, secretory vesicles, or lysosomes. Using an integrated biochemical and electron microscopic approach, we have shown that the formation of post Golgi vesicles carrying proteins destined to both plasma membrane domains of epithelial cells requires the activation of an ArF-like GTP-binding protein that serves to promote the assembly of the protein coat necessary to deform the donor membrane and generate a vesicle. The formation of the post Golgi vesicles also requires the participation of a Golgi membrane-associated Protein Kinase C, but not its phosphorylating activity. Other authors have shown that this is also the case for the PKC activation of the enzyme phospholipase D, which generates phosphatidic acid from phosphatidyl choline and may be involved in remodeling of membranes. We have been able to dissect the process of post Golgi vesicle generation into two sequential stages, one of coat assembly and bud formation, and a subsequent one of vesicle scission. The first stage can occur at 20 degrees C and requires the activation of the Arf protein necessary for coat assembly. The second stage does not require nucleotides or an energy supply, but requires cytosolic proteins, and in particular, an NEM sensitive membrane scission promoting activity that operates only at a higher temperature of incubation. Because various PKC inhibitors blocked vesicle scission without preventing bud formation, we propose that the PKC is required for the activation of a PLD in the TGN, which leads to remodeling of the donor membrane and the severing of connections between the emerging vesicles and the membranes
— id: 12450, year: 1996, vol: 20, page: 287, stat: Journal Article,

Sequence of the rat PB-inducible CYP2B1 promoter
Shaw PM; Edigkaufer M; Doehmer J; Adesnik M
1996 Feb 7;1305(1-2):54-58, Biochimica & biophysica acta
The 5' flanking region of the rat PB-inducible CYP2B1 gene was isolated and the sequence from +27 to -3878 was determined. This sequence contains several putative binding sites for the liver-enriched factors HNF3 as well as an AP1, two NF-kappa B and several possible STAT sites. The promoter sequence of the CYP2B1 gene was compared to that of the CYP2B2 sequence, published by Hoffman et al. ((1992) Gene Exp. 2, 353-362) and was found to be almost identical up to -2300 bp, beyond which it diverges significantly from the remaining published sequence of CYP2b2 gene. Transient transfection experiments in the differentiated hepatoma cell line, FGC4, showed that the 3.9 kb promoter was expressed, however, an increase in reporter activity was not observed in the presence of phenobarbital
— id: 12645, year: 1996, vol: 1305, page: 54, stat: Journal Article,

The production of post-Golgi vesicles requires a protein kinase C-like molecule, but not its phosphorylating activity
Simon JP; Ivanov IE; Adesnik M; Sabatini DD
1996 Oct;135(2):355-370, Journal of cell biology
We have recently described a system that recreates in vitro the generation of post-Golgi vesicles from purified Golgi fractions obtained from virus-infected MDCK cells in which the vesicular stomatitis virus-G envelope glycoprotein had been allowed to accumulate in vivo in the TGN. Vesicle formation, monitored by the release of the viral glycoprotein, was shown to require the activation of a GTP-binding ADP ribosylation factor (ARF) protein that promotes the assembly of a vesicle coat in the TGN, and to be regulated by a Golgi-associated protein kinase C (PKC)-like activity. We have now been able to dissect the process of post-Golgi vesicle generation into two sequential stages, one of coat assembly and bud formation, and another of vesicle scission, neither of which requires an ATP supply. The first stage can occur at 20 degrees C, and includes the GTP-dependent activation of the ARF protein, which can be effected by the nonhydrolyzable nucleotide analogue GTP gamma S, whereas the second stage is nucleotide independent and can only occur at a higher temperature of incubation. Cytosolic proteins are required for the vesicle scission step and they cannot be replaced by palmitoyl CoA, which is known to promote, by itself, scission of the coatomer-coated vesicles that mediate intra-Golgi transport. We have found that PKC inhibitors prevented vesicle generation, even when this was sustained by GTP gamma S and ATP levels reduced far below the K(m) of PKC. The inhibitors suppressed vesicle scission without preventing coat assembly, yet to exert their effect, they had to be added before coat assembly took place. This indicates that a target of the putative PKC is activated during the bud assembly stage of vesicle formation, but only acts during the phase of vesicle release. The behavior of the PKC target during vesicle formation resembles that of phospholipase D (PLD), a Golgi-associated enzyme that has been shown to be activated by PKC, even in the absence of the latter's phosphorylating activity. We therefore propose that during coat assembly, PKC activates a PLD that, during the incubation at 37 degrees C, promotes vesicle scission by remodeling the phospholipid bilayer and severing connections between the vesicles and the donor membrane
— id: 8519, year: 1996, vol: 135, page: 355, stat: Journal Article,

The in vitro generation of post-Golgi vesicles carrying viral envelope glycoproteins requires an ARF-like GTP-binding protein and a protein kinase C associated with the Golgi apparatus
Simon JP; Ivanov IE; Shopsin B; Hersh D; Adesnik M; Sabatini DD
1996 Jul 12;271(28):16952-16961, Journal of biological chemistry
We have developed a system that recreates in vitro the generation of post-Golgi vesicles from an isolated Golgi fraction prepared from vesicular stomatitis virus- or influenza virus-infected Madin-Darby canine kidney or HepG2 cells. In this system, vesicle generation is temperature- and ATP-dependent and requires a supply of cytosolic proteins, including an N-ethylmaleimide-sensitive factor distinct from NSF. Cytosolic proteins obtained from yeast were as effective as mammalian cytosolic proteins in supporting vesicle formation and had the same requirements. The vesicles produced (50-80 nm in diameter) are depleted of the trans Golgi marker sialyltransferase, contain the viral glycoprotein molecules with their cytoplasmic tails exposed, and do not show an easily recognizable protein coat. Vesicle generation was inhibited by brefeldin A, which indicates that it requires the activation of an Arf-like GTP-binding protein that promotes assembly of a vesicle coat. Vesicles formed in the presence of the nonhydrolyzable GTP analogue guanosine 5'-3-O-(thio)triphosphate retained a nonclathrin protein coat resembling that of COP-coated vesicles, and sedimented more rapidly in a sucrose gradient than the uncoated ones generated in its absence. This indicates that GTP hydrolysis is not required for vesicle generation but that it is for vesicle uncoating. The activity of a Golgi-associated protein kinase C (PKC) was found to be necessary for the release of post-Golgi vesicles, as indicated by the capacity of a variety of inhibitors and antibodies to PKC to suppress it, as well as by the stimulatory effect of the PKC activator 12-O-tetradecanoylphorbol-13-acetate
— id: 7048, year: 1996, vol: 271, page: 16952, stat: Journal Article,

Characterization of rab binding proteins that may mediate the actions of rab11 and rab8
Ren, M.; Zeng, J.; Gravotta, D.; Morimoto, T.; Rosenfled, M.; De Lemos-Chiarandini, C.; Tempsti, P.; Erdjument-Bromage, H.; Lui, M.; Adesnik, M.; Sabatini, D. D.
1995 ;6(SUPPL.):119A-119A, Molecular biology of the cell
— id: 104643, year: 1995, vol: 6, page: 119A, stat: Journal Article,

CHARACTERIZATION OF RAB BINDING-PROTEINS THAT MAY MEDIATE THE ACTIONS OF RAB11 AND RAB8
REN, M; ZENG, J; GRAVOTTA, D; MORIMOTO, T; ROSENFELD, M; DELEMOSCHIARANDINI, C; TEMPST, P; ERDJUMENTBROMAGE, H; LUI, M; ADESNIK, M; SABATINI, DD
1995 NOV ;6(23):690-690, Molecular biology of the cell
— id: 52664, year: 1995, vol: 6, page: 690, stat: Journal Article,

The biogenesis of membranes and organelles
Sabatini DD; Adesnik M
The metabolic and molecular bases of inherited disease New York : McGraw-Hill, Health Professions Division, 1995,
— id: 3525, year: 1995, vol: , page: ?, stat: Chapter,

A PKC-like activity is involved in the release of post Golgi vesicles
Simon, J.-P.; Shopsin, B.; Hersh, D.; Ivanov, I. E.; Adesnik, M.; Sabatini, D. D.
1995 ;6(SUPPL.):397A-397A, Molecular biology of the cell
— id: 104644, year: 1995, vol: 6, page: 397A, stat: Journal Article,

Rat liver cytochrome P450 2B3: structure of the CYP2B3 gene and immunological identification of a constitutive P450 2B3-like protein in rat liver
Jean, A; Reiss, A; Desrochers, M; Dubois, S; Trottier, E; Trottier, Y; Wirtanen, L; Adesnik, M; Waxman, D J; Anderson, A
1994 Aug;13(8):781-792, DNA & cell biology
The cytochrome P450 2B subfamily in the rat contains an estimated eight to eleven members at the genomic level. Synthesis in the liver of the prototypic forms P450 2B1 and P450 2B2 is dramatically induced by phenobarbital. The 1.9-kb mRNA for P450 2B3, a third member of the P450 2B subfamily, is constitutively present in rat liver but is not inducible by phenobarbital. We have now cloned and sequenced exonic sequences corresponding to the entire 2B3 mRNA and determined their exon-intron structure, which is identical to that of CYP2B1/CYP2B2 and other CYP2B genes. A putative CYP2B3 transcription start site was identified and CYP2B3 5'- and 3'-flanking sequences were compared to those of CYP2B1 and CYP2B2. CYP2B3, like CYP2B1 and CYP2B2, has a modified TATA box preceding the transcription start site and lacks the canonical polyadenylation signal preceding the poly(A) site. A 2B3 expression vector, pMT2-2B3, directed the synthesis in COS-1 cells of an approximately 50-kD protein detectable on Western blots with a polyclonal antibody and with one of four monoclonal antibodies raised against 2B1 but not with a polyclonal antibody raised against P450 PB6. The 2B3 protein migrated with a slightly higher electrophoretic mobility than 2B1 and comigrated with a protein detected by anti-2B1 antibodies in liver microsomes from untreated rats. The results indicate that a 2B3-like protein is present in rat liver and that it is distinct from P450 PB6 and other known constitutive rat hepatic P450s
— id: 106303, year: 1994, vol: 13, page: 781, stat: Journal Article,

Sequence and activity of the rat PB-inducible aldehyde dehydrogenase promoter
Shaw PM; Adesnik M
1994 Jun 21;1218(2):242-244, Biochimica & biophysica acta
The 5' flanking region of the rat PB inducible aldehyde dehydrogenase gene was isolated and the sequence from +42 to -1339 was determined. This sequence contains several putative binding sites for the liver-enriched factors HNF3 and DBP as well as a GRE and several possible AP1 sites. A TATA and CCAAT motif were assigned at positions -26 and -53. A promoter construct containing the -1339 bp of the aldehyde dehydrogenase 5' flanking region was active when transfected into both H411 and HepG2 liver cell lines
— id: 12962, year: 1994, vol: 1218, page: 242, stat: Journal Article,

