Suppressor analysis of fimbrin (Sac6p) overexpression in yeast.
Suppressor analysis of fimbrin (Sac6p) overexpression in yeast.
复制标题
酵母中菌丝蛋白 (Sac6p) 过度表达的抑制子分析。
DOI：
10.1093/genetics/151.4.1287
复制
发表时间：
1999
期刊：
影响因子：
3.3
通讯作者：
Adams,AE
中科院分区：
文献类型：
--
作者：
Sandrock,TM;Brower,SM;Toenjes,KA;Adams,AE
酵母菌丝蛋白 (Sac6p) 是一种肌动蛋白丝成束蛋白，过度表达时会致命。为了确定这种致死性的基础，我们寻找可以抑制这种致死性的突变。总共分离并分析了 1326 个抑制突变。由于预计绝大多数突变只会将 Sac6p 的表达降低至可耐受的水平，因此设计了快速筛选来消除这些突变。通过降低细胞中 Sac6p 的水平，总共发现了 1324 个突变。其余 2 个突变均位于肌动蛋白基因中，并导致新的变化 G48V (act1-20) 和 K50E (act1-21)。这些突变抑制了过度表达 SAC6 的 ACT1 细胞中细胞骨架组织和细胞形态的缺陷。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参与 Sac6p 结合的肌动蛋白区域，它们导致肌动蛋白与 Sac6p 的亲和力降低。这些结果表明，这两种突变很可能通过降低体内肌动蛋白对 Sac6p 的亲和力来抑制。这项研究表明应该可以使用这种类型的抑制分析来识别其他物理相互作用的蛋白质对，并表明有可能识别这些蛋白质彼此相互作用的位点。
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.