Holtzman D A, Wertman K F, Drubin D G
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
J Cell Biol. 1994 Jul;126(2):423-32. doi: 10.1083/jcb.126.2.423.
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.
我们开发了一种体内策略，用于鉴定肌动蛋白与肌动蛋白结合蛋白功能相互作用所需的氨基酸。该方法基于这样一种假设：特异性损害与肌动蛋白结合蛋白相互作用的肌动蛋白突变将导致与编码该肌动蛋白结合蛋白的基因中的无效突变相似的表型。我们在芽殖酵母中分析了21种肌动蛋白突变，并发现肌动蛋白亚结构域1的特定区域与肌动蛋白丝束集蛋白丝切蛋白的相互作用有关。结果表明，该肌动蛋白亚结构域中的突变与酵母丝切蛋白基因（SAC6）的无效等位基因一样，与ABP1和SLA2基因的无效突变组合时是致死的，而与SLA1基因的无效突变组合时是存活的。对act1 - 120肌动蛋白（E99A，E100A）进行的生化实验证实了丝切蛋白 - 肌动蛋白相互作用存在缺陷。酵母肌动蛋白基因的突变等位基因与SAC6、ABP1、SLA1和SLA2基因的无效等位基因之间的遗传相互作用还表明，这21种肌动蛋白突变的影响是多样的，并且首次使七个假野生型肌动蛋白等位基因中的四个与野生型基因得以区分，为肌动蛋白不同表面之间的功能冗余提供了证据。
