Belmont L D, Patterson G M, Drubin D G
Department of Molecular and Cell Biology, University of California, Berkeley CA 94720-3202, USA.
J Cell Sci. 1999 May;112 ( Pt 9):1325-36. doi: 10.1242/jcs.112.9.1325.
We have generated 9 site-specific mutations in Saccharomyces cerevisiae actin. These mutants display a variety of phenotypes when expressed in vivo, including slow actin filament turnover, slow fluid-phase endocytosis, and defects in actin organization. Actin mutation D157E confers resistance to the actin-sequestering drug, latrunculin A. Latrunculin A inhibits nucleotide exchange on wild-type yeast actin but not on D157E actin, suggesting that this residue is part of the latrunculin A binding site. We have refined our earlier map of the phalloidin binding site on actin, demonstrating a requirement for residue G158 in addition to D179 and R177. The nine new actin mutants as well as a large collection of existing actin mutants were also used to identify the putative binding site of another actin binding drug, tolytoxin, on actin. The actin alleles that result in decreased sensitivity to this drug cluster at a site near the nucleotide-binding pocket. Actin purified from one of these mutants has a reduced affinity for tolytoxin. In addition, tolytoxin causes a 2.4-fold increase in the t1/2 of ATP exchange, further suggesting that this drug binds near the nucleotide-binding pocket of actin. We note that the binding sites for latrunculin A, phalloidin, and tolytoxin all map close to the actin nucleotide binding pocket.
我们在酿酒酵母肌动蛋白中产生了9个位点特异性突变。这些突变体在体内表达时表现出多种表型，包括肌动蛋白丝周转缓慢、液相内吞作用缓慢以及肌动蛋白组织缺陷。肌动蛋白突变体D157E对肌动蛋白隔离药物拉特罗毒素A具有抗性。拉特罗毒素A抑制野生型酵母肌动蛋白上的核苷酸交换，但不抑制D157E肌动蛋白上的核苷酸交换，这表明该残基是拉特罗毒素A结合位点的一部分。我们完善了之前关于肌动蛋白上鬼笔环肽结合位点的图谱，证明除了D179和R177外，还需要残基G158。这9个新的肌动蛋白突变体以及大量现有的肌动蛋白突变体也被用于确定另一种肌动蛋白结合药物甲苯毒素在肌动蛋白上的假定结合位点。对该药物敏感性降低的肌动蛋白等位基因聚集在核苷酸结合口袋附近的一个位点。从其中一个突变体中纯化的肌动蛋白与甲苯毒素的亲和力降低。此外，甲苯毒素使ATP交换的t1/2增加2.4倍，进一步表明该药物结合在肌动蛋白的核苷酸结合口袋附近。我们注意到，拉特罗毒素A、鬼笔环肽和甲苯毒素的结合位点都定位在靠近肌动蛋白核苷酸结合口袋的位置。
