The translocation of proteins across the membrane of the rough endoplasmic reticulum (ER) is the first step in secretion. Previous work in this and other laboratories has identified a number of cytosolic and membrane activities, proteins, and genes that participate in translocation. The proposed research will focus on biochemical studies that are designed to gain a more complete and unified understanding of the passage of a preprotein through the ER membrane, relying on the ability to reconstitute the process from purified proteins and lipids. The system will continue to be yeast, where molecular genetics provides in vivo verification of biochemical results. Initial efforts will concentrate on identifying proteins for which activities have been defined. These include: 1) the saturable preprotein binding to the membrane observed in vitro in the absence of ATP (preprotein receptor); and 2) the requirement for ATP hydrolysis within the membrane (translocation ATPase). The products of the SEC61-63 genes, as well as the translocation ATPase, the preprotein receptor and the yeast docking protein will be employed in the identification of the minimum number of components necessary to achieve translocation in a reconstituted system. To this end, new approaches will be used whose goal is to identify participants in the translocation reaction, and to simultaneously produce the chemical amounts needed for effective reconstitution and further biochemical and genetic analysis. By adding affinity-tags, recombinant preproteins and translocation-relevant membrane proteins, will be used as """"""""fishhooks"""""""" to recover functional complexes from the yeast in vitro system and from intact cells. These complexes will be tested for their ability to reconstitute translocation, or one of its subreactions, in liposomes prior to being dissected biochemically. The roles played by individual proteins will then be tested by depleting them from the extracts used to prepare active liposomes, and by manipulating expression of their genes in vivo. The same affinity-tagged preproteins will be translated in yeast lysates, in chemical amounts, to isolate the unknown cytosolic proteins that are involved in post- translational translocation in yeast. Current studies on proteins that mediate ribosome binding will be continued, with an emphasis on understanding the role of such proteins (and ribosome binding in general) in the translocation process. To achieve this, putative ribosome receptors will be depleted from extracts of membrane proteins that are used to reconstitute translocation in liposomes. This will serve to elucidate the role of a given protein in ribosome binding, but more importantly, in translocation. Ribosome binding will also be investigated in yeast so that its role in translocation can be analyzed by genetic means.
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