CYTOSOL/VESICLE/VACUOLAR PROTEIN DEGRADATION PATHWAY

Project: Research project

Project Details

Description

Protein degradation is important for cell cycle control, signal
transduction and cell growth. Abnormal protein degradation has been
implicated in metabolic disorders, cancer development and muscular
dystrophy.

A novel pathway of protein degradation in the yeast vacuole has been
established in our lab. They key gluconeogenic enzyme, fructose-1.6-
biophosphatase (FBPase), is targeted from the cytosol to the yeast
lysosome (vacuole) for degradation when Saccharomyces cerevisiae are
replenished with glucose. Our long term goal is to understand the FBPase
degradation pathway. We have reconstituted this glucose-regulated
targeting pathway using semi-intact cells, purified FBPase, an ATP
regenerating system and cytosol. FBPase is targeted to the vacuole in the
reconstituted system. We have isolated 33 vid (vacuolar import and
degradation) mutants defective in the glucose-induced degradation of
FBPase. Mutant analysis led to the hypothesis that FBPase is targeted from
the cytosol to the intermediate vesicle and then the vacuole for
degradation. We have purified a novel FBPase-associated vesicle to near
homogeneity. We cloned the VID24 gene involved in vesicle targeting to the
vacuole. Vid24p is synthesized and localized to the vesicles.

Our specific aims are: (1) Reconstitution of FBPase import into the
vesicle using the vid24-1 mutant. We will examine whether the imported
FTPase is indeed targeted to the intermediate vesicles. We will divide
vid1-vid13 which accumulates FBPase in the cytosol into functional
subgroups. (2) Cloning of the VID genes. We plan to clone the VID genes
using the colony blotting procedure and study the expression and
localization of the Vid proteins. As an alternative approach, we will
clone the VID15 gene which is tightly linked to the URA3 gene by
chromosomal walking. (3) Purification of cytosolic proteins required for
FBPase import into the vesicles. We will use the vid1-vid13 mutants that
contain defective cytosolic factor(s) and add fractionated wild type
cytosol to identify the fractions that complement the mutant cytosol
defect. If we identify such protein, we will make mutants and prepare
cytosol from the mutants to test whether the cytosol is defective in
FBPase import in vitro. We will examine whether FBPase import into the
vesicles is regulated by ATP or GTP hydrolysis.
StatusFinished
Effective start/end date9/1/988/31/99

Funding

  • National Institute of General Medical Sciences

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