The goal of this project is to understand in detail how newly synthesized proteins which must be secreted from cells or inserted into biological membranes are identified and directed to transport sites in the endoplasmic reticulum (ER) or the cytoplasmic membrane (in prokaryotic cells). The project combines biochemical and genetic approaches to investigate the role of the signal recognition particle ribonucleoprotein (SRP), the signal recognition particle receptor (SRP receptor), and their bacterial homologs in this process. Previous studies have indicated that SRP and the SRP receptor are key components of the cellular machinery responsible for the """"""""initiation"""""""" phase of protein export. We expect that in addition to augmenting our understanding of protein sorting, this project will provide insight into the regulation of multi-step pathways, the function of proteins that have broad substrate specificies, and the functional capabilities of RNA. Previous work indicated that the """"""""signal sequences"""""""" which earmark proteins for entry into the ER bind to a site in the COOH-terminal domain of the 54kd subunit of mammalian SRP (SRP54). Recent experiments, however, suggest that sequences in the NH2-terminal domain, which contains a GTPase activity, might also play a role in signal sequence binding. We have introduced point mutations into a conserved motif in the NH2-terminal domain of SRP54 which does not correspond to sequences found in any other GTPase. The mutations do not affect GTP binding or hydrolysis, but rather reduce signal sequence binding activity. We are now examining the mutants in established biochemical assays to determine the mechanism by which sequences in the NH2-terminal domain of the protein participate in signal sequence binding. Because of methodological limitations to studying the SRP targeting pathway in mammalian cells, we have begun to study homologs of SRP and the SRP receptor in E. coli. The ease of performing genetic experiments in this organism is one reason that it is an attractive model system. We have used both site-directed mutagenesis and phenotypic selection to obtain mutants of homologs of SRP54 (""""""""Ffh""""""""), SRP RNA (""""""""4.5S RNA"""""""") and the SRP receptor (""""""""FtsY"""""""") which have distinctive phenotypes. We are using these mutants as tools to study the SRP pathway in detail. Genetic analysis of Ffh has supported our previous proposal that conserved methionine residues in its COOH-terminal domain play a role in signal sequence binding and has provided evidence that it functions as part of a complex with 4.5S RNA. Cell fractionation studies have also indicated that a majority of Ffh molecules are associated with membrane bound polysomes, consistent with the notion that like eukaryotic SRP54, Ffh has a targeting function in E. coli.
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