Sandrock T M, Brower S M, Toenjes K A, Adams A E
Department of Molecular and Cellular Biology, Life Sciences South, University of Arizona, Tucson, Arizona 85721, USA.
Genetics. 1999 Apr;151(4):1287-97. doi: 10.1093/genetics/151.4.1287.
Yeast fimbrin (Sac6p) is an actin filament-bundling protein that is lethal when overexpressed. To identify the basis for this lethality, we sought mutations that can suppress it. A total of 1326 suppressor mutations were isolated and analyzed. As the vast majority of mutations were expected to simply decrease the expression of Sac6p to tolerable levels, a rapid screen was devised to eliminate these mutations. A total of 1324 mutations were found to suppress by reducing levels of Sac6p in the cell. The remaining 2 mutations were both found to be in the actin gene and to make the novel changes G48V (act1-20) and K50E (act1-21). These mutations suppress the defect in cytoskeletal organization and cell morphology seen in ACT1 cells that overexpress SAC6. These findings indicate that the lethal phenotype caused by Sac6p overexpression is mediated through interaction with actin. Moreover, the altered residues lie in the region of actin previously implicated in the binding of Sac6p, and they result in a reduced affinity of actin for Sac6p. These results indicate that the two mutations most likely suppress by reducing the affinity of actin for Sac6p in vivo. This study suggests it should be possible to use this type of suppressor analysis to identify other pairs of physically interacting proteins and suggests that it may be possible to identify sites where such proteins interact with each other.
酵母丝束蛋白（Sac6p）是一种肌动蛋白丝束集蛋白，过度表达时具有致死性。为了确定这种致死性的基础，我们寻找了能够抑制它的突变。共分离并分析了1326个抑制突变。由于绝大多数突变预计只是将Sac6p的表达降低到可耐受水平，因此设计了一种快速筛选方法来消除这些突变。共发现1324个突变通过降低细胞中Sac6p的水平来抑制。其余2个突变均位于肌动蛋白基因中，并产生了新的变化G48V（act1-20）和K50E（act1-21）。这些突变抑制了在过度表达SAC6的ACT1细胞中观察到的细胞骨架组织和细胞形态缺陷。这些发现表明，Sac6p过度表达引起的致死表型是通过与肌动蛋白相互作用介导的。此外，改变的残基位于先前与Sac6p结合有关的肌动蛋白区域，它们导致肌动蛋白对Sac6p的亲和力降低。这些结果表明，这两个突变最有可能通过降低体内肌动蛋白对Sac6p的亲和力来抑制。这项研究表明，使用这种类型的抑制分析来鉴定其他物理相互作用的蛋白对应该是可行的，并表明有可能鉴定出这些蛋白相互作用的位点。
