Processing and trafficking of proteins traversing the secretory and endocytic pathways in Saccharomyces cerevisiae
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An i&barbelow;nsulin-c&barbelow;ontaining f&barbelow;usion p&barbelow;rotein (ICFP) has been expressed in yeast to study the regulation of proprotein processing and secretion. In this system, insulin derived by processing of the ICFP proprotein is delivered to the vacuole, whereas mutants that impair processing enhance immunoreactive insulin secretion. Of the six yeast mutants obtained by genetic screen that cause enhanced insulin secretion, the weakest phenotype is lte1 (L&barbelow;ow-T&barbelow;emperature E&barbelow; ssential) while the strongest phenotype is vps35 ( V&barbelow;acuolar P&barbelow;rotein S&barbelow; orting-35). My dissertation further characterizes the roles of these two mutants in secretory and endosomal protein processing and trafficking.;Lte1p: is an essential protein for completion of mitosis at temperatures <15°C, and it functions to positively regulate GTPase that is essential to the cell cycle. I find that lte1 mutant cells exhibit defective processing of proalpha factor; this phenotype is rescued by expression of full-length Lte1p but not a stable truncation missing the GEF domain. Overexpression of GTPase Vps21p can rescues both the proalpha factor processing defect and the low-temperature growth defect of lte1 cells, while vps21 mutant creates a synthetic defect with lte1 mutant in proalpha factor processing and growth at 17°C. The data create new links between a mitotic GEF and a GTPase regulating secretory and endocytic pathways.;Vps35: is a protein component of the retromer complex. Human Vps35p diverges greatly from yeast Vps35p at the C-terminal end, but their N-termini show a high degree of similarity. I found that while a chimera comprised of the N-terminal region of hVps35 fused to the C-terminal region of yVps35 could improve vacuolar protein sorting in a vps35 knockout strain. But direct expressing hVps35 cause dominant-negative defect for vacuolar protein sorting, the mechanism appears to include substantial loss of other retromer components yVps26p and yVps29p. Introduction of the single R107W point mutation of hVps35 caused complete loss of the dominant-negative defect, and eliminated the deficiency of yVps26p and yVps29p. The data suggest the presence of an N-terminal interaction domain in both yVps35 and hVps35 that is involved in assembly of the yVps26p/yVps29p subcomplex.
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