The proposed research is a combined biochemical and genetic approach to isolate and characterize the components of the secretory apparatus responsible for protein translocation and subcellular compartmentalization in the eukaryotic organism Saccharomyces cerevisiae. The initial aims of the project focus on the isolation and characterization of leader (signal) peptidase, the enzyme responsible for removal of amino-terminal leader sequences from precursors of secreted and membrane proteins. The first goal is to isolate the structural gene coding for yeast leader peptidase (YLP). This will be the first example of the isolation of a gene coding for a component of the eukaryotic secretory pathway. Several approaches which take advantage of possible structural and functional similarities between prokaryotic and eukaryotic leader peptidases are proposed to isolate the YLP gene. The cloned YLP gene will be used to construct strains which overproduce leader peptidase. These strains will be used to purify the enzyme to homogeneity and determine its subcellular location. Furthermore, the isolated gene will provide a unique opportunity to search for information within the structure of leader peptidase which is responsible for maintaining the enzyme in its proper subcellular compartment. Elucidation of this mechanism will be an important contribution to understanding protein localization in eukaryotes. The isolated YLP gene will also serve as the basis for an analysis of the genetics of translocation and protein sorting in yeast. Mutations in yeast leader peptidase will define the role of this enzyme in membrane assembly and secretion. The isolation of extragenic suppressors of lethal YLP mutants will define ancillary components of the secretory pathway which are responsible for moving newly made proteins into and across membranes. The analysis of these additional components will clarify our understanding of the complex mechanisms of self-assembly in eukaryotic organisms.
| Young, M R; Andreadis, J; Hu, L T et al. (1990) Expression of acid phosphatase-beta-galactosidase hybrid proteins prevents translocation by depleting a soluble factor. J Biol Chem 265:19824-32 |
| Wolfe, P B (1988) PHO5-LACZ hybrid proteins block translocation of native acid phosphatase in Saccharomyces cerevisiae. J Biol Chem 263:6908-15 |
