Adams A E, Cooper J A, Drubin D G
Department of Molecular and Cellular Biology, Life Sciences South, University of Arizona, Tuscon 85721.
Mol Biol Cell. 1993 May;4(5):459-68. doi: 10.1091/mbc.4.5.459.
To understand the role of the actin cytoskeleton in cell physiology, and how actin-binding proteins regulate the actin cytoskeleton in vivo, we and others previously identified actin-binding proteins in Saccharomyces cerevisiae and studied the effect of null mutations in the genes for these proteins. A null mutation of the actin gene (ACT1) is lethal, but null mutations in the tropomyosin (TPM1), fimbrin (SAC6), Abp1p (ABP1), and capping protein (CAP1 and CAP2) genes have relatively mild or no effects. We have now constructed double and triple mutants lacking 2 or 3 of these actin-binding proteins, and studied the effect of the combined mutations on cell growth, morphology, and organization of the actin cytoskeleton. Double mutants lacking fimbrin and either Abp1p or capping protein show negative synthetic effects on growth, in the most extreme case resulting in lethality. All other combinations of double mutations and the triple mutant lacking tropomyosin, Abp1p, and 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 specificity by overexpression of capping protein and Abp1p in strains lacking fimbrin. Thus, while overexpression of these proteins has deleterious effects on actin organization in wild-type strains, no synthetic phenotype was observed in the absence of fimbrin. We draw two important conclusions from these results. First, since mutations in pairs of actin-binding protein genes cause inviability, the actin cytoskeleton of yeast does not contain a high degree of redundancy. Second, the lack of structural and functional homology among these genetically redundant proteins (fimbrin and capping protein or Abp1p) indicates that they regulate the actin cytoskeleton by different mechanisms. Determination of the molecular basis for this surprising conclusion will provide unique insights into the essential mechanisms that regulate the actin cytoskeleton.
为了解肌动蛋白细胞骨架在细胞生理学中的作用，以及肌动蛋白结合蛋白如何在体内调节肌动蛋白细胞骨架，我们和其他研究人员之前在酿酒酵母中鉴定了肌动蛋白结合蛋白，并研究了这些蛋白基因的缺失突变效应。肌动蛋白基因（ACT1）的缺失突变是致死性的，但原肌球蛋白（TPM1）、丝束蛋白（SAC6）、Abp1p（ABP1）和封端蛋白（CAP1和CAP2）基因的缺失突变具有相对较轻的影响或无影响。我们现在构建了缺失其中2种或3种肌动蛋白结合蛋白的双突变体和三突变体，并研究了组合突变对细胞生长、形态以及肌动蛋白细胞骨架组织的影响。缺失丝束蛋白和Abp1p或封端蛋白的双突变体对生长表现出负向合成效应，在最极端的情况下会导致致死性。双突变体的所有其他组合以及缺失原肌球蛋白、Abp1p和封端蛋白的三突变体都是可存活的，并且它们的表型与单突变体相似或仅略为严重。因此，合成表型具有高度特异性。我们通过在缺失丝束蛋白的菌株中过表达封端蛋白和Abp1p证实了这种特异性。因此，虽然这些蛋白的过表达对野生型菌株中的肌动蛋白组织有有害影响，但在没有丝束蛋白的情况下未观察到合成表型。我们从这些结果中得出两个重要结论。第一，由于肌动蛋白结合蛋白基因对的突变导致不可存活，酵母的肌动蛋白细胞骨架不包含高度冗余。第二，这些遗传冗余蛋白（丝束蛋白和封端蛋白或Abp1p）之间缺乏结构和功能同源性，表明它们通过不同机制调节肌动蛋白细胞骨架。确定这一惊人结论的分子基础将为调节肌动蛋白细胞骨架的基本机制提供独特见解。