Hepatocyte nuclear factor 3 is a major determinant of CYP2C6 promoter activity in hepatoma cells
Shaw PM; Weiss MC; Adesnik M
1994 Jul;46(1):79-87, Molecular pharmacology
Cytochrome P450 2C6 (CYP2C6) is a developmentally regulated, constitutively expressed form of rat liver microsomal cytochrome P450 that in the liver of adult male rats is induced to a limited extent by phenobarbital. The gene is not expressed at detectable levels in the lung, kidney, or brain. It is expressed and inducible by phenobarbital in differentiated Reuber hepatoma cells that express many hepatocyte-specific genes but not in dedifferentiated derivatives lacking the majority of hepatocyte-specific functions. A 505-base pair proximal segment of the CYP2C6 promoter is highly efficient in driving transcription of a linked chloramphenicol acetyltransferase reporter gene in the differentiated rat hepatoma cell line FGC4, is much less effective in a related dedifferentiated variant H5, and has no measurable activity in nonhepatic C33 human cervical carcinoma cells. The activity of the CYP2C6 promoter in the differentiated hepatoma cells is strongly dependent on hepatocyte nuclear factor (HNF)3, which acts at a complex site just upstream of the TATA motif. Transactivation experiments show that the D-site-binding protein (DBP) may also contribute to CYP2C6 promoter activity, via a site that is adjacent to the proximal HNF3 site. A substantial contribution to promoter activity by the base pair -505 to -316 segment is observed in FGC4 and H5 cells but not in HepG2 cells; deletion of this segment causes a marked diminution in promoter activity only in the former two cell lines. Although footprinting experiments have permitted the definition of three protein binding sites in this region (two HNF3 and one unidentified), mutation of these sites does not diminish promoter activity. The functionally important cis sequences in this region therefore remain to be defined. In HepG2 cells the distal region does not contribute to promoter activity. This most likely accounts for the low promoter activity in HepG2 and implies a deficiency in the relevant trans-acting factor(s)
— id: 12950, year: 1994, vol: 46, page: 79, stat: Journal Article,

Actin microfilaments play a critical role in endocytosis at the apical but not the basolateral surface of polarized epithelial cells
Gottlieb TA; Ivanov IE; Adesnik M; Sabatini DD
1993 Feb;120(3):695-710, Journal of cell biology
Treatment with cytochalasin D, a drug that acts by inducing the depolymerization of the actin cytoskeleton, selectively blocked endocytosis of membrane bound and fluid phase markers from the apical surface of polarized MDCK cells without affecting the uptake from the basolateral surface. Thus, in MDCK cell transformants that express the VSV G protein, cytochalasin blocked the internalization of an anti-G mAb bound to apical G molecules, but did not reduce the uptake of antibody bound to the basolateral surface. The selective effect of cytochalasin D on apical endocytosis was also demonstrated by the failure of the drug to reduce the uptake of 125I-labeled transferrin, which occurs by receptor-mediated endocytosis, via clathrin-coated pits, almost exclusively from the basolateral surface. The actin cytoskeleton appears to play a critical role in adsorptive as well as fluid phase apical endocytic events, since treatment with cytochalasin D prevented the apical uptake of cationized ferritin, that occurs after the marker binds to the cell surface, as well as uptake of Lucifer yellow, a fluorescent soluble dye. Moreover, the drug efficiently blocked infection of the cells with influenza virus, when the viral inoculum was applied to the apical surface. On the other hand, it did not inhibit the basolateral uptake of Lucifer yellow, nor did it prevent infection with VSV from the basolateral surface, or with influenza when this virus was applied to monolayers in which the formation of tight junctions had been prevented by depletion of calcium ions. EM demonstrated that cytochalasin D leads to an increase in the number of coated pits in the apical surface where it suppresses the pinching off of coated vesicles. In addition, in drug-treated cells cationized ferritin molecules that were bound to microvilli were not cleared from the microvillar surface, as is observed in untreated cells. These findings indicate that there is a fundamental difference in the process by which endocytic vesicles are formed at the two surfaces of polarized epithelial cells and that the integrity and/or the polymerization of actin filaments are required at the apical surface. Actin filaments in microvilli may be part of a mechanochemical motor that moves membrane components along the microvillar surface towards intermicrovillar spaces, or provides the force required for converting a membrane invagination or pit into an endocytic vesicle within the cytoplasm
— id: 13277, year: 1993, vol: 120, page: 695, stat: Journal Article,

Prenylated proteins play a critical role in transport from the TGN to both cell surface domains of MDCK cells
Gravotta, D.; Mayer, A.; Adesnik, M.; Sabatini, D. D.
1993 ;4(SUPPL.):209A-209A, Molecular biology of the cell
— id: 104645, year: 1993, vol: 4, page: 209A, stat: Journal Article,

Membranes
Sabatini, David D; Louvard, Daniel; Adesnik, Milton; Higgins, CF
London : Current Biology, 1993,
— id: 1683, year: 1993, vol: , page: , stat: ,

The phenobarbital-induced transcriptional activation of cytochrome P-450 genes is blocked by the glucocorticoid-progesterone antagonist RU486
Shaw PM; Adesnik M; Weiss MC; Corcos L
1993 Oct;44(4):775-783, Molecular pharmacology
Several of the hepatic microsomal cytochromes P450 can be induced by various drugs and xenobiotics, among them the barbiturate phenobarbital. Rat hepatoma cells (Fao and its derivatives) respond to phenobarbital or dexamethasone treatment with an increased accumulation of CYP2C6 mRNA and thus provide a culture system to investigate the mechanisms involved. Examination of the kinetics of CYP2C6 mRNA induction revealed that the response to dexamethasone is rapid, whereas induction by phenobarbital occurs only slowly after an 8-10-hr lag. Run-on transcription measurements demonstrated that phenobarbital treatment led to a 3-4-fold increase in CYP2C6 gene transcription. Surprisingly, induction by phenobarbital of both accumulation of CYP2C6 mRNA and transcription of the gene was blocked by the antiprogestin-antiglucocorticoid RU486, suggesting the involvement of a steroid receptor in the induction process. Transfection of promoter constructs containing a reporter gene whose expression is driven by a 1.4-kilobase 5' flanking segment of the CYP2B1 or CYP2B2 genes, which are highly inducible by phenobarbital in rat liver, led to > 3-fold increases in reporter gene activity in the presence of the drug. Again, phenobarbital induction was prevented by RU486. The RU486 inhibition of the phenobarbital induction of both the endogenous CPY2C6 gene and the transfected CYP2B1 and CYP2B2 promoter constructs leads us to propose a model whereby the drug acts indirectly to cause the accumulation of an endogenous steroid, and this molecule, acting via its receptor, would be the direct inducer of cytochromes P450. Whether or not this model proves to be correct, the results presented here provide the first evidence of the involvement of a steroid receptor in phenobarbital induction
— id: 13068, year: 1993, vol: 44, page: 775, stat: Journal Article,

An in vitro system for studying the biogenesis and function of Golgi-derived clathrin-coated vesicles
Simon, J.-P.; Ivanov, I. E.; De Lemos-Chiarandini, C.; Adesnik, M.; Sabatini, D. D.
1993 ;4(SUPPL.):321A-321A, Molecular biology of the cell
— id: 104646, year: 1993, vol: 4, page: 321A, stat: Journal Article,

CELL-FREE TRANSPORT OF VIRAL MEMBRANE-GLYCOPROTEINS FROM THE GOLGI-APPARATUS TO THE PLASMA-MEMBRANE OF POLARIZED CELLS
SIMON, JP; MAYER, A; GRAVOTTA, D; IVANOV, I; ADESNIK, M; SABATINI, DD
1993 JAN 26 ;36(1):263-263, Journal of cellular biochemistry
— id: 54369, year: 1993, vol: 36, page: 263, 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,

INDIRECT TRANSCRIPTIONAL ACTIVATION BY PHENOBARBITAL BLOCKED BY ANTIGLUCOCORTICOIDS
CORCOS L; SHAW P; WEISS M; ADESNIK M; DRINKWATER N
1992 ;4(16 PART C):33-33, Journal of cellular biochemistry. Supplement
— id: 104648, year: 1992, vol: 4, page: 33, stat: Journal Article,

Sequence of a novel cytochrome CYP2B cDNA coding for a protein which is expressed in a sebaceous gland, but not in the liver
Friedberg, T; Grassow, M A; Bartlomowicz-Oesch, B; Siegert, P; Arand, M; Adesnik, M; Oesch, F
1992 Nov 1;287 ( Pt 3):775-783, Biochemical journal
The major phenobarbital-inducible rat hepatic cytochromes P-450, CYP2B1 and CYP2B2, are the paradigmatic members of a cytochrome P-450 gene subfamily that contains at least seven additional members. Specific oligonucleotide probes for these genomic members of the CYP2B subfamily were used to assess their tissue-specific expression. In Northern-blot analysis a probe specific to gene 4 (which is designated now as CYP2B12) hybridized to a single mRNA present in the preputial gland, an organ which is used as a model for sebaceous glands, but did not hybridize to mRNA isolated from the liver or from five other tissues of untreated or Aroclor 1254-treated rats. The cDNA sequence for the CYP2B12 RNA was determined from overlapping cDNA clones and contained a long open reading frame of 1476 bp. The nucleotide sequence of the CYP2B12 cDNA was 85% similar to the sequence of the CYP2B1 cDNA in its coding region and was different from any CYP2B cDNA characterized until now. The cDNA-derived primary structure of the CYP2B12 protein contains a signal sequence for its insertion into the endoplasmic reticulum and the putative haem-binding site characteristic of cytochromes P-450. A part of the potential haem pocket of CYP2B12 was identical with a similar structure in a bacterial protocatechuate dioxygenase. In immunoblot analysis of preputial-gland microsomes, antibodies against CYP2B1 recognized a single abundant protein with a lower apparent molecular mass than that of CYP2B1. Our results demonstrate that the CYP2B12 protein has the potential to be enzymically active and are the first demonstration that a member of the CYP2B subfamily is expressed exclusively and at high levels in an extrahepatic organ
— id: 106304, year: 1992, vol: 287 ( Pt 3), page: 775, stat: Journal Article,

MUTATION OF THE TYROSINE-CONTAINING ENDOCYTIC SIGNAL IN THE VSV G-PROTEIN IMPAIRS BASOLATERAL BUT NOT APICAL ENDOCYTOSIS IN MDCK CELLS
GOTTLIEB, T; KRUPPA, J; KRUPPA, A; ADESNIK, M; SABATINI, D
1992 SEP ;3(4):A303-A303, Molecular biology of the cell
— id: 51868, year: 1992, vol: 3, page: A303, 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,

Membranes
Louvard D; Adesnik M; Sabatini DD
1992 ;4:569-572, Current opinion in cell biology
— id: 55894, year: 1992, vol: 4, page: 569, stat: Journal Article,

CELL-FREE RECONSTITUTION OF THE POLARIZED TRANSPORT OF A VIRAL GLYCOPROTEIN FROM THE TGN TO THE BASOLATERAL PLASMA-MEMBRANE OF MDCK CELLS
MAYER, A; IVANOV, I; ADESNIK, M; SABATINI, D
1992 SEP ;3(4):A307-A307, Molecular biology of the cell
— id: 51869, year: 1992, vol: 3, page: A307, stat: Journal Article,

ROLE OF CHARGED AMINO-ACIDS WITHIN THE SEGMENTS FLANKING THE HYDROPHOBIC CORE OF A SIGNAL ANCHOR SEQUENCE IN DETERMINING THE TRANSMEMBRANE DISPOSITION OF A PROTEIN
MONIER, S; SABATINI, D; ADESNIK, M
1992 SEP ;3(4):A125-A125, Molecular biology of the cell
— id: 51862, year: 1992, vol: 3, page: A125, stat: Journal Article,

INVITRO FORMATION OF POST GOLGI VESICLES CARRYING VIRAL ENVELOPE GLYCOPROTEINS
SIMON, JP; IVANOV, I; RINDLER, M; ADESNIK, M; SABATINI, DD
1992 SEP ;3(4):A308-A308, Molecular biology of the cell
— id: 51870, year: 1992, vol: 3, page: A308, 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,

