PROTEIN TRANSLOCATION IN YEAST

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Our primary objective is understanding the mechanism by which certain classes of proteins are selectively targeted to and translocated across the endoplasmic reticulum membrane in the yeast Saccharomyces cerevisiae. Yeast components of the cellular machinery that catalyzes this process will be biochemically identified, characterized and purified. In subsequent genetic analyses the cloned genes will be perturbed and the effects studied in vivo and in vitro. Specifically: i) A yeast in vitro protein translocation assay will be used to purify soluble, as well as membrane-bound translocation factors. Further in vitro assays will be designed to elucidate their function. The genes encoding such factors will be cloned using molecular probes generated to the purified material. ii) Additionally, we will purify and clone the yeast analogs of those components that are already well defined in the mammalian protein translocation system, namely SRP and SRP receptor, which are expected to be present also in yeast. iii) Genes identified in i) or ii) will be disrupted and the phenotype of the resulting null alleles will be characterized. Once a secretion-impaired phenotype has been identified, it will be used to isolate conditional alleles. iv) Interacting components will be identified by isolation of extragenic suppressors of either ts or cs or of in vitro engineered mutations. v) The 'reverse genetic' approaches outlined above will be complemented by a biochemical characterization of mutants isolated by classical genetic means by screening for alterations in the yeast translocation machinery. Ultimately, the power of a combined biochemical and genetic approach should lead to a complete molecular description of the protein translocation machinery and its essential and modulatory constituents, and allow us to gain an understanding of the mechanism of protein movement across membranes. The proposed research is clearly of a most basic nature and there is no doubt that it will be of profound significance for an understanding of physiology and pathology at the cellular and molecular level.