Presently, there is only limited and indirect in vivo evidence supporting any of the proposed functions of the signal recognition particle (SRP) deduced from in vitro studies. Specific objectives of this project are to confirm an in vivo role in secretion for SRP, to examine in vivo translational arrest mediated by SRP, and to initiate studies of regulation of 7S RNA synthesis. These studies will be done with the genetically tractable yeast, Yarrowia lipolytica. It has two SCR genes coding for functional 7S RNAs, and it secretes high levels of a protease (AEP). Mutated genes coding for SRP components will be introduced into Y. lipolytica, and the effects on synthesis and translocation of secretory proteins determined by pulse-chase immunoprecipitation experiments using AEP-related polypeptides as reporter molecules. Three interrelated lines of research will be conducted. First, scr1-1, a mutation of SCR1 which results in translational arrest of AEP synthesis, will be further characterized. Second, SRP will be purified, sizes of the protein subunits determined, partial amino acid sequences of the Y. lipolytica homologues of Srp14p and Srp54p obtained, and the SRP14 and SRP54 genes cloned and sequenced. Conditional mutations will be isolated, and their effects on AEP synthesis and secretion determined. Third, SCR regulation will be studied by determining the role of a 5' consensus sequence on SCR1 and SCR2 RNA synthesis. The presumed function of the signal recognition particle, as deduced from in vitro studies, is to recognize the amino-terminal signal peptide leader sequence of nascent proteins destined for sequestration in the endoplasmic reticulum, and facilitate protein translocation to that organelle. SRP binds to the signal peptide of the nascent protein, transiently arresting further translation. The peptide-bound SRP then binds to a receptor on the cytoplasmic side of the endoplasmic reticulum membrane; this results in dissociation of the peptide from the SRP at the site of translocation across the ER membrane. The experiments to be carried out will extend our understanding of the function of SRP and provide information derived from in vivo studies. SRP and the secretory process in general is well-studied in yeasts, particularly S. cerevisiae and S. pombe, and it has become clear that there are significant differences between those two yeasts in this process. Yarrowia lipolytica, which is not closely related to either of the better-studied yeasts, has a very high secretory capacity and is currently being developed as a cellular factory for the commercial production of genetically-engineered proteins. A better understanding of the secretion process in this yeast is therefore significant not only because of the basic information that can be derived, but also because of the potential contribution to biotechnology through improved commercial yields of genetically- engineered proteins.