Potentiation of the inductive effect of phenobarbital on cytochrome P450 mRNAs by cannabidiol
Deutsch, D G; Tombler, E R; March, J E; Lo, S H; Adesnik, M
1991 Oct 24;42(10):2048-2053, Biochemical pharmacology
— id: 106305, year: 1991, vol: 42, page: 2048, stat: Journal Article,

Membranes
Sabatini DD; Louvard D; Adesnik M
1991 Aug;3(4):575-579, Current opinion in cell biology
— id: 55807, year: 1991, vol: 3, page: 575, stat: Journal Article,

The human dioxin-inducible NAD(P)H: quinone oxidoreductase cDNA-encoded protein expressed in COS-1 cells is identical to diaphorase 4
Shaw PM; Reiss A; Adesnik M; Nebert DW; Schembri J; Jaiswal AK
1991 Jan 1;195(1):171-176, European journal of biochemistry
NAD(P)H: quinone oxidoreductase (NQO1) is believed to be protective against cancer and toxicity caused by exposure to quinones and their metabolic precursors. This enzyme catalyzes the two-electron reduction of compounds, compared with one-electron reduction mediated by NADPH: cytochrome-P450 oxidoreductase which produces toxic and mutagenic free radicals. Recently we cloned and sequenced the cDNA encoding human 2.3,7,8-tetrachlorodibenzo-p-dioxin (dioxin)-inducible cytosolic NQO1 [Jaiswal et al. (1988) J. Biol. Chem. 263, 13572-13578] and provided preliminary evidence that this enzyme may correspond to diaphorase 4, an enzymatic activity present in various tissues that catalyzes the reduction of a variety of quinones by both NADH and NADPH [Edwards et al. (1980) Biochem. J. 187, 429-436]. In the present report we characterize the catalytic properties of the protein encoded by the NQO1 cDNA. The enzyme was synthesized in monkey kidney COS-1 cells transfected with a pMT2-based expression plasmid containing the NQO1 cDNA. Western blot analysis of the transfected cells using an antibody against rat liver cytosolic NQO1 revealed a 31-kDa band that was not detected in nontransfected cells. This band corresponded to a polypeptide with the same electrophoretic mobility as the endogenous NQO1 protein detected in the human hepatoblastoma (Hep-G2) cells with the same antibody. The immunoreactive protein detected in human Hep-G2 cells was induced approximately fourfold by exposure of the cultures to dioxin, an increase commensurate with the increased in quinone oxidoreductase activity. These studies suggest that the protein encoded by NQO1 cDNA is indeed similar, if not identical, to the dioxin-inducible protein band detected in human Hep-G2 cells. Further characterization of the product of NQO1 cDNA, which was present at approximately 20-30-fold higher levels in transfected COS cells than the endogenous product in uninduced human Hep-G2 cells indicated that it had very high capacity (greater than 1000-fold over background) to catalyze the reduction of 2.6-dichloroindophenol and menadione. Besides these two commonly used substrates for quinone reductase, the expressed NQO1 protein also effectively metabolized 2,6-dimethylbenzoquinone, methylene blue, p-benzoquinone, 1,4-naphthoquinone, 2-methyl-1,4-benzoquinone, with the latter being the most potent electron acceptor at 50 microM concentration of the substrate
— id: 14190, year: 1991, vol: 195, page: 171, 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,

A PROCEDURE FOR THE SELECTIVE PERMEABILIZATION OF THE APICAL OR BASOLATERAL PLASMA MEMBRANE DOMAINS OF EPITHELIAL CELLS THAT ALLOWS THE STUDY OF MEMBRANE PROTEIN TRANSPORT
GRAVOTTA D; ADESNIK M; SABATINI D D
1990 ;111(5 PART 2):325A-325A, Journal of cell biology
— id: 104649, year: 1990, vol: 111, page: 325A, stat: Journal Article,

TRANSPORT OF INFLUENZA HA FROM THE TGN TO APICAL SURFACE OF MDCK CELLS TAKES PLACE IN TWO STEPS THAT ARE DIFFERENTIALLY AFFECTED BY GTP-GAMMA-S
GRAVOTTA D; ADESNIK M; SABATINI D D
1990 ;111(5 PART 2):326A-326A, Journal of cell biology
— id: 104650, year: 1990, vol: 111, page: 326A, stat: Journal Article,

Transport of influenza HA from the trans-Golgi network to the apical surface of MDCK cells permeabilized in their basolateral plasma membranes: energy dependence and involvement of GTP-binding proteins
Gravotta D; Adesnik M; Sabatini DD
1990 Dec;111(6 Pt 2):2893-2908, Journal of cell biology
A procedure employing streptolysin O to effect the selective permeabilization of either the apical or basolateral plasma membrane domains of MDCK cell monolayers grown on a filter support was developed which permeabilizes the entire monolayer, leaves the opposite cell surface domain intact, and does not abolish the integrity of the tight junctions. This procedure renders the cell interior accessible to exogenous macromolecules and impermeant reagents, permitting the examination of their effects on membrane protein transport to the intact surface. The last stages of the transport of the influenza virus hemagglutinin (HA) to the apical surface were studied in pulse-labeled, virus-infected MDCK cells that were incubated at 19.5 degrees C for 90 min to accumulate newly synthesized HA in the trans-Golgi network (TGN), before raising the temperature to 35 degrees C to allow synchronized transport to the plasma membrane. In cells permeabilized immediately after the cold block, 50% of the intracellular HA molecules were subsequently delivered to the apical surface. This transport was dependent on the presence of an exogenous ATP supply and was markedly inhibited by the addition of GTP-gamma-S at the time of permeabilization. On the other hand, the GTP analogue had no effect when it was added to cells that, after the cold block, were incubated for 15 min at 35 degrees C before permeabilization, even though at this time most HA molecules were still intracellular and their appearance at the cell surface was largely dependent on exogenous ATP. These findings indicate that GTP-binding proteins are involved in the constitutive process that effects vesicular transport from the TGN to the plasma membrane and that they are charged early in this process. Transport of HA to the cell surface could be made dependent on the addition of exogenous cytosol when, after permeabilization, cells were washed to remove endogenous cytosolic components. This opens the way towards the identification of cell components that mediate the sorting of apical and basolateral membrane components in the TGN and their polarized delivery to the cell surface
— id: 14265, year: 1990, vol: 111, page: 2893, stat: Journal Article,

Nucleotide and deduced amino acid sequence of a human cDNA (NQO2) corresponding to a second member of the NAD(P)H:quinone oxidoreductase gene family. Extensive polymorphism at the NQO2 gene locus on chromosome 6
Jaiswal, A K; Burnett, P; Adesnik, M; McBride, O W
1990 Feb 20;29(7):1899-1906, Biochemistry
NAD(P)H:quinone oxidoreductases (NQOs) are flavoproteins that catalyze the oxidation of NADH or NADPH by various quinones and oxidation-reduction dyes. We have previously described a complementary DNA that encodes a dioxin-inducible cytosolic form of human NAD(P)H:quinone oxidoreductase (NQO1). In the present report we describe the nucleotide sequence and deduced amino acid sequence for a cDNA clone that is likely to encode a second form of NAD(P)H:quinone oxidoreductase (NQO2) which was isolated by screening a human liver cDNA library by hybridization with a NQO1 cDNA probe. The NQO2 cDNA is 976 nucleotides long and encodes a protein of 231 amino acids (Mr = 25,956). The human NQO2 cDNA and protein are 54% and 49% similar to human liver cytosolic NQO1 cDNA and protein, respectively. COS1 cells transfected with NQO2 cDNA showed a 5-7-fold increase in NAD(P)H:quinone oxidoreductase activity as compared to nontransfected cells when either 2,6-dichlorophenolindophenol or menadione was used as substrate. Western blot analysis of the expressed NQO1 and NQO2 cDNA proteins showed cross-reactivity with rat NQO1 antiserum, indicating that NQO1 and NQO2 proteins are immunologically related. Northern blot analysis shows the presence of one NQO2 mRNA of 1.2 kb in control and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treated human hepatoblastoma Hep-G2 cells and that TCDD treatment does not lead to enhanced levels of NQO2 mRNA as it does for NQO1 mRNA. Southern blot analysis of human genomic DNA suggests the presence of a single gene approximately 14-17 kb in length. The NQO2 gene locus is highly polymorphic as indicated by several restriction fragment length polymorphisms detected with five different restriction enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 106307, year: 1990, vol: 29, page: 1899, stat: Journal Article,

Glucocorticoid regulation of a phenobarbital-inducible cytochrome P-450 gene: the presence of a functional glucocorticoid response element in the 5'-flanking region of the CYP2B2 gene
Jaiswal, A K; Haaparanta, T; Luc, P V; Schembri, J; Adesnik, M
1990 Jul 25;18(14):4237-4242, Nucleic acids research
The rat cytochrome P450 CYP2B2 gene encodes one of the two major phenobarbital-inducible forms of hepatic microsomal cytochrome P-450. The sequence of a 1.4 Kb DNA segment from the 5' flanking region of this region [Jaiswal, A., Rivkin, E. and Adesnik, M. Nucl. Acids. Res. 15: 6755 (1987)] reveals the presence of a pentadecameric oligonucleotide sequence, located approximately 1.3 Kb upstream of the transcription initiation site, which is highly similar to the sequences of glucocorticoid response elements (GREs) that mediate the hormone-dependent transcriptional activation of many other genes. The putative GRE in the CYP2B2 gene 5' flanking region is shown to be functional by demonstrating that segments of DNA that contain it, including one that is only 25bp long, are capable of conferring dexamethasone inducibility on a chloramphenicol acetyltransfer-ase gene whose transcription is driven by the Herpes virus thymidine kinase gene promoter. Moreover, binding of a protein contained in a rat liver nuclear extract to a 25 bp synthetic DNA segment that contains the putative GRE was demonstrated in a gel mobility shift assay. This binding was specifically competed away by a DNA segment that contains the murine mammary tumor virus long terminal repeat which encompasses several well characterized GRE elements. The implications of these findings for the in vivo regulation of the P450IIB2 gene by glucocorticoids are discussed
— id: 106306, year: 1990, vol: 18, page: 4237, stat: Journal Article,

Hormonal regulation of levels of the messenger RNA encoding hepatic P450 2c (IIC11), a constitutive male-specific form of cytochrome P450
Janeczko, R; Waxman, D J; Le Blanc, G A; Morville, A; Adesnik, M
1990 Feb;4(2):295-303, Molecular endocrinology
A cDNA clone for rat hepatic cytochrome P450 2c (gene product IIC11) was isolated and used to study the sex specificity, expression during development, and hormonal regulation of the mRNA encoding this protein in rat liver. P450 2c mRNA levels were about 16-fold higher in males than in females and were only slightly increased in male rats after administration of phenobarbital, a drug that dramatically raises the levels of mRNAs encoding several other members of the P450 II family. In contrast to the mRNA encoding P450 f (gene product IIC7), which increases gradually over the first 6 weeks of life, P450 2c mRNA showed a dramatic increase at puberty, between 4.5-5.5 weeks of life. The roles of sex steroids and GH in controlling this male-specific, developmentally regulated mRNA were then examined. A dependence on adult androgen was demonstrated by the 2- to 4-fold decrease in P-450 2c mRNA levels after castration of adult male rats and their restoration to normal by administration of the synthetic androgen methyltrienolone. Prolonged treatment (15 days) of ovariectomized female rats with this androgen also increased the levels of P450 2c mRNA and its encoded testosterone 16 alpha-hydroxylase to those of intact males. In male rats treated with estradiol valerate, mRNAs for P450 2c and alpha 2u-globulin, a major male-specific hepatic secretory protein that is under complex hormonal control, fell to negligible levels. None of these hormonal perturbations had a detectable effect on the levels of PB-1 (gene product IIC6) mRNA, which is not expressed in a sex-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 106308, year: 1990, vol: 4, page: 295, stat: Journal Article,

