Bénédetti H, Raths S, Crausaz F, Riezman H
Biozentrum of the University of Basel, Switzerland.
Mol Biol Cell. 1994 Sep;5(9):1023-37. doi: 10.1091/mbc.5.9.1023.
Two Saccharomyces cerevisiae mutants, end3 and end4, defective in the internalization step of endocytosis, have previously been isolated. The END3 gene was cloned by complementation of the temperature-sensitive growth defect caused by the end3 mutation and the END3 nucleotide sequence was determined. The END3 gene product is a 40-kDa protein that has a putative EF-hand Ca(2+)-binding site, a consensus sequence for the binding of phosphotidylinositol 4,5-bisphosphate (PIP2), and a C-terminal domain containing two homologous regions of 17-19 aa. The EF-hand consensus and the putative PIP2-binding sites are seemingly not required for End3 protein function. In contrast, different portions of the End3p N-terminal domain, and at least one of the two repeated regions in its C-terminus, are required for End3p activity. Disruption of the END3 gene yielded cells with the same phenotype as the original end3 mutant. An end3ts allele was obtained and this allowed us to demonstrate that End3p is specifically involved in the internalization step of endocytosis. In addition, End3p was shown to be required for proper organization of the actin cytoskeleton and for the correct distribution of chitin at the cell surface.
先前已分离出酿酒酵母的两个突变体end3和end4，它们在内吞作用的内化步骤中存在缺陷。通过对由end3突变引起的温度敏感生长缺陷进行互补克隆了END3基因，并确定了END3核苷酸序列。END3基因产物是一种40 kDa的蛋白质，具有一个假定的EF手型Ca(2+)结合位点、一个磷脂酰肌醇4,5-二磷酸（PIP2）结合的共有序列，以及一个包含两个17 - 19个氨基酸同源区域的C末端结构域。End3蛋白功能似乎不需要EF手型共有序列和假定的PIP2结合位点。相反，End3p N末端结构域的不同部分以及其C末端两个重复区域中的至少一个对于End3p活性是必需的。END3基因的破坏产生了与原始end3突变体具有相同表型的细胞。获得了一个end3ts等位基因，这使我们能够证明End3p特异性参与内吞作用的内化步骤。此外，已表明End3p对于肌动蛋白细胞骨架的正确组织以及几丁质在细胞表面的正确分布是必需的。
