Geli M I, Wesp A, Riezman H
Biozentrum of the University of Basel, CH-4056 Basel, Switzerland.
EMBO J. 1998 Feb 2;17(3):635-47. doi: 10.1093/emboj/17.3.635.
The uptake step of receptor-mediated endocytosis in yeast is dependent on the calcium binding protein calmodulin (Cmd1p). In order to understand the role that Cmd1p plays, a search was carried out for possible targets among the genes required for the internalization process. Co-immunoprecipitation, two-hybrid and overlay assays demonstrated that Cmd1p interacts with Myo5p, a type I unconventional myosin. Analysis of the endocytic phenotype and the Cmd1p-Myo5p interaction in thermosensitive cmd1 mutants indicated that the Cmd1p-Myo5p interaction is required for endocytosis in vivo. However, the Cmd1p-Myo5p interaction requirement was partially overcome by deleting the calmodulin binding sites (IQ motifs) from Myo5p, suggesting that these motifs inhibit Myo5p function. Additionally, genetic and biochemical evidence obtained with a collection of cmd1 mutant alleles strongly suggests that Cmd1p plays an additional role in the internalization step of receptor-mediated endocytosis in yeast.
酵母中受体介导的内吞作用的摄取步骤依赖于钙结合蛋白钙调蛋白（Cmd1p）。为了了解Cmd1p所起的作用，对内化过程所需基因中可能的靶点进行了搜索。免疫共沉淀、双杂交和覆盖分析表明，Cmd1p与I型非常规肌球蛋白Myo5p相互作用。对温度敏感的cmd1突变体的内吞表型和Cmd1p-Myo5p相互作用的分析表明，Cmd1p-Myo5p相互作用在体内内吞作用中是必需的。然而，通过从Myo5p中删除钙调蛋白结合位点（IQ基序），部分克服了对Cmd1p-Myo5p相互作用的需求，这表明这些基序抑制了Myo5p的功能。此外，用一系列cmd1突变等位基因获得的遗传和生化证据强烈表明，Cmd1p在酵母受体介导的内吞作用的内化步骤中还起着额外的作用。