Sequence requirements for cytochrome P-450IIB1 catalytic activity. Alteration of the stereospecificity and regioselectivity of steroid hydroxylation by a simultaneous change of two hydrophobic amino acid residues to phenylalanine
Aoyama, T; Korzekwa, K; Nagata, K; Adesnik, M; Reiss, A; Lapenson, D P; Gillette, J; Gelboin, H V; Waxman, D J; Gonzalez, F J
1989 Dec 15;264(35):21327-21333, Journal of biological chemistry
The phenobarbital-inducible P-450 forms IIB1 and IIB2 are identical in sequence except for 14 amino acid differences within the carboxyl-terminal half of the molecule. IIB1 has about a 5-10-fold higher turnover number for most monooxygenase substrates examined although the substrate specificities of both enzymes are virtually identical. Both P-450s oxygenate testosterone to yield the 16 alpha-hydroxy, 16 beta-hydroxy, 17-keto, and 16 beta-hydroxy, 17-keto metabolites as major products. A variant IIB2 cDNA, isolated from an uninduced rat liver lambda gt11 library, and when expressed in Hep G2 cells using a vaccinia virus vector, was found to code for a protein that produced the 16 alpha-hydroxy and 17-keto metabolites of testosterone but no 16 beta-hydroxylated products. Although the published sequences of IIB1 and IIB2 are identical within the N-terminal halves of the proteins, sequence analysis of the variant cDNA revealed two amino acid substitutions in this region; Leu58----Phe and I1e114----Phe. When these two amino acid changes were incorporated into IIB1, via construction of a chimeric cDNA, the resultant expressed enzyme did not catalyze the 16 beta-hydroxylation of testosterone or androstenedione. Formation of the 16 alpha-hydroxy and 17-keto metabolites, however, was only slightly reduced compared with the parent IIB1. A IIB1 protein that possessed only the I1e114----Phe replacement catalyzed the production of all four testosterone metabolites with only slightly different product ratios compared with the parent enzyme. The substrate specificity of a IIB1 variant containing only the Leu58----Phe replacement could not be determined, since that protein did not accumulate in cells infected with the corresponding recombinant vaccinia virus. These data suggest that two distinct amino acid residues located within the amino-terminal fourth of IIB1 and IIB2 can affect substrate orientation at the active site
— id: 106309, year: 1989, vol: 264, page: 21327, stat: Journal Article,

The expression of cytochrome P450IIB1 in Saccharomyces cerevisiae results in an increased mutation frequency when exposed to cyclophosphamide
Black, S M; Ellard, S; Meehan, R R; Parry, J M; Adesnik, M; Beggs, J D; Wolf, C R
1989 Nov;10(11):2139-2143, Carcinogenesis
A recombinant plasmid containing a full length cDNA encoding the rat cytochrome P450IIB1 under the control of the Saccharomyces cerevisiae ADC1 promoter was constructed and transformed into the yeast strain KY118. The encoded P450IIB1 protein was produced at a level of between 0.1 and 0.2% of total yeast cellular protein (0.068 nmol/mg total cellular protein). This protein was localized in the microsomal fraction and had activity towards the substrate benzyloxyresorufin, the activity being 0.16 nmol resorufin produced/min/mg microsomal protein. When exposed to the anticancer drug cyclophosphamide the mutation frequency, as determined by the development of resistance to the arginine analogue canavanine, increased in a dose-dependent manner over a control strain and was up to 16-fold higher at the highest doses used
— id: 106310, year: 1989, vol: 10, page: 2139, stat: Journal Article,

A sorting signal for the basolateral delivery of the vesicular stomatitis virus (VSV) G protein lies in its luminal domain: analysis of the targeting of VSV G-influenza hemagglutinin chimeras
Compton T; Ivanov IE; Gottlieb T; Rindler M; Adesnik M; Sabatini DD
1989 Jun;86(11):4112-4116, Proceedings of the National Academy of Sciences of the United States of America
When synthesized in polarized epithelial cells, the envelope glycoproteins hemagglutinin of influenza and G of vesicular stomatitis virus are targeted to the apical and basolateral plasma membranes, respectively. To determine which portions of these transmembrane proteins contain information necessary for their sorting, the behavior of two different G-hemagglutinin chimeric polypeptides, consisting of all or nearly all the luminal portion of the vesicular stomatitis virus G protein linked to C-terminal segments of influenza hemagglutinin that included its transmembrane and cytoplasmic domains, was studied in MDCK cells transformed with the corresponding cDNAs. Both chimeras were transported from the endoplasmic reticulum to the Golgi apparatus and from there to the cell surface with the same rapid kinetics as the intact G protein. By using a cell surface immunoprecipitation assay with monolayers cultured on permeable filters that allows the recovery of labeled protein molecules present in each cell surface domain, it was found that both chimeric proteins as well as the intact G protein were delivered almost exclusively to the basolateral surface. This polarized distribution of the polypeptides did not change during a subsequent 90-min chase period, although during this time a large fraction of the glycoprotein molecules underwent degradation. In addition, a small fraction of the cell surface-associated glycoprotein molecules shed their ectoplasmic segments into the basolateral compartment, apparently as a result of a proteolytic cleavage. Immunofluorescence on transverse frozen sections and immunoelectron microscopy revealed a prominent accumulation of the chimeric polypeptides in the lateral cell membranes, with lesser amounts on the basal and apical surfaces. These results indicate that information specifying the basolateral transport of the G glycoprotein is located within the first 426 N-terminal amino acids of its ectoplasmic portion
— id: 10609, year: 1989, vol: 86, page: 4112, 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,

The P450 superfamily: updated listing of all genes and recommended nomenclature for the chromosomal loci
Nebert, D W; Nelson, D R; Adesnik, M; Coon, M J; Estabrook, R W; Gonzalez, F J; Guengerich, F P; Gunsalus, I C; Johnson, E F; Kemper, B
1989 Jan-Feb;8(1):1-13, DNA
In this update we provide a list of the 71 P450 genes and the four P450 pseudogenes that have been characterized as of September 30, 1988. The chromosomal locations of many of these genes are also summarized. A modest revision of the initially proposed nomenclature of the P450 superfamily (Nebert et al., DNA 6, 1-11, 1987) is described specifically for the human and mouse chromosomal loci. The motivation for this revision is to conform to the rules of nomenclature for human and mouse genes. Recommendations for the naming of chromosomal loci include the root symbol 'CYP' for human ('Cyp' for mouse), denoting 'cytochrome P450.' We recommend that this root also be used for other organisms. For a chromosomal locus, the root symbol is followed by an Arabic numeral designating the P450 family, a letter indicating the subfamily, and an Arabic numeral representing the individual gene within the family or subfamily. Numbers of the individual genes usually will be assigned in the order the genes are identified. This system is consistent with our earlier proposed nomenclature for P450 families and gene products from all eukaryotes and prokaryotes
— id: 106312, year: 1989, vol: 8, page: 1, stat: Journal Article,

Brain cytochromes P-450 are responsive to phenobarbital and tricyclic amines
Strobel, H W; Cattaneo, E; Adesnik, M; Maggi, A
1989 Mar-Apr;21(2):169-175, Pharmacological research
Cytochrome P-450 form b has been detected for the first time in rat brain by cross-hybridization of poly(A+) RNA with a radiolabelled cDNA probe. The cross-hybridizable material was easily detectable in rats after treatment with phenobarbital but not in untreated rats. Moreover, treatment of rats with amitriptyline or imipramine, tricyclic antidepressants in wide therapeutic use for depression, markedly increases the amount of RNA species hybridizing with P-450b cDNA probe in comparison to untreated controls. These observations raise the issue of a possible role for brain cytochromes P-450 in the metabolism of drugs such as the tricyclic antidepressants
— id: 106311, year: 1989, vol: 21, page: 169, stat: Journal Article,

Construction of mutant and chimeric genes using the polymerase chain reaction
Vallette F; Mege E; Reiss A; Adesnik M
1989 Jan 25;17(2):723-733, Nucleic acids research
In the polymerase chain reaction (PCR) the specific amplification of a small segment of DNA within a complex DNA sample is effected by repeated cycles of DNA denaturation and enzymatic synthesis primed by two oligonucleotides complementary to regions within opposite strands of the DNA. In this report a simple and efficient method is described in which PCR methodology is used to introduce specific mutations into a double stranded DNA molecule. In this procedure a supercoiled plasmid DNA serves as template for a PCR in which a primer bearing the mutated sequence is incorporated into the amplified product. The presence of convenient restriction sites in the mutagenic primer and in the amplified DNA permit direct replacement of a wild type DNA segment with the mutated segment by treating the PCR mixture with the appropriate restriction endonucleases followed by DNA ligase. Using this procedure, a single amino acid replacement, a 16 amino acid deletion and a replacement of four amino acids with a twelve amino acid segment from another membrane protein were introduced into the amino terminal signal segment of rat hepatic cytochrome P450b (P450IIB1)
— id: 10741, year: 1989, vol: 17, page: 723, 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,

EXPRESSION OF CYTOCHROME-P-450 CDNAS IN YEAST
BLACK, S; MEEHAN, RR; BEGGS, JD; MILES, JS; ADESNIK, M; WOLF, CR
1988 ;58(2):236-236, British journal of cancer
— id: 104652, year: 1988, vol: 58, page: 236, stat: Journal Article,

Stable expression of rat cytochrome P-450IIB1 cDNA in Chinese hamster cells (V79) and metabolic activation of aflatoxin B1
Doehmer, J; Dogra, S; Friedberg, T; Monier, S; Adesnik, M; Glatt, H; Oesch, F
1988 Aug;85(16):5769-5773, Proceedings of the National Academy of Sciences of the United States of America
V79 Chinese hamster fibroblasts are widely used for mutagenicity testing but have the serious limitation that they do not express cytochromes P-450, which are needed for the activation of many promutagens to mutagenic metabolites. A full-length cDNA clone encoding the monooxygenase cytochrome P-450IIB1 under control of the simian virus 40 early promoter was constructed and cointroduced with the selection marker neomycin phosphotransferase (conferring resistance to G418) into V79 Chinese hamster cells. G418-resistant cells were selected, established as cell lines, and tested for cytochrome P-450IIB1 expression and enzymatic activity. Two cell lines (SD1 and SD3) were found that stably produce cytochrome P-450IIB1. Although purified cytochromes P-450 possess monooxygenase activity only after reconstitution with cytochrome P-450 reductase and phospholipid, the gene product of the construct exhibited this activity. This implies that the gene product is intracellularly localized in a way that allows access to the required components. If compared with V79 cells, the mutation rate for the hypoxanthine phosphoribosyltransferase (HPRT) locus in SD1 cells is markedly increased when exposed to aflatoxin B1, which is activated by this enzyme
— id: 106313, year: 1988, vol: 85, page: 5769, stat: Journal Article,

