PROTEIN TRANSLOCATION IN YEAST

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The primary objective of the proposed research is to understand, mechanistically, how the correct proteins are targeted to and translocated across the endoplasmic reticulum membrane in the simple eukaryote Saccharomyces cerevisiae. We will test our current hypothesis that there are two parallel targeting pathways, one dependent on the signal recognition particle (SRP), the other one SRP-independent. Our aim is to identify all the gene products catalyzing the reactions in both pathways, and to understand their molecular function and importance in vivo. Specifically, i) we will identify and sequence the genes encoding all subunits of yeast SRP and SRP receptor; ii) we will use in vitro protein translocation assays or assays for partial reactions to analyze in detail their mechanism of action. We aim to identify novel components functioning in targeting and translocation and clone their genes; iii) we will use random mutagenesis to isolate conditional (cs, ts) mutations in SRP, SRP receptor and newly identified components. These mutations and site- directed mutations in predicted GTP binding sites, and in regions of predicted RNA-RNA, RNA-protein or protein-protein interactions will be used to analyze in vivo and in vitro the role of the gene products; iv) we will isolate additional genes involved in protein targeting and translocation using improved genetic selections. Such mutations will be analyzed as to their involvement in either the SRP-dependent or the SRP-independent pathway; v) we will use suppressor analysis of mutations in each pathway to identify interacting gene products. The proposed biochemical and genetic analyses aim at a precise molecular understanding of the protein targeting and translocation process. We wish to learn which aspects of the process are unique to yeast and which aspects can be generalized to other eukaryotic cells and bacteria. Ultimately, we hope that through the power of a combined genetic and biochemical approach, we will identify the cellular constituents of the protein translocation machinery that are essential for translocation, as well as those that are modulatory.