UNEXPECTED COMBINATIONS OF NULL MUTATIONS IN GENES ENCODING THE ACTINCYTOSKELETON ARE LETHAL IN YEAST

To understand the role of the actin cytoskeleton in cell physiology, a nd how actin-binding proteins regulate the actin cytoskeleton in vivo, we and others previously identified actin-binding proteins in Sacchar omyces cerevisiae and studied the effect of null mutations in the gene s for these proteins. A null mutation of the actin gene (ACT1) is leth al, but null mutations in the tropomyosin (TPM1), fimbrin (SAC6), Abp1 p (ABP1), and capping protein (CAP1 and CAP2) genes have relatively mi ld or no effects. We have now constructed double and triple mutants la cking 2 or 3 of these actin-binding proteins, and studied the effect o f the combined mutations on cell growth, morphology, and organization of the actin cytoskeleton. Double mutants lacking fimbrin and either A bp1p or capping protein show negative synthetic effects on growth, in the most extreme case resulting in lethality. All other combinations o f double mutations and the triple mutant lacking tropomyosin, Abp1p, a nd capping protein, are viable and their phenotypes are similar to or only slightly more severe than those of the single mutants. Therefore, the synthetic phenotypes are highly specific. We confirmed this speci ficity by overexpression of capping protein and Abp1p in strains lacki ng fimbrin. Thus, while overexpression of these proteins has deleterio us effects on actin organization in wild-type strains, no synthetic ph enotype was observed in the absence of fimbrin. We draw two important conclusions from these results. First, since mutations in pairs of act in-binding protein genes cause inviability, the actin cytoskeleton of yeast does not contain a high degree of redundancy. Second, the lack o f structural and functional homology among these genetically redundant proteins (fimbrin and capping protein or Abp1p) indicates that they r egulate the actin cytoskeleton by different mechanisms. Determination of the molecular basis for this surprising conclusion will provide uni que insights into the essential mechanisms that regulate the actin cyt oskeleton.