CLONING OF A COMPLEMENTARY DNA ENCODING A RAT LIVER POLYPEPTIDE BEARING COMMON ANTIGENIC DETERMINANTS TO A LYSOSOMAL-ENDOSOMAL MEMBRANE PROTEIN
HYMAN C; MORIMOTO T; ADESNIK M; SABATINI D D; ROSENFELD M
1988 ;107(6 PART 3):343A-343A, Journal of cell biology
— id: 104653, year: 1988, vol: 107, page: 343A, stat: Journal Article,

Human dioxin-inducible cytosolic NAD(P)H:menadione oxidoreductase. cDNA sequence and localization of gene to chromosome 16
Jaiswal AK; McBride OW; Adesnik M; Nebert DW
1988 Sep 25;263(27):13572-13578, Journal of biological chemistry
NAD(P)H:menadione oxidoreductase (NMOR1) is a flavoprotein that catalyzes the two-electron reduction of various redox dyes and quinones. It has been proposed that this enzyme may have a protective effect against cancer caused by quinones and their metabolic precursors. We show that tetrachlorodibenzo-p-dioxin (TCDD) treatment of the human hepatoblastoma cell line Hep-G2 produces a 5-fold induction of NMOR activity. Several overlapping human NMOR1 cDNAs were isolated from a human liver lambda gt 11 expression library, and their composite sequence corresponds to an mRNA of 2448 nucleotides containing a continuous open reading frame encoding a protein of 274 residues (molecular weight, 30,880). The corresponding human NMOR1 mRNA has an unusually long 3'-untranslated region (1679 base pairs) with four potential polyadenylation signals (I-IV) at positions 986, 1460, 1838, and 2419 and a single copy of human Alu repetitive sequence between polyadenylation sites II and III. Southern blot analysis of human genomic DNA suggests the presence of a single NMOR1 gene approximately 10 kilobases (KB) in length. The use of three of the aforementioned polyadenylation signals is likely to account for the three different species (2.7, 1.7, and 1.2 kb) of mRNA hybridizing to NMOR1 cDNA in Hep-G2 cells. Indeed several partial cDNA clones were isolated that corresponded to the mRNA derived by use of the proximal polyadenylation signal. Interestingly, the longest (2.7 kb) mRNA species was induced severalfold by TCDD, whereas the other two mRNAs (1.7 and 1.2 kb) were induced to a much lesser extent by TCDD treatment. The human NMOR1 cDNA and protein are 83 and 85% similar to rat liver cytosolic NMOR1 cDNA and protein, respectively. Southern analysis of DNA from 54 human x mouse and 39 human x hamster somatic cell hybrids shows that the NMOR1 gene resides on human chromosome 16
— id: 10959, year: 1988, vol: 263, page: 13572, stat: Journal Article,

Human cytochrome P-450 PB-1: a multigene family involved in mephenytoin and steroid oxidations that maps to chromosome 10
Meehan, R R; Gosden, J R; Rout, D; Hastie, N D; Friedberg, T; Adesnik, M; Buckland, R; van Heyningen, V; Fletcher, J; Spurr, N K
1988 Jan;42(1):26-37, American journal of human genetics
The cytochrome P-450 monooxygenase system possesses catalytic activity toward many exogenous compounds (e.g., drugs, insecticides, and polycyclic aromatic hydrocarbons) and endogenous compounds (e.g., steroids, fatty acids, and prostaglandins). Multiple forms of cytochrome P-450 with different substrate specificities have been isolated. In the present paper we report the isolation and sequence of a cDNA clone for the human hepatic cytochrome P-450 responsible for mephenytoin (an anticonvulsant) oxidation. The mephenytoin cytochrome P-450 is analogous to the rat cytochrome P-450 form termed PB-1 (family P450C2C). We also report that human PB-1 is encoded by one of a small family of related genes all of which map to human chromosome 10q24.1-10q24.3. The endogenous role of this enzyme appears to be in steroid oxidations. This cytochrome P-450 family does not correspond to any of the hepatic cytochrome P-450 gene families previously mapped in humans
— id: 106315, year: 1988, vol: 42, page: 26, stat: Journal Article,

Chromosomal organization of the cytochrome P450-2C gene family in the mouse: a locus associated with constitutive aryl hydrocarbon hydroxylase
Meehan, R R; Speed, R M; Gosden, J R; Rout, D; Hutton, J J; Taylor, B A; Hilkens, J; Kroezen, V; Hilgers, J; Adesnik, M
1988 Apr;85(8):2662-2666, Proceedings of the National Academy of Sciences of the United States of America
Cytochromes P-450 represent a superfamily of enzymes with a central role in the metabolism of drugs, chemical toxins, and carcinogens. We have used genetic analysis to establish the complexity and catalytic function of a recently identified constitutively expressed murine hepatic cytochrome P-450 encoded by P450-2C. Southern blotting analysis shows that there are at least seven or eight genes within this family in the mouse and rat and that DNA restriction fragment length variants between different mouse inbred strains are observed. Analysis of recombinant inbred strains derived from these parent strains shows (i) these genes are clustered within 1 centimorgan, (ii) this gene family does not correspond to any of the known cytochrome P-450 loci or map near any well-characterized genomic markers, and (iii) this gene family segregates to within 1-2 centimorgans of a locus controlling constitutive aryl hydrocarbon hydroxylase activity in mice. With use of Chinese hamster/mouse somatic cell hybrids, the P450-2C locus was assigned to a region of mouse chromosome 19 that appears to be syntenic with the previously mapped human P450C2C locus on human chromosome 10. By in situ hybridization to mitotic mouse chromosomes, we have localized this region to the tip of chromosome 19. These results are discussed in relation to the physiological roles of this P-450 family in foreign compound metabolism and steroid oxidations
— id: 106314, year: 1988, vol: 85, page: 2662, 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,

BIOSYNTHESIS OF IL-1 ALPHA IN CULTURED MACROPHAGES AND TRANSFECTED CELLS
MOORE B; ZHANG Y; ADESNIK M; SABATINI D D
1988 ;107(6 PART 3):699A-699A, Journal of cell biology
— id: 104654, year: 1988, vol: 107, page: 699A, 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,

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,

Nonpolarized secretion of truncated forms of the influenza hemagglutinin and the vesicular stomatitus virus G protein from MDCK cells
Gonzalez A; Rizzolo L; Rindler M; Adesnik M; Sabatini DD; Gottlieb T
1987 Jun;84(11):3738-3742, Proceedings of the National Academy of Sciences of the United States of America
The demonstration that the envelope glycoproteins G of vesicular stomatitus virus and hemagglutinin of influenza virus synthesized in polarized epithelial cells transfected with the corresponding genes are effectively segregated to the basolateral or apical plasma membrane domains, respectively, implies that the information determining this segregation resides within the structures of the proteins themselves. To localize the sorting information within these proteins, the polarity of secretion of truncated hemagglutinin and G glycoproteins secreted from confluent monolayers of MDCK cells transformed with vectors containing the corresponding truncated cDNAs was examined. It was found that, even though the transformed cells continued to secrete a major endogenous glycoprotein exclusively from the apical surface, the modified viral glycoproteins were secreted in a nonpolarized fashion from both sides of the monolayers. These observations suggest that important information for the sorting of the viral glycoprotein is contained within their membrane anchoring or cytoplasmic segments or that, if sorting signals are luminally located, these signals must be present in a conformation that is not attainable when the polypeptides are not attached to the membrane
— id: 55809, year: 1987, vol: 84, page: 3738, stat: Journal Article,

Sorting Of Plasma Membrane And Secretory Proteins In Cultured Polarized Epithelial Cells
Gottlieb TA; Gonzalez A; Beaudry G; Rindler MJ; Adesnik M; Sabatini DD
Molecular mechanisms in the regulation of cell behavior New York : Liss, 1987,
— id: 5236, year: 1987, vol: , page: 187, stat: Chapter,

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,

5' flanking sequence of the gene for rat hepatic cytochrome P450e
Jaiswal, A K; Rivkin, E; Adesnik, M
1987 Aug 25;15(16):6755-6755, Nucleic acids research
— id: 106316, year: 1987, vol: 15, page: 6755, stat: Journal Article,

The P450 gene superfamily: recommended nomenclature
Nebert, D W; Adesnik, M; Coon, M J; Estabrook, R W; Gonzalez, F J; Guengerich, F P; Gunsalus, I C; Johnson, E F; Kemper, B; Levin, W
1987 Feb;6(1):1-11, DNA
A nomenclature for the P450 gene superfamily is proposed based on evolution. Recommendations include Roman numerals for distinct gene families, capital letters for subfamilies, and Arabic numerals for individual genes. An updating of this list, which presently includes 65 entries, will be required every 1-2 years. Assignment of orthologous genes is presently uncertain in some cases--between widely diverged species and especially in the P450II family due to the large number of genes. As more is known, it might become necessary to change some gene assignments that are based on our present knowledge
— id: 106317, year: 1987, vol: 6, page: 1, stat: Journal Article,

Molecular Aspects Of Cytochrome P-450 Monooxygenases Characterization Of Some Constitutively Expressed Forms
Wolf C R; Meechan R; Burke M D; Adams D J; Oesch F; Friedberg T; Adesnik M; Hastie N
Drug metabolism from molecules to man New York : Taylor & Francis, 1987,
— id: 5222, year: 1987, vol: , page: 14, stat: Chapter,

Genes for cytochrome P-450 and their regulation
Adesnik, M; Atchison, M
1986 ;19(3):247-305, CRC Critical reviews in biochemistry
The capacity of the liver microsomal mixed-function oxidase system to metabolize a wide variety of exogenous as well as endogenous compounds reflects the participation of multiple forms of the terminal oxidase, cytochrome P-450, which have different broad, but overlapping, substrate specificities. Several of these isozymes accumulate in the liver after exposure of animals to specific inducing agents. Recent studies employing recombinant DNA techniques to investigate the genetic and evolutionary relatedness of various cytochrome P-450 isozymes as well as the molecular basis for the induction phenomenon are described. The conclusions from these investigations are presented in the context of the substantial body of data obtained from the characterization of specific cytochrome P-450 isozymes and from studies on the induction of specific isozymes or enzymatic activities during development or after treatment of animals with various inducing agents
— id: 106320, year: 1986, vol: 19, page: 247, stat: Journal Article,

SYNTHESIS AND SORTING TO LYSOSOMES OF BETA-GLUCURONIDASE IN TRANSFECTED CELLS
ANDY, R; ROSENFELD, M; ADESNIK, M; SABATINI, D
1986 NOV ;103(5):A359-A359, Journal of cell biology
— id: 41324, year: 1986, vol: 103, page: A359, stat: Journal Article,

