Protein export in yeast is a complex process requiring many gene products and a series of membrane-bounded organelles. This proposal addresses the function of 10 secretory (SEC) gene products required for the final stages of secretion: vesicle maturation, vesicle transport and vesicle fusion with the plasma membrane. Three specific questions will be addressed: 1) Which gene products interact physically with the late sec gene products? The interaction of two gene products can be detected genetically by pseudoreversion analysis. A deleterious mutation affecting one protein can be compensated by a corresponding mutation in an interacting protein. We will isolate such extragenic suppressors of the late sec genes and construct a map of gene product interactions. 2) In what order do the genes function and how do they depend on one another? Cold-sensitive sec mutants will be isolated and combined pairwise with the temperature-sensitive sec mutants. The transport of the periplasmic protein invertase will be followed in the double mutants as the temperature is shifted from one restrictive condition to the other. These reciprocal shifts will rigorously define the order and dependency relationships among the sec gene product functions. 3) Where are these gene products localized? The late sec genes will be cloned and fused to the lacZ gene. Antibody will be raised to the hybrid protein purified from bacterial extracts. A combination of indirect immunofluorescence and subcellular fractionation will be used to localize the sec gene products.
Showing the most recent 10 out of 66 publications
