Synthesis, Assembly and Sorting of Membrane Proteins Research Summary The endoplasmic reticulum (ER) plays a central role in the synthesis of a large number of proteins which are then distributed to different subcellular compartments. Polypeptides are inserted into ER membranes during the course of their elongation in membrane-bound ribosomes, cotranslationally modified and, when their synthesis is completed, they may form oligomeric complexes. Our research is concerned with the functional organization of ER membranes, the process of assembly of membrane proteins into oligomeric complexes, and the biogenetic relationship between the ER and other subcellular membrane systems. Of special interest are the components of the polypeptide translocation apparatus in the ER which are involved in signal sequence recognition, insertion, translocation and in the cotranslational processing of proteins synthesized in membrane bound polysomes. We have discovered two transmembrane glycoproteins, ribophorins I and II, which, together with membrane proteins that include 48 kD (OST48) and 12 kD (Dad1), form an oligomeric complex that functions in the cotranslational N-glycosylation of the growing nascent chains. Using biophysical approaches such as fluorescent recovery after photobleaching (FRAP), we have investigated mechanisms by which the membrane proteins forming the translocation apparatus are retained in the ER, while other membrane proteins exit from this organelle. We have found that microtubules that bind to the ER-specific membrane protein CLIMP-63 interfere with the lateral mobility of membrane bound polysomes in the lipid bilayer of ER membranes. (Supported by NSF) Related to our interest in the oligomerization of membrane proteins are studies on the assembly and the intracellular transport of four membrane proteins (uropakins Ia, Ib, II and III) that are expressed in the bladder epithelium (urothelium). These membrane proteins are made on membrane bound polysomes, and the co- and posttranslationally processed proteins accumulate in a post-Golgi compartment where they assemble into two dimensional crystals that have have the appearance of asymmetric unit membranes (AUMs). Eventually, these AUMs cover the whole surface of fusiform vesicles (FVs). These AUM storage organelles, that belong to the class of lysosome-related organelles, can fuse in a regulated fashion with the apical surface of urothelial cells. Our research is concerned with the elucidation of mechanisms that function in the delivery of these highly specialized membranes to the apical surface of the bladder epithelium, and in the regulation of endocytic processes that result in the degradation of AUMs. For these studies we use aside from ultrastructural, biochemical and molecular biology approaches also mouse strains that have specific genes deleted (Rab27b), or carry mutations that affect lysosome-related organelles and represent mouse models for Hermansky-Pudlack Syndrome. (Supported by NIH)