Gene conversion in a cytochrome P-450 gene family
Atchison, M; Adesnik, M
1986 Apr;83(8):2300-2304, Proceedings of the National Academy of Sciences of the United States of America
The mRNAs encoding the two major phenobarbital-inducible forms of cytochrome P-450 of rat liver, P-450b and P-450e, are remarkably similar (98% homologous) in nucleotide sequence, but the distribution of differences within them is not random. While the 5' halves (approximately equal to 1 kilobase) appear to be identical, there are 36 divergent residues in the remaining sequences of the two mRNAs, with 14 differences residing in two short highly divergent segments, which in the P-450e gene are located within exon 7. DNA sequence analysis of portions of a number of P-450b/e-related genes provides strong evidence that at least one of the short divergent segments is the result of a recent gene conversion event between an ancestor to the cytochrome P-450e gene and a related donor P-450 gene of unknown function. The sequence data also suggest that extensive gene conversion has occurred within all the members of this gene family in the region including exons 7 and 8 and the intron between them, with a resultant homogenization of those sequences relative to other portions of the genes. Genomic Southern blotting analysis demonstrates that the presence of an apparent 'constant' region in the 5' halves of the P-450b and P-450e mRNAs does not reflect a rearrangement in somatic cells of a germ-line DNA configuration. It is therefore proposed that it, too, is a consequence of a very recent gene conversion event between ancestors of the genes encoding both proteins or of an unequal crossing-over between them. On the basis of these and other data we propose that gene conversion represents an important evolutionary mechanism for the generation of related cytochrome P-450 isozymes in which regions of extraordinary sequence similarity and dissimilarity are intermixed. The gene conversion mechanism would account for some of the overlaps in substrate specificities of distantly related P-450s as well as for substantial differences in catalytic properties between closely related members of the same P-450 protein family
— id: 106319, year: 1986, vol: 83, page: 2300, stat: Journal Article,

POLARIZED DELIVERY AND RECYCLING IN MDCK CELLS OF A CHIMERIC PROTEIN CONTAINING THE LUMINAL PORTION OF VSV-G AND THE CYTOPLASMIC AND TRANSMEMBRANE DOMAIN OF INFLUENZA HA
Compton, T; Gottlieb, T; Rindler, M; Adesnik, M; Sabatini, DD
1986 NOV ;103(5):A7-A7, Journal of cell biology
— id: 51125, year: 1986, vol: 103, page: A7, stat: Journal Article,

EFFECT OF DRUGS OF ABUSE ON CYTOCHROME-P-450 MESSENGER-RNA LEVELS
Deutsch, DG; Schutzbank, TE; March, JE; Adesnik, M
1986 May;45(6):1852-1852, Federation Proceedings (Federation of American Societies for Experimental Biology)
— id: 31061, year: 1986, vol: 45, page: 1852, stat: Journal Article,

Isolation and characterization of cDNA clones for cytochromes P-450 immunochemically related to rat hepatic P-450 form PB-1
Friedberg, T; Waxman, D J; Atchison, M; Kumar, A; Haaparanta, T; Raphael, C; Adesnik, M
1986 Dec 2;25(24):7975-7983, Biochemistry
Rat hepatic cytochrome P-450 PB-1 is a prominent constitutive P-450 form whose levels increase approximately 2-3 fold upon phenobarbital administration. Antibodies raised against this protein recognized two major proteins in immunoblots of rat liver microsomal proteins and precipitated comparable amounts of two electrophoretically separable hepatic mRNA translation products. The levels of the two mRNAs encoding these polypeptides were increased substantially upon phenobarbital administration. The anti-PB-1 antibodies were used to screen a cDNA library, and two distinct cDNA clones, pTF-1 and pTF-2, were isolated. These clones contain inserts of 1227 and 410 base pairs, respectively, and show 80% nucleic acid sequence homology in their region of overlap. The DNA sequences of these clones show 54% sequence homology to the corresponding portions of the mRNA encoding P-450 PB-4, a major phenobarbital-inducible form of rat liver P-450, and can be optimally aligned with the PB-4 sequence without introducing insertions or deletions. The level of hepatic mRNA which hybridizes to clone pTF-2 increases approximately 2-4-fold after phenobarbital treatment, whereas mRNA which hybridizes to pTF-1 does not change in concentration after this treatment. mRNA, which hybridizes to pTF-1, is, however, 4-fold more abundant in livers of female rats than in livers of male rats.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 106318, year: 1986, vol: 25, page: 7975, stat: Journal Article,

Secretion of endogenous and exogenous proteins from polarized MDCK cell monolayers
Gottlieb TA; Beaudry G; Rizzolo L; Colman A; Rindler M; Adesnik M; Sabatini DD
1986 Apr;83(7):2100-2104, Proceedings of the National Academy of Sciences of the United States of America
Confluent monolayers of MDCK (Madin-Darby canine kidney) cells provide a widely used system to study the biogenesis of epithelial cell polarity. We now report that these cells are also capable of the vectorial constitutive secretion of a major endogenous product, a glycoprotein of 81 kDa, which is released into the medium from the apical surface within 30 min of its synthesis. This release represents a bona fide exocytotic secretory process and is not the result of proteolytic cleavage of a plasma membrane-associated precursor since, in cells treated with chloroquine, a protein indistinguishable from the mature secretory product accumulated intracellularly. In contrast to the vectorial secretion of the endogenous product, a variety of exogenous exocrine and endocrine proteins synthesized in MDCK cells transfected with the corresponding genes were secreted from both the apical and basolateral surfaces. These included proteins such as rat growth hormone, chicken oviduct lysozyme, bovine gastric prochymosin, and rat salivary gland alpha 2u-globulin, which in their cells of origin are secreted via a regulated pathway, as well as the liver form of the alpha 2u-globulin and the immunoglobulin kappa chain, which are normally released constitutively. These results demonstrate the existence of secretory pathways that lead to both surfaces of MDCK cells and are accessible to the foreign secretory products. They are consistent with the operation of a sorting mechanism in which the polarized secretion of the endogenous product is effected through the recognition of signals that prevent its random distribution within the fluid phase in the cellular endomembrane system
— id: 35728, year: 1986, vol: 83, page: 2100, stat: Journal Article,

Sorting and endocytosis of viral glycoproteins in transfected polarized epithelial cells
Gottlieb TA; Gonzalez A; Rizzolo L; Rindler MJ; Adesnik M; Sabatini DD
1986 Apr;102(4):1242-1255, Journal of cell biology
Previous studies (Rindler, M. J., I. E., Ivanov, H. Plesken, and D. D. Sabatini, 1985, J. Cell Biol., 100: 136-151; Rindler, M. J., I. E. Ivanov, H. Plesken, E. J. Rodriguez-Boulan, and D. D. Sabatini, 1984, J. Cell Biol., 98: 1304-1319) have demonstrated that in polarized Madin-Darby canine kidney cells infected with vesicular stomatitis virus (VSV) or influenza virus the viral envelope glycoproteins G and HA are segregated to the basolateral and apical plasma membrane domains, respectively, where budding of the corresponding viruses takes place. Furthermore, it has been shown that this segregation of the glycoproteins reflects the polarized delivery of the newly synthesized polypeptides to each surface domain. In transfection experiments using eukaryotic expression plasmids that contain cDNAs encoding the viral glycoproteins, it is now shown that even in the absence of other viral components, both proteins are effectively segregated to the appropriate cell surface domain. In transfected cells, the HA glycoprotein was almost exclusively localized in the apical cell surface, whereas the G protein, although preferentially localized in the basolateral domains, was also present in lower amounts, in the apical surfaces of many cells. Using transfected and infected cells, it was demonstrated that, after reaching the cell surface, the G protein, but not the HA protein, undergoes interiorization by endocytosis. Thus, in the presence of chloroquine, a drug that blocks return of interiorized plasma membrane proteins to the cell surface, the G protein was quantitatively trapped in endosome- or lysosome-like vesicles. The sequestration of G was a rapid process that was completed in many cells by 1-2 h after chloroquine treatment. The fact that in transfected cells the surface content of G protein was not noticeably reduced during a 5-h incubation with cycloheximide, a protein synthesis inhibitor that did not prevent the effect of chloroquine, implies that normally, G protein molecules are not only interiorized but are also recycled to the cell surface
— id: 35729, year: 1986, vol: 102, page: 1242, stat: Journal Article,

Small basic proteins of myelin from central and peripheral nervous systems are encoded by the same gene
Mentaberry A; Adesnik M; Atchison M; Norgard EM; Alvarez F; Sabatini DD; Colman DR
1986 Feb;83(4):1111-1114, Proceedings of the National Academy of Sciences of the United States of America
Peripheral nervous system (PNS) and central nervous system (CNS) rodent myelins, which are produced by different cell types, share common morphological and functional characteristics although their major integral membrane proteins are completely different. Both types of myelin however, contain sets of four myelin basic proteins (MBPs), which share similar immunochemical and electrophoretic properties. We have isolated and characterized cDNA clones corresponding to the rat mRNAs encoding the small MBPs (SMBPs) found in both CNS and PNS myelin. Sequence analysis of these clones indicate that SMBPs in both divisions of the nervous system are encoded by the same nucleotide sequences, which suggests that they are the products of the same gene expressed in both oligodendrocyte and Schwann cells. In dot-blot hybridization experiments with the CNS SMBP cDNA as a probe, it was shown that there is a 20-fold higher level of MBP mRNA in a CNS myelin fraction than in total brainstem mRNA. It also was found that in optic and sciatic nerves, which contain oligodendrocytes and Schwann cells respectively, there are higher levels (4-fold and 2-fold, respectively) of MBP mRNA than in brainstem. Blot-hybridization experiments showed that a probe derived from the coding region of the rat SMBP cDNA hybridizes to an homologous mRNA (approximately equal to 2.6 kilobases) present in human optic nerve, which is not detectable with a probe derived from the 3' untranslated region. This conservation of coding-region sequences is in accord with the highly homologous amino acid sequences reported for the MBPs in the two species
— id: 55810, year: 1986, vol: 83, page: 1111, 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,

ISOLATION AND CHARACTERIZATION OF CDNA CLONES FOR RIBOPHORIN-I - COMPLETE CODING SEQUENCE AND INVITRO SYNTHESIS AND INSERTION OF THE ENCODING PRODUCT INTO ER MEMBRANES
ORT, V; PRAKASH, K; COLMAN, DR; ROSENFELD, MG; ADESNIK, M; SABATINI, DD; KREIBICH, G
1986 NOV ;103(5):A65-A65, Journal of cell biology
— id: 41317, year: 1986, vol: 103, page: A65, stat: Journal Article,

CHROMATIN STRUCTURE OF PHENOBARBITAL-INDUCIBLE RAT CYTOCHROME- P-450 GENES
Thyagarajan, S; Adesnik, M; Bustin, M
1986 May;45(6):1855-1855, Federation Proceedings (Federation of American Societies for Experimental Biology)
— id: 31062, year: 1986, vol: 45, page: 1855, stat: Journal Article,

NON-POLARIZED SECRETION OF FOREIGN SECRETORY PROTEINS IN TRANSFECTED MDCK CELLS
BEAUDRY G; GOTTLIEB T; ADESNIK M; RINDLER M; SABATINI D D
1985 ;101(5 PART 2):183A-183A, Journal of cell biology
— id: 104656, year: 1985, vol: 101, page: 183A, stat: Journal Article,

Intracellular Sorting And Distinct Recycling Patterns Of Viral Glycoproteins In Polarized Epithelial Cells
Rizzolo L J; Gonzalez A; Gottlieb T A; Finidori J; Ivanov I E; Rindler M J; Adesnik MB; Sabatini DD
Protein transport and secretion Cold Spring Harbor, N.Y. : Cold Spring Harbor Laboratory, 1985,
— id: 5223, year: 1985, vol: , page: 147, stat: Chapter,

