GENETIC &BIOCHEMICAL STUDIES OF YEAST CLATHRIN FUNCTION

Project: Research project

Description

The long term objective is to gain insight into the mechanisms by which
intracellula sorting and traffic of membranes and membrane-associated
components are controlled within cells. Many proteins are localized in
specific regions or organelles and movement to and from these regions
within cells requires the budding, transport and fusion of membrane
vesicles. The focus concerns the role that clathrin and clathrin coated
vesicles (CV) serve in these processes. This will be examined by a
biochemical, cellular and molecular genetic dissection of the clathrin
system in S. cerevisiae. The complete yeast clathrin heavy chain gene will be cloned, using segments
of the gene that have already been isolated, and the DNA sequence
determined. The phenotype of cells lacking clathrin heavy chains will be assessed by
deleting the gene to determine whether clathrin is an essential gene
product and/or whether clathrin is required for cell processes, such as
endocytosis, secretion, vacuole formation, mating and sporulation. The effect of overexpression of clathrin in cells will be studied. This is
of interest because clathrin is a structural protein which assembles with
other proteins in the initiation of specific membrane vesiculation, and the
coordinate synthesis and stoichiometry of these molecules in cells may be
required to maintain functional clathrin activity. Point mutations in the clathrin gene will be isolated to extend the
analysis of the clathrin system. It will be possible to define specific
amino acid sequednces in the clathrin heavy chain which are essential for
coated vesicle formation and inteaction with clathrin associated proteins.
Extragenic suppressors of these clahtrin heavy chain mutants will be
isolated, which will help to identify gene products that physically
interact with clathrin or proteins that may substitute for clathrin. Molelcular genetic studies will be complemented by further biochemical
characterization. A battery of monoclonal antibodies that bind to yeast
clathrin heavy chains will be studied to define structural and functional
domains on clathrin. These antibodies will also be used to identify
proteins that co-assemble with clathrin, to determine the ratio of clathrin
in CV (or cages) to unassembled forms in cells and to aid in the
characteriztion of mutant clathrin heavy chains.
StatusFinished
Effective start/end date8/1/868/31/90

Funding

  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health

Fingerprint

Clathrin
Molecular Biology
Yeasts
Clathrin Heavy Chains
Genes
Membranes
Proteins
Vesicular Transport Adaptor Proteins
Vacuoles
Point Mutation
Organelles
Saccharomyces cerevisiae
Dissection

ASJC

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)