Shiflett Shelly L, Ward Diane McVey, Huynh Dinh, Vaughn Michael B, Simmons Jennifer C, Kaplan Jerry
Department of Pathology, Division of Cell Biology and Immunology, University of Utah Health Science Center, Salt Lake City, Utah 84132-2501, USA.
J Biol Chem. 2004 Mar 19;279(12):10982-90. doi: 10.1074/jbc.M312669200. Epub 2003 Dec 29.
We identified VTA1 in a screen for mutations that result in altered vacuole morphology. Deletion of VTA1 resulted in delayed trafficking of the lipophilic dye FM4-64 to the vacuole and altered vacuolar morphology when cells were exposed to the dye 5-(and 6)-carboxy-2',7'-dichlorofluorescein diacetate (CDCFDA). Deletion of class E vacuolar protein sorting (VPS) genes, which encode proteins that affect multivesicular body formation, also showed altered vacuolar morphology upon exposure to high concentrations of CDCFDA. These results suggest a VPS defect for Deltavta1 cells. Deletion of VTA1 did not affect growth on raffinose and only mildly affected carboxypeptidase S sorting. Turnover of the surface protein Ste3p, the a-factor receptor, was affected in Deltavta1 cells with the protein accumulating on the vacuolar membrane. Likewise the alpha-factor receptor Ste2p accumulated on the vacuolar membrane in Deltavta1 cells. We demonstrated that many class E VPS deletion strains are hyper-resistant to the cell wall disruption agent calcofluor white. Deletion of VTA1 or VPS60, another putative class E gene, resulted in calcofluor white hypersensitivity. A Vta1p-green fluorescent protein fusion protein transiently associated with a Pep12p-positive compartment. This localization was altered by deletion of many of the class E VPS genes, indicating that Vta1p binds to endosomes in a manner dependent on the assembly of the endosomal sorting complexes required for transport. Membrane-associated Vta1p co-purified with Vps60p, suggesting that Vta1p is a class E Vps protein that interacts with Vps60p on a prevacuolar compartment.
我们在一个导致液泡形态改变的突变筛选中鉴定出了VTA1。VTA1的缺失导致亲脂性染料FM4-64向液泡的运输延迟，并且当细胞暴露于染料5-(和6)-羧基-2',7'-二氯荧光素二乙酸酯(CDCFDA)时，液泡形态发生改变。编码影响多泡体形成的蛋白质的E类液泡蛋白分选(VPS)基因的缺失，在暴露于高浓度的CDCFDA时也显示出液泡形态的改变。这些结果表明Δvta1细胞存在VPS缺陷。VTA1的缺失不影响在棉子糖上的生长，仅轻微影响羧肽酶S的分选。表面蛋白Ste3p（α-因子受体）的周转在Δvta1细胞中受到影响，该蛋白积累在液泡膜上。同样，α-因子受体Ste2p在Δvta1细胞中也积累在液泡膜上。我们证明许多E类VPS缺失菌株对细胞壁破坏剂荧光增白剂具有超抗性。VTA1或另一个假定的E类基因VPS60的缺失导致对荧光增白剂超敏。一个Vta1p-绿色荧光蛋白融合蛋白与一个Pep12p阳性区室短暂相关。这种定位因许多E类VPS基因的缺失而改变，表明Vta1p以依赖于运输所需的内体分选复合物组装的方式与内体结合。膜相关的Vta1p与Vps60p共同纯化，表明Vta1p是一种E类Vps蛋白，在液泡前区室与Vps60p相互作用。