Biosynthesis and intracellular sorting of growth hormone-viral envelope glycoprotein hybrids
Rizzolo LJ; Finidori J; Gonzalez A; Arpin M; Ivanov IE; Adesnik M; Sabatini DD
1985 Oct;101(4):1351-1362, Journal of cell biology
Various aspects of the biogenetic mechanisms that are involved in the insertion of nascent plasma membrane proteins into the endoplasmic reticulum (ER) membrane and their subsequent distribution through the cell have been investigated. For these studies chimeric genes that encode hybrid proteins containing carboxy-terminal portions of the influenza virus hemagglutinin (154 amino acids) or the vesicular stomatitis virus envelope glycoprotein (G) (60 amino acids) linked to the carboxy terminus of a nearly complete secretory polypeptide, growth hormone (GH), were used. In in vitro transcription-translation experiments, it was found that the insertion signal in the GH portion of the chimeras led to incorporation of the membrane protein segments into the ER membrane. Effectively, GH became part of the luminal segment of membrane proteins of which only very small segments, corresponding to the cytoplasmic portions of the G or HA proteins, remained exposed on the surface of the microsomes. When the chimeric genes were expressed in transfected cells, the products, as expected, failed to be secreted and remained cell-associated. These results support the assignment of a halt transfer role to segments of the membrane polypeptides that include their transmembrane portions. The hybrid polypeptide containing the carboxy-terminal portion of HA linked to GH accumulated in a juxtanuclear region of the cytoplasm within modified ER cisternae, closely apposed to the Golgi apparatus. The location and appearance of these cisternae suggested that they represent overdeveloped transitional ER elements and thus may correspond to a natural way station between the ER and the Golgi apparatus, in which further transfer of the artificial molecules is halted. The GH-G hybrid could only be detected in transfected cells treated with chloroquine, a drug that led to its accumulation in the membranes of endosome or lysosome-like cytoplasmic vesicles. Although the possibility that the chimeric protein entered such vesicles directly from the Golgi apparatus cannot be ruled out, it appears more likely that it was first transferred to the cell surface and was then internalized by endocytosis
— id: 55811, year: 1985, vol: 101, page: 1351, 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,

INTRACELLULAR-TRANSPORT OF GROWTH HORMONE-MEMBRANE PROTEIN HYBRIDS
RIZZOLO, LJ; ADESNIK, M; SABATINI, DD
1984 ;99(4):A100-A100, Journal of cell biology
— id: 50830, year: 1984, vol: 99, page: A100, stat: Journal Article,

A cytochrome P-450 multigene family. Characterization of a gene activated by phenobarbital administration
Atchison, M; Adesnik, M
1983 Sep 25;258(18):11285-11295, Journal of biological chemistry
The capacity of the liver microsomal mixed function oxidase system to metabolize a great variety of exogenous as well as endogenous compounds is thought to reflect the participation of multiple forms of the terminal oxidase, cytochrome P-450, which have different broad, but overlapping, substrate specificities. Several of these isozymes accumulate in the liver after exposure of animals to specific inducing agents. In order to approach the questions of the genetic basis for the existence of multiple cytochrome P-450 isozymes and the molecular mechanisms of the induction process, we have used a cloned cDNA for a major phenobarbital-induced form of cytochrome P-450 to identify and characterize thirteen distinct rat genomic clones containing segments of six different genes. Only three clones, representing overlapping segments of a single gene, hybridized to the cloned cDNA or to phenobarbital-induced mRNA under conditions of high stringency. In vitro transcription studies with isolated rat liver nuclei indicated that only this gene, but not the genes represented by the other genomic clones, appears to be markedly activated by phenobarbital treatment of rats. Although there are a small number of differences, DNA sequence analysis of the eight exons of the gene present in these genomic clones indicates that they encode residues 58 to 491 (the COOH terminus) of cytochrome P-450e, a major phenobarbital-induced isozyme. It appears that the other cloned genes may contain only a small region of very strong homology to the cytochrome P-450e gene, a region which includes the exon encoding a tridecapeptide which is also present in two dissimilar forms of rabbit liver cytochrome P-450, one constitutive and one induced by phenobarbital. Southern blotting analysis of rat liver DNA indicates that the rat genome may contain two additional genes which are very closely related to the cytochrome P-450e gene but which we have not yet isolated from the genomic library. One of these genes is likely to encode cytochrome P-450b, the major phenobarbital induced form of cytochrome P-450 whose mRNA is greater than 95% homologous to that encoding cytochrome P-450e
— id: 106321, year: 1983, vol: 258, page: 11285, stat: Journal Article,

A CYTOCHROME-P-450 MULTIGENE FAMILY - CHARACTERIZATION OF A GENE ACTIVATED BY PHENOBARBITAL ADMINISTRATION
ATCHISON, M; ADESNIK, M
1983 ;258(18):1285-1295, Journal of biological chemistry
— id: 40511, year: 1983, vol: 258, page: 1285, 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,

Cloned cytochrome P-450 cDNA. Nucleotide sequence and homology to multiple phenobarbital-induced mRNA species
Kumar, A; Raphael, C; Adesnik, M
1983 Sep 25;258(18):11280-11284, Journal of biological chemistry
Phenobarbital (PB) treatment of rats of various strains leads to the accumulation of liver mRNAs which encode two or three immunochemically related but electrophoretically separable cytochrome P-450 polypeptides. These mRNAs hybridize efficiently to a single cloned cDNA derived from mRNA of PB-treated rats and, therefore, must have extensive sequence homology. The nucleotide sequence of this cloned cDNA was determined and shown to encode the COOH-terminal 211 amino acids of one of the major cytochrome P-450 isozymes induced in rat liver by PB. Together with the recently reported sequence data of Fujii-Kuriyama et al. (Fujii-Kuriyama, Y., Mizukami, Y., Kawajiri, K., Sagawa, K., and Muramatsu, M. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2793-2797) for cloned rat cytochrome P-450 cDNA, our data suggest that differences between two closely related P-450 isozymes are restricted to the COOH-terminal half of the polypeptides, with highly divergent regions flanking a tridecapeptide which has been previously shown to be highly conserved in two dissimilar forms of rabbit liver cytochrome P-450. The significance of other interesting features of the cDNA sequence such as a second long (409 residues) open frame, an unusual poly(A) addition signal, and the absence of long hydrophobic stretches in the encoded polypeptide is discussed
— id: 106322, year: 1983, vol: 258, page: 11280, stat: Journal Article,

CLONED CYTOCHROME-P-450 CDNA - NUCLEOTIDE-SEQUENCE AND HOMOLOGY TO MULTIPLE PHENOBARBITAL-INDUCED MESSENGER-RNA SPECIES
KUMAR, A; RAPHAEL, C; ADESNIK, M
1983 ;258(18):1280-1284, Journal of biological chemistry
— id: 40510, year: 1983, vol: 258, page: 1280, 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,

The accumulation of distinct mRNAs for the immunochemically related cytochromes P-450c and P-450d in rat liver following 3-methylcholanthrene treatment
Morville, A L; Thomas, P; Levin, W; Reik, L; Ryan, D E; Raphael, C; Adesnik, M
1983 Mar 25;258(6):3901-3906, Journal of biological chemistry
Treatment of rats with 3-methylcholanthrene leads not only to a marked accumulation in the liver of translatable mRNA coding for a 56-kilodalton polypeptide representing cytochrome P-450c, the major 3-methylcholanthrene-induced cytochrome P-450 of rat liver, but also to the accumulation of comparable amounts of mRNA encoding a 52-kilodalton polypeptide which is immunoprecipitated with antibodies prepared against rat liver cytochrome P-450c. Further electrophoretic and immunochemical characterization of the latter translation product demonstrates that it corresponds to cytochrome P-450d, the major isosafrole-induced form of rat liver cytochrome P-450. The mRNAs for cytochromes P-450c and P-450d can be completely separated by electrophoresis in denaturing agarose gels and have chain lengths of approximately 4000 and 2000 nucleotides, respectively. These two mRNAs do not show detectable sequence homology to the mRNAs coding for the major phenobarbital-induced forms of cytochrome P-450 (P-450b and P-450e) since in Northern blotting experiments they fail to hybridize under conditions of low to moderate stringency to cloned probes for the latter mRNAs
— id: 106323, year: 1983, vol: 258, page: 3901, 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,

Genes For Cytochrome P-450 And Their Regulation
Atchison M; Ryvkin E; Lippman A; Raphael C; Adesnik M
From gene to protein : translation into biotechnology New York : Academic Press, 1982,
— id: 5221, year: 1982, vol: , page: 507, stat: Chapter,

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,

Mechanism of induction of cytochrome P-450 by phenobarbital
Adesnik, M; Bar-Nun, S; Maschio, F; Zunich, M; Lippman, A; Bard, E
1981 Oct 25;256(20):10340-10345, Journal of biological chemistry
Treatment of rats with phenobarbital (PB) leads to a substantial increase in the hepatic levels of translatable polysomal poly(A) + cytochrome P-450 mRNA. An enriched fraction of P-450 mRNA was obtained by agarose gel electrophoresis and used to prepare a cDNA probe by differential hybridization to total mRNA from control and PB-treated rats. The majority of the sequences within the probe hybridized to recombinant DNA plasmids which contained a bona fide P-450 cDNA insert identified by positive hybridization selection and in vitro translation. The cDNA probe was used to demonstrate that PB treatment leads to a 30-fold increase in polysomal P-450 mRNA, which is not due to more efficient utilization of previously existing mRNA but to the appearance of new messenger in the cytoplasm. The induction of cytoplasmic P-450 mRNA by PB was rapid, with increases detected within 3 h of PB injection and steady state levels reached in approximately 20 h. The data suggest that the increase in cytoplasmic P-450 protein levels observed after PB treatment may be totally accounted for by an enhanced rate of synthesis resulting from translation of higher cytoplasmic levels of its specific mRNA. The P-450 mRNA was almost exclusively segregated into the membrane-bound polysome compartment was expected for an mRNA coding for an integral membrane protein of the endoplasmic reticulum
— id: 106324, year: 1981, vol: 256, page: 10340, stat: Journal Article,

Segregation of specific classes of messenger RNA into free and membrane-bound polysomes
Adesnik, M; Maschio, F
1981 Feb;114(2):271-284, European journal of biochemistry
Analysis of mRNA populations from rat liver rough microsomes and free polysomes by homologous and heterologous cDNA . mRNA hybridization shows that the two mRNA populations are distinct, demonstrating that specific mRNA classes are efficiently segregated for translation in association with endoplasmic reticulum membranes. We estimate that approximately 90% of the mRNA in membrane-bound polysomes contains a diverse set of messengers with a minimum of 500--2000 different species although approximately 5--8 messengers may constitute 25--30% of the mRNA mass. The complexity of the mRNA population of free polysomes appears to be comparable to that estimated for total liver poly(A) + mRNA by other investigators, and is likely to be substantially greater than that of the bulk of bound mRNA. In addition, mRNA in free polysomes lacks the high abundance class characteristic of mRNA-bound polysomes. The substantial complexity of the bound mRNA population suggests that the segregation of polysomes in rough microsomes is not limited to a small class specialized in manufacturing secretory proteins, but extends to polysomes engaged in the synthesis of proteins for intracellular distribution. The segregation of specific messengers into the free and membrane-bound classes was abolished when polysome disassembly was induced by administration of ethionine. Thus, messenger RNA molecules themselves lacked the capacity for segregation, although they contain information for segregation which is expressed during translation. These findings are consistent with the presence of signal sequences in nascent polypeptides which determine the attachment of ribosomes to endoplasmic reticulum membranes
— id: 106325, year: 1981, vol: 114, page: 271, 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,

