MOLECULAR ANALYSIS OF THE YEAST ACTIN CYTOSKELETON

The actin cytoskeleton has been implicated in a large variety of motile
functions, as well as in the structural and mechanical properties of the
cytoplasmic matrix. Except in muscle cells, however, very little is known
about its composition, roles, or mechanism of action. The proposed research
is designed to determine at the molecular level how the actin cytoskeleton
functions in the yeast Saccharomyces cerevisiae. Yeast can be readily
manipulated genetically, and should allow rapid and detailed analysis of
these complex processes. Because actin is highly conserved from yeast to
man, what is learned from these studies is likely to apply to higher cells,
where defective actin cytoskeleton function is implicated in pathological
conditions such as muscular dystrophy and neoplastic transformation. The
following specific questions will be addressed: 1. How does SAC6p, an actin-binding protein, function in the cytoskeleton?
Conditional-lethal mutations in the SAC6 gene will be produced by in vitro
mutagenesis. Biochemical and physiological analyses of a full spectrum of
mutants will then allow activities observed in vitro to be correlated with
functions seen in vivo. 2. What residues or domains are important for function of SAC6p and its
higher-cell homologs? Fine-structure mapping of mutations will be used to
identify regions of the gene associated with various C6p activities.
Specific hypotheses concerning sequences such as the putative actin-binding
domain (a highly conserved 25 amino acid sequence found in a number of
actin-binding proteins) will be tested by site-specific mutagenesis.
Information from these studies will be used in an analysis of human plastin
and chicken fimbrin, which are 40% identical to SAC6p. 3. What other proteins are involved in the function of the actin
cytoskeleton? The diversity of actin cytoskeletal function must be mediated
by non-actin proteins that regulate actin assembly, establish interactions
between the cytoskeleton and other cell structures, and generate the force
for intracellular motility. A variety of genetic methods will be used to
identify additional components of the actin cytoskeleton, and proteins that
interact with it.