Lisa Belmont and David Drubin
Department of Molecular and Cellular Biology, University of California, Berkeley, CA 94720-3220 USA
Actin monomers bind ATP, and this nucleotide is hydrolized subsequent to assembly. Release of phosphate from actin filaments and exchange of nucleotide on actin monomers are thought to be key regulatory steps in vivo. In order to directly test the role of nucleotide hydrolysis and exchange in vivo, I have performed site directed mutagenesis on the nucleotide binding pocket of yeast actin. Of the mutants characterized so far, one, in which V159 is mutated to N (V159N) results in an increased number of actin cables when expressed as the sole actin source in yeast. This mutation also confers resistance to the actin destabilizing drug, latrunculin A (LatA). D157E appears to slightly increase actin assembly, and confers resistance to LatA. Yeast expressing G158A actin appear to have fewer actin cables and are more sensitive to LatA. These mutant actins are being purified from yeast so that the polymerization properties, nucleotide exchange, ATP hydrolysis rates, and phosphate release can be measured. If biochemical analysis demonstrates that these actins have altered nucleotide hydrolysis or exchange, it will provide an opportunity to directly observe the role of nucleotide hydrolysis and exchange in vivo.