Erythrocyte membrane protein band 3: its biosynthesis and incorporation into membranes
Sabban E; Marchesi V; Adesnik M; Sabatini DD
1981 Dec;91(3 Pt 1):637-646, Journal of cell biology
Band 3, a transmembrane protein that provides the anion channel of the erythrocyte plasma membrane, crosses the membrane more than once and has a large amino terminal segment exposes on the cytoplasmic side of the membrane. The biosynthesis of band 3 and the process of its incorporation into membranes were studied in vivo in erythroid spleen cells of anemic mice and in vitro in protein synthesizing cell-free systems programmed with polysomes and messenger RNA (mRNA). In intact cells newly synthesized band 3 is rapidly incorporated into intracellular membranes where it is glycosylated and it is subsequently transferred to the plasma membrane where it becomes sensitive to digestion by exogenous chymotrypsin. The appearance of band 3 in the cell surface is not contingent upon its glycosylation because it proceeds efficiently in cells treated with tunicamycin. The site of synthesis of band 3 in bound polysomes was established directly by in vitro translation experiments with purified polysomes or with mRNA extracted from them. The band-3 polypeptide synthesized in an mRNA-dependent system had the same electrophoretic mobility as that synthesized in cells treated with tunicamycin. When microsomal membranes were present during translation, the in vitro synthesized band-3 polypeptide was cotranslationally glycosylated and inserted into the membranes. This was inferred from the facts that when synthesis was carried out in the presence of membranes the product had a lower electrophoretic mobility and showed partial resistance to protease digestion. Our observations indicate that the primary translation product of band-3 mRNA is not proteolytically processed either co- or posttranslationally. It is, therefore, proposed that the incorporation of band 3 into the endoplasmic reticulum (ER) membrane is initiated by a permanent insertion signal. To account for the cytoplasmic exposure of the amino terminus of the polypeptide we suggest that this signal is located within the interior of the polypeptide. a mechanism that explains the final transmembrane disposition of band 3 in the plasma membrane as resulting from the mode of its incorporation into the ER is presented
— id: 55812, year: 1981, vol: 91, page: 637, stat: Journal Article,

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,

Biosynthesis of erythrocyte membrane protein band 3 in DMSO-induced Friend erythroleukemia cells
Sabban EL; Sabatini DD; Marchesi VT; Adesnik M
1980 Aug;104(2):261-268, Journal of cellular physiology
The major integral membrane protein of red blood cells, the mouse equivalent of human band 3, was purified and used to raise a specfic antiserum. The murine protein resembles its human counterpart in several of its properties, including susceptibility to digestion by chymotrypsin added to intact cells and an ability to bind to concanavalin A. The synthesis of 35S-labeled band 3 was detected in Friend erythroleukemia cells treated with DMSO by immuneprecipitation followed by SDS gel electrophoresis and fluorography. Induction with DMSO led to a greater than tenfold increase in the synthesis of band 3 and maximal synthesis was reached 3 to 4 days after the beginning of induction
— id: 55813, year: 1980, vol: 104, page: 261, stat: Journal Article,

SYNTHESIS AND COTRANSLATIONAL PROCESSING OF ERYTHROCYTE- MEMBRANE PROTEIN BAND-3 BY MICROSOMAL-MEMBRANES
Sabban, E; Marchesi, V; Adesnik, M; Sabatini, DD
1980 ;87(2):A307-A307, Journal of cell biology
— id: 28089, year: 1980, vol: 87, page: A307, stat: Journal Article,

BIOSYNTHESIS OF MURINE ERYTHROCYTE MEMBRANE PROTEIN BAND 3
SABBAN E; SABATINI D; ADESNIK M; MARCHESI V
1979 ;83(2 PART 2):437A-437A, Journal of cell biology
— id: 104657, year: 1979, vol: 83, page: 437A, stat: Journal Article,

Induction of erythroid differentiation in Friend leukemia cells by bromodeoxyuridine
Adesnik, M; Snitkin, H
1978 Jun;95(3):307-317, Journal of cellular physiology
Treatment of Friend leukemia cells with BrdU, the thymidine analog which interferes with DMSO induced differentiation in these cells as well as the expression of differentiated character in many other cell systems, is capable of inducing erythroid differentiation. Globin mRNA, as assayed by hybridization to globin cDNA, increases 2.5- to 30-fold after appropriate treatment with BrdU. This effect was observed with several different subclones of three independent Friend tumor cell lines. After BrdU treatment, globin mRNA content may reach up to 10-20% of the levels in DMSO induced cultures. The induction of erythroid differentiation is also apparent when accumulated heme content or the appearance of benzidine positive cells is monitored. One Friend cell line (745) we examined was not induced by BrdU although it incorporated an amount of BrdU into its DNA comparable to that incorporated by the other cell lines. In addition, BrdU did interfere with DMSO induction in this cell line. These results suggest that two different mechanisms may be operative in regulating erythroid differentiation in Friend leukemia cells. While BrdU interferes with the mechanism activated by DMSO treatment, this analog could independently activate an alternative mechanism
— id: 106326, year: 1978, vol: 95, page: 307, stat: Journal Article,

Retention of mRNA on the endoplasmic reticulum membranes after in vivo disassembly of polysomes by an inhibitor of initiation
Adesnik M; Lande M; Martin T; Sabatini DD
1976 Oct;71(1):307-313, Journal of cell biology
Membrane-bound ribosomes and messenger RNA remained associated with the microsomal membranes of human fibroblasts after cultures were treated with Verrucarin A, an inhibitor of initiation which led to extensive run-off of ribosomes from polysomal structures. When a membrane fraction from Verrucarin-treated cells containing such inactive ribosomes and mRNA was suspended in a medium of high salt concentration, extensive release of ribosomal subunits occurred without the need for puromycin. The mRNA nevertheless remained associated with the membranes. These results add support to the conclusion that, in human fibroblasts, mRNA is bound directly to ER membranes, independently of the ribosomes and nascent polypeptide chains
— id: 55814, year: 1976, vol: 71, page: 307, stat: Journal Article,

IMPAIRED MESSENGER-RNA BIOGENESIS IN SENESCENT HUMAN FIBROBLASTS
Hadjiolov, A; Adesnik, M; Lande, M; Sabatini, D
1975 ;67(2):A151-A151, Journal of cell biology
— id: 28625, year: 1975, vol: 67, page: A151, stat: Journal Article,

Direct association of messenger RNA with microsomal membranes in human diploid fibroblasts
Lande MA; Adesnik M; Sumida M; Tashiro Y; Sabatini DD
1975 Jun;65(3):513-528, Journal of cell biology
Messenger RNA (mRNA) of membrane-bound polysomes in a membrane fraction of WI-38 cells remains associated with the microsomal membranes even after ribosomes and their nascent polypeptide chains are removed by using puromycin in a high salt buffer or by disassembling the ribosomes in a medium of high ionic strength lacking magnesium. mRNA either was specifically labeled in the presence of actinomycin D, or it was recognized by virtue of its affinity for oligo-dT. Poly A segments in bound mRNAs have an electrophoretic mobility in acrylamide gels which is characteristic of cytoplasmic mRNAs and corresponds to 150-200 adenyl residues. Extensive RNase treatment did not lead to release of the poly A segments of membrane-associated mRNA molecules either from an intact membrane fraction or from a membrane fraction previously stripped of ribosomes. On the other hand, RNase treatment led to the release and digestion of the nonpoly A segments of the mRNA molecules, indicating that the site of attachment of mRNA to the ER membranes is located near or at the 3' end of the molecule which contains the poly A. A direct association of mRNAs and endoplasmic reticulum membranes is considered in a modelto explain the assembly of bound polysomes and protein synthesis in a membrane-associated apparatus
— id: 55815, year: 1975, vol: 65, page: 513, 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,

SYNTHESIS OF 2 SECRETORY PROTEINS DURING AGING OF HUMAN DI PLOID CELLS
FRIEDMAN E; FALCOFF E; ADESNIK M; SABATINI D
1974 ;9(5):351-352, In vitro
— id: 104658, year: 1974, vol: 9, page: 351, stat: Journal Article,

DIRECT ASSOCIATION OF MESSENGER-RNA WITH MICROSOMAL-MEMBRANES IN HUMAN DIPLOID FIBROBLASTS
Lande, M; Sumida, M; Tashiro, Y; Adesnik, M; Sabatini, D
1974 ;63(2):A183-A183, Journal of cell biology
— id: 28424, year: 1974, vol: 63, page: A183, stat: Journal Article,

Further evidence on the nuclear origin and transfer to the cytoplasm of polyadenylic acid sequences in mammalian cell RNA
Jelinek, W; Adesnik, M; Salditt, M; Sheiness, D; Wall, R; Molloy, G; Philipson, L; Darnell, J E
1973 Apr 15;75(3):515-532, Journal of molecular biology
— id: 106327, year: 1973, vol: 75, page: 515, stat: Journal Article,

Biogenesis and characterization of histone messenger RNA in HeLa cells
Adesnik, M; Darnell, J E
1972 Jun 28;67(3):397-406, Journal of molecular biology
— id: 106329, year: 1972, vol: 67, page: 397, stat: Journal Article,

Evidence that all messenger RNA molecules (except histone messenger RNA) contain Poly (A) sequences and that the Poly(A) has a nuclear function
Adesnik, M; Salditt, M; Thomas, W; Darnell, J E
1972 Oct 28;71(1):21-30, Journal of molecular biology
— id: 106328, year: 1972, vol: 71, page: 21, stat: Journal Article,

Poly Acrylamide Gel Electrophoresis Of Viral RNA
Adesnik M
1971 ;67(3):125-177, Methods in virology
— id: 104714, year: 1971, vol: 67, page: 125, stat: Journal Article,

CORRECTION
ADESNIK, M
1971 ;58(2):641-&, Journal of molecular biology
— id: 104659, year: 1971, vol: 58, page: 641, stat: Journal Article,

Polyadenylic acid sequences: role in conversion of nuclear RNA into messenger RNA
Darnell, J E; Philipson, L; Wall, R; Adesnik, M
1971 Oct 29;174(8):507-510, Science
Polyadenylic acid [poly(A)] segments containing 150 to 250 nucleotides appear to be covalently linked to heterogeneous nuclear RNA (HnRNA) and messenger RNA (mRNA) in eucaryotic cells. The poly(A) is synthesized in the nucleus, and is probably linked initially to HnRNA that is ultimately transported as mRNA to the cytoplasm. Studies with inhibitors of RNA or poly(A) synthesis indicate that synthesis of poly(A) segments is independent of transcription. The poly(A) marker may prove useful to elucidate mRNA modification and transport in eucaryotic cells.s
— id: 106330, year: 1971, vol: 174, page: 507, stat: Journal Article,

Polysomal distribution of late bacteriophage T4 messenger RNA
Adesnik, M; Klagsbrun, M
1970 Nov 28;54(1):155-158, Journal of molecular biology
— id: 106331, year: 1970, vol: 54, page: 155, stat: Journal Article,

SYNTHESIS AND DEGRADATION OF LACTOSE OPERON MESSENGER RNA IN E-COLI
ADESNIK, M; LEVINTHA.C
1970 ;35(2):451-&, Cold Spring Harbor symposia on quantitative biology
— id: 104660, year: 1970, vol: 35, page: 451, stat: Journal Article,

RNA metabolism in T4-infected Escherichia coli
Adesnik, M; Levinthal, C
1970 Mar 14;48(2):187-208, Journal of molecular biology
— id: 106332, year: 1970, vol: 48, page: 187, stat: Journal Article,

Synthesis and maturation of ribosomal RNA in Escherichia coli
Adesnik, M; Levinthal, C
1969 Dec 14;46(2):281-303, Journal of molecular biology
— id: 106333, year: 1969, vol: 46, page: 281, stat: Journal Article,