Characterization of Novel Yeast Endosome to Vacuole Mutants by Electron and Fluorescent Microscopy

Abstract

The vacuole of yeast, Saccharomyces cerevisae, is equivalent to the mammalian lysosome. Several genes and gene products have been identified in the vesicular transport of soluble hydrolases to the vacuole via an endosomal compartment. This postendosomal stage of vacuolar trafficking delivering biosynthetic, endocytosed, and autophagic/Cvt cargo specifically to the vacuole remains one of the least defined stages of vesicular transport. We have previously developed an immunological screen for mutants that internally accumulate a pre-vacuolar intermediate of the vacuolar protease, Carboxypeptidase Y (CPY), in a PEP4-independent manner while exhibiting normal secretion phenotypes. We hypothesize that such mutants would be defective at endosome to vacuole step of delivery, hence env mutants. in order to morphologically characterize our novel set of mutants we performed electron, fluorescent and Nomarski microscopy analysis.

Preliminary studies revealed pleiotrophic phenotypes with respect to vacuole structure by Nomarski and fluorescent microscopy. FM-464 uptake and staining assays shows punctate staining throughout the cell in mut79 and mut72.