Characterization of mutants defective in the late stages of protein sorting to the yeast vacuole (Saccharomyces cerevisiae).

摘要

The vacuole of the yeast Saccharomyces cerevisiae plays an important role in cell physiology, participating in macromolecular degradation, pH- and ion-homeostasis, and functioning as a storage compartment of ions. Most of what is known today regarding the biosynthetic pathway of vacuolar delivery through an endosomal intermediate was achieved by localization studies of vacuolar hydrolases. One vacuolar hydrolase that has been consistently used as a marker protein is carboxypeptidase Y (CPY). Our laboratory has developed a screening assay that specifically selects for mutants that accumulate internally the p2CPY intermediate form of the protein. We attempted a complete morphological characterization of five endosome to v&barbelow;acuole mutants (env) by electron, fluorescent, and light microscopy. mut72 exhibited the presence of aberrant tubular structures, accumulation cf membrane compartments within the vacuole and accumulation of endocytic dyes in large intermediate structures. mut78 exhibited abnormal patterns of vacuolar staining, with dark staining aggregates and wild type kinetics of FM4-64 internalization. mut79 exhibited accumulation of membrane vesicles in the cytoplasm and accumulation of FM4-64 into early endosomal compartments. mut165 exhibited abnormal vacuolar staining patterns at both the permissive and non-permissive temperatures and delayed kinetics of FM4-64 internalization at the non-permissive temperature. mut186 exhibited abnormal vacuolar morphology at both the permissive and non-permissive temperature, and accumulation of FM4-64 into early endosomal structures at the non-permissive temperature. In order to clone the defective genes, genetic analyses were performed that confirmed at least 3 complementation groups and the recessive nature of the alleles. Next, high efficiency transformation methods were tested for gene cloning, by transforming wild type cells with a plasmid containing a yeast genomic library.