Characterization of the yeast tricalbins: membrane-bound multi-C2-domain proteins that form complexes involved in membrane trafficking
Characterization of the yeast tricalbins: membrane-bound multi-C2-domain proteins that form complexes involved in membrane trafficking
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DOI：
10.1007/s00018-004-4029-8
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发表时间：
2004-05-01
影响因子：
8
通讯作者：
Schulz, TA
中科院分区：
文献类型：
--
作者：
Creutz, CE;Snyder, SL;Schulz, TA
在一项酵母基因组序列的调查中，发现了三个同源基因编码蛋白质，每个基因都有三个C2结构域和一个明显的跨膜结构域靠近N端。建议将这些蛋白命名为tricalbins，对应于开放阅读框YOR086c （TCB1）、YNL087w （TCB2）和YML072c （TCB3）。针对tricalbin 2的c端部分提出了一种抗血清，并在Western blots上证明了相应的蛋白在酵母中表达，并且在130 kDa处表现为高分子量带，在39 kDa和46 kDa处表现为较小的片段。由全长三卡尔宾2与绿色荧光蛋白融合组成的融合蛋白在细胞中表达，并发现从细胞表面运输到液泡附近的细胞内囊泡。与tricalbin 2 c端部分的双杂交相互作用筛选表明，tricalbin 2结合了tricalbin 1和tricalbin 3的c端部分，表明三白蛋白可能在体内形成异源二聚体。Tricalbin 2还与多效耐药转录因子Pdr1p的激活域相互作用。tralbin基因的组合破坏表明，tcb2单突变体或tcb1、tcb3双突变体液泡形态发生改变，对环己亚胺过敏。筛选三卡宾突变体环己亚胺敏感性的单拷贝抑制因子得到了RSP5，该基因编码一种含c2结构域的泛素偶联连接酶，对受体介导和液相内吞作用至关重要。结果表明，三白蛋白在膜转运事件中起多聚体的作用，并可能为其他生物中多c2结构域蛋白（如突触tagmins）的作用提供见解。
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.