Kamińska Joanna, Gajewska Beata, Hopper Anita K, Zoładek Teresa
Department of Genetics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.
Mol Cell Biol. 2002 Oct;22(20):6946-8. doi: 10.1128/MCB.22.20.6946-6958.2002.
Rsp5p is an ubiquitin-protein ligase of Saccharomyces cerevisiae that has been implicated in numerous processes including transcription, mitochondrial inheritance, and endocytosis. Rsp5p functions at multiple steps of endocytosis, including ubiquitination of substrates and other undefined steps. We propose that one of the roles of Rsp5p in endocytosis involves maintenance and remodeling of the actin cytoskeleton. We report the following. (i) There are genetic interactions between rsp5 and several mutant genes encoding actin cytoskeletal proteins. rsp5 arp2, rsp5 end3, and rsp5 sla2 double mutants all show synthetic growth defects. Overexpressed wild-type RSP5 or mutant rsp5 genes with lesions of some WW domains suppress growth defects of arp2 and end3 cells. The defects in endocytosis, actin cytoskeleton, and morphology of arp2 are also suppressed. (ii) Rsp5p and Sla2p colocalize in abnormal F-actin-containing clumps in arp2 and pan1 mutants. Immunoprecipitation experiments confirmed that Rsp5p and Act1p colocalize in pan1 mutants. (iii) Rsp5p and Sla2p coimmunoprecipitate and partially colocalize to punctate structures in wild-type cells. These studies provide the first evidence for an interaction of an actin cytoskeleton protein with Rsp5p. (iv) rsp5-w1 mutants are resistant to latrunculin A, a drug that sequesters actin monomers and depolymerizes actin filaments, consistent with the fact that Rsp5p is involved in actin cytoskeleton dynamics.
Rsp5p是酿酒酵母的一种泛素蛋白连接酶，它参与了包括转录、线粒体遗传和内吞作用在内的众多过程。Rsp5p在内吞作用的多个步骤中发挥作用，包括底物的泛素化和其他未明确的步骤。我们提出，Rsp5p在内吞作用中的一个作用涉及肌动蛋白细胞骨架的维持和重塑。我们报告如下：（i）rsp5与几个编码肌动蛋白细胞骨架蛋白的突变基因之间存在遗传相互作用。rsp5 arp2、rsp5 end3和rsp5 sla2双突变体均表现出合成生长缺陷。过表达的野生型RSP5或具有一些WW结构域损伤的突变型rsp5基因可抑制arp2和end3细胞的生长缺陷。arp2的内吞作用、肌动蛋白细胞骨架和形态缺陷也得到抑制。（ii）在arp2和pan1突变体中，Rsp5p和Sla2p共定位于异常的含F-肌动蛋白的团块中。免疫沉淀实验证实，在pan1突变体中Rsp5p和Act1p共定位。（iii）在野生型细胞中，Rsp5p和Sla2p通过免疫共沉淀，部分共定位于点状结构。这些研究为肌动蛋白细胞骨架蛋白与Rsp5p之间的相互作用提供了首个证据。（iv）rsp5-w1突变体对Latrunculin A具有抗性，Latrunculin A是一种螯合肌动蛋白单体并使肌动蛋白丝解聚的药物，这与Rsp5p参与肌动蛋白细胞骨架动力学这一事实相符。
