Moorthy Balaji T, Sharma Anupam, Boettner Douglas R, Wilson Thomas E, Lemmon Sandra K
Department of Molecular and Cellular Pharmacology, University of Miami, Miller School of Medicine, Miami, FL.
Department of Pathology, University of Michigan Medical School, Ann Arbor, MI.
G3 (Bethesda). 2019 Mar 7;9(3):867-877. doi: 10.1534/g3.118.200782.
Clathrin is a major coat protein involved in vesicle formation during endocytosis and transport in the endosomal/trans Golgi system. Clathrin is required for normal growth of yeast and in some genetic backgrounds deletion of the clathrin heavy chain gene () is lethal. Our lab defined a locus referred to as " uppressor of lathrin eficiency" (). In the presence of the allele ("v" - viable), yeast cells lacking clathrin heavy chain survive but grow slowly, are morphologically abnormal and have many membrane trafficking defects. In the presence of ("i"- inviable), causes lethality. As a strategy to identify , we used pooled linkage analysis and whole genome sequencing. Here, we report that () is the locus. is synthetic lethal with ; whereas a deletion of its paralog, , is not synthetic lethal with clathrin deficiency. Like Pal1, Pal2 has two NPF motifs that are potential binding sites for EH domain proteins such as the early endocytic factor Ede1, and Pal2 associates with Ede1 Also, GFP-tagged Pal2p localizes to cortical patches containing other immobile phase endocytic coat factors. Overall, our data show that is the locus and the Pal2 protein has characteristics of an early factor involved in clathrin-mediated endocytosis.
网格蛋白是一种主要的包被蛋白，参与内吞作用过程中的囊泡形成以及在内体/反式高尔基体系统中的运输。酵母的正常生长需要网格蛋白，在某些遗传背景下，网格蛋白重链基因（）的缺失是致死的。我们实验室定义了一个位点，称为“网格蛋白效率的uppressor”（）。在存在等位基因（“v” - 可存活）的情况下，缺乏网格蛋白重链的酵母细胞能够存活但生长缓慢，形态异常且有许多膜运输缺陷。在存在（“i” - 不可存活）的情况下，会导致致死性。作为鉴定的一种策略，我们使用了混合连锁分析和全基因组测序。在这里，我们报告（）是位点。与是合成致死的；而其旁系同源物的缺失与网格蛋白缺陷不是合成致死的。与Pal1一样，Pal2有两个NPF基序，它们是EH结构域蛋白（如早期内吞因子Ede1）的潜在结合位点，并且Pal2与Ede1相关联。此外，绿色荧光蛋白标记的Pal2p定位于含有其他固定相内吞包被因子的皮质斑块。总体而言，我们的数据表明是位点，并且Pal2蛋白具有参与网格蛋白介导的内吞作用的早期因子的特征。
