MAPPING ACTIN SURFACES REQUIRED FOR FUNCTIONAL INTERACTIONS IN-VIVO

An in vivo strategy to identify amino acids of actin required for func tional interactions with actin-binding proteins was developed. This ap proach is based on the assumption that an actin mutation that specific ally impairs the interaction with an actin-binding protein will cause a phenotype similar to a null mutation in the gene that encodes the ac tin-binding protein. 21 actin mutations were analyzed in budding yeast , and specific regions of actin subdomain 1 were implicated in the int eraction with fimbrin, an actin filament-bundling protein. Mutations i n this actin subdomain were shown to be, like a null allele of the yea st fimbrin gene (SAC6), lethal combination with null mutations in the ABP1 and SLA2 genes, and viable in combination with a null mutation in the SW gene. Biochemical experiments with act1-120 actin (E99A,E100A) verified a defect in the fimbrin-actin interaction. Genetic interacti ons between mutant alleles of the yeast actin gene and null alleles of the SAC6, ABP1, SLA1, and SLA2 genes also demonstrated that the effec ts 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.