The targeting of proteins across or into the endoplasmic reticulum (ER) is the first step in secretion and in the assembly of many membranes. With the exception of SRP, docking protein (SRP receptor) and the signal peptidase, little is known about the components involved or the requirements of this process. This is due, in large part, to the difficulty of further dissecting and reconstituting such a complex process. Augmenting a biochemical approach with a genetic one would allow further, and potentially more rapid, progress to be made in this area. Yeast cells, whose secretory pathway closely resembles that of higher eukaryotes, have the obvious advantage that they can be easily manipulated genetically. Recently targeting to the ER has been accomplished in a cell-free system derived from yeast. The objective of the research described in this proposal is the exploitation of yeast genetics, in combination with our existing biochemical expertise, to identify and characterize the components that mediate targeting to, and the translocation across, the membrane of the ER. We propose to refine the homologous yeast cell-free system to enable the isolation of cytosolic and membrane proteins involved in translocation. Important cytosolic components will be identified in a lysate-dependent post-translational assay. Biochemical and immunological methods will be employed to analyze the role of rough ER-specific proteins in translocation. Verification of the participation of these proteins in the secretory process will be accomplished in vivo by gene disruption techniques that are feasible in yeast. In parallel, we will select for new secretory mutants, defective in targeting and translocation, by the expression of a crucial cytoplasmic enzyme as a signal sequence-bearing chimera. Mutants produced in this way will then be characterized biochemically in the in vitro system.
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