Creutz C E, Snyder S L, Schulz T A
Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908, USA.
Cell Mol Life Sci. 2004 May;61(10):1208-20. doi: 10.1007/s00018-004-4029-8.
In a survey of yeast genomic sequences encoding calcium- and phospholipid-binding C2 domains, three homologous genes were identified that encode proteins that each have three C2 domains and an apparent transmembrane domain near the N terminus. The name tricalbins is suggested for these proteins, corresponding to the open reading frames YOR086c (TCB1), YNL087w (TCB2), and YML072c (TCB3). An antiserum was raised against the C-terminal portion of tricalbin 2 and used on Western blots to demonstrate that the corresponding protein is expressed in yeast and appears as a high-molecular-weight band at 130 kDa with smaller fragments at 39 kDa and 46 kDa. A fusion protein consisting of full length tricalbin 2 fused to the green fluorescent protein was expressed in cells and found to traffic from the cell surface to intracellular vesicles near the vacuole. A two-hybrid interaction screen with the C-terminal portion of tricalbin 2 indicated that tricalbin 2 binds the C-terminal portions of tricalbins 1 and 3 suggesting that the tricalbins may form heterodimers in vivo. Tricalbin 2 also interacted with the activation domain of the pleiotropic drug resistance transcription factor Pdr1p. Combinatorial disruptions of the tricalbin genes revealed that tcb2 single mutants or tcb1, tcb3 double mutants have an altered vacuole morphology and are hypersensitive to cycloheximide. A screen for single-copy suppressors of the cycloheximide sensitivity of tricalbin mutants yielded RSP5, which encodes a C2-domain-containing, ubiquitin-conjugating ligase essential for receptor-mediated and fluid phase endocytosis. The results suggest that the tricalbins function as multimers in membrane-trafficking events and may provide insights into the roles of multi-C2-domain proteins, such as the synaptotagmins, in other organisms.
在一项对编码钙结合和磷脂结合C2结构域的酵母基因组序列的调查中，鉴定出了三个同源基因，它们编码的蛋白质各自具有三个C2结构域，并且在N端附近有一个明显的跨膜结构域。建议将这些蛋白质命名为三钙结合蛋白，对应于开放阅读框YOR086c（TCB1）、YNL087w（TCB2）和YML072c（TCB3）。制备了针对三钙结合蛋白2 C端部分的抗血清，并用于蛋白质免疫印迹，以证明相应的蛋白质在酵母中表达，呈现为130 kDa的高分子量条带，还有39 kDa和46 kDa的较小片段。由全长三钙结合蛋白2与绿色荧光蛋白融合而成的融合蛋白在细胞中表达，并发现其从细胞表面运输到液泡附近的细胞内小泡。用三钙结合蛋白2的C端部分进行的双杂交相互作用筛选表明，三钙结合蛋白2与三钙结合蛋白1和3的C端部分结合，这表明三钙结合蛋白可能在体内形成异二聚体。三钙结合蛋白2还与多药耐药转录因子Pdr1p的激活结构域相互作用。三钙结合蛋白基因的组合破坏显示，tcb2单突变体或tcb1、tcb3双突变体具有改变的液泡形态，并且对环己酰亚胺高度敏感。对三钙结合蛋白突变体环己酰亚胺敏感性的单拷贝抑制子筛选得到了RSP5，它编码一种含C2结构域的泛素结合连接酶，这对于受体介导的和液相内吞作用至关重要。结果表明，三钙结合蛋白在膜运输事件中作为多聚体发挥作用，并可能为多C2结构域蛋白（如突触结合蛋白）在其他生物体中的作用提供见解。
