Mapping actin surfaces required for functional interactions in vivo.
An in vivo strategy to identify amino acids of actin required for functional interactions with actin-binding proteins was developed. This approach is based on the assumption that an actin mutation that specifically impairs the interaction with an actin-binding protein will cause a phenotype similar to a null mutation in the gene that encodes the actin-binding protein. 21 actin mutations were analyzed in budding yeast, and specific regions of actin subdomain 1 were implicated in the interaction with fimbrin, an actin filament-bundling protein. Mutations in this actin subdomain were shown to be, like a null allele of the yeast fimbrin gene (SAC6), lethal in combination with null mutations in the ABP1 and SLA2 genes, and viable in combination with a null mutation in the SLA1 gene. Biochemical experiments with act1-120 actin (E99A, E100A) verified a defect in the fimbrin-actin interaction. Genetic interactions between mutant alleles of the yeast actin gene and null alleles of the SAC6, ABP1, SLA1, and SLA2 genes also demonstrated that the effects of the 21 actin mutations are diverse and allowed four out of seven pseudo-wild-type actin alleles to be distinguished from the wild-type gene for the first time, providing evidence for functional redundancy between different surfaces of actin.
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
D A
DA
Holtzman
K F
KF
Wertman
D G
DG
Drubin
D051339:Actin Depolymerizing Factors; D000199:Actins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics; D003598:Cytoskeletal Proteins; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005800:Genes, Fungal / Q000235:genetics; D005804:Genes, Lethal; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010641:Phenotype; D029701:Saccharomyces cerevisiae Proteins; D013489:Suppression, Genetic / Q000235:genetics*; D015003:Yeasts / Q000235:genetics*
C062612:ABP1 protein, S cerevisiae; D051339:Actin Depolymerizing Factors; D000199:Actins; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D005656:Fungal Proteins; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; C082265:SLA1 protein, S cerevisiae; C082266:SLA2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C026742:plastin