Slac2-a/melanophilin contains multiple PEST-like sequences that are highly sensitive to proteolysis
Slac2-a/melanophilin contains multiple PEST-like sequences that are highly sensitive to proteolysis
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DOI：
10.1074/jbc.401791200
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发表时间：
2004-05-21
影响因子：
4.8
通讯作者：
Itoh, T
中科院分区：
文献类型：
--
作者：
Fukuda, M;Itoh, T
缺乏 C2 结构域 -a (Slac2-a)/亲黑素的类突触结合蛋白同源物最近被确定为小 GTP 酶 Rab27A 和基于肌动蛋白的运动蛋白肌球蛋白 Va 之间的“缺失环节”。 (2003) Mol. Cell. 23, 5245-5255)，该复合物的调节机制（即复合物的组装和分解）从未被阐明。在这项研究中，我们发现 Slac2-a 和密切相关的异构体 Slac2-c/MyRIP 在 C 末端的肌球蛋白 Va 和肌动蛋白结合域中包含多个 PEST 样序列（蛋白质快速降解的潜在信号）。我们发现，Slac2-a 的 C 端结构域在体外对低浓度的蛋白酶（如胰蛋白酶和钙蛋白酶）高度敏感，而 N 端 Rab27A 结合结构域对这些蛋白酶高度耐药。我们进一步发现，内源性钙蛋白酶选择性地裂解黑素细胞中的 Slac2-a，但不裂解 Rab27A 或肌球蛋白 Va。突变体 Slac2-a 缺乏位于肌球蛋白 Va 和肌动蛋白结合结构域之间界面的 PEST 样序列之一（DeltaPEST；氨基酸 399-405），无论在体外还是在黑素细胞中都比野生型蛋白更稳定。与野生型 Slac2-a 相比，具有 N 末端绿色荧光蛋白标签的突变型 Slac2-a-DeltaPEST 的表达通常会诱导黑素体的核周聚集（类似于 40% 的转染细胞）。我们的研究结果表明，Slac2-a 的蛋白质降解是黑素细胞中黑素体正确分布的重要过程。
The synaptotagmin-like protein homologue lacking C2 domains-a (Slac2-a)/melanophilin was recently identified as the "missing link" between the small GTPase Rab27A and the actin-based motor protein myosin Va. Although formation of a tripartite protein complex by three molecules had been shown to be required for proper melanosome distribution in melanocytes (Kuroda, T. S., Ariga, H., and Fukuda, M. ( 2003) Mol. Cell. Biol. 23, 5245-5255), the regulatory mechanisms of the complex (i.e. assembly and disassembly of the complex) had never been elucidated. In this study, we discovered that Slac2-a and a closely related isoform, Slac2-c/MyRIP, contain multiple PEST-like sequences ( potential signals for rapid protein degradation) in the myosin Va- and actin-binding domains at the C terminus. We found that the C-terminal domain of Slac2-a is highly sensitive to low concentrations of proteases, such as trypsin and calpain, in vitro, whereas the N-terminal Rab27A-binding domain is highly resistant to these proteases. We further found that endogenous calpains selectively cleave Slac2-a, but not Rab27A or myosin Va, in melanocytes. A mutant Slac2-a lacking one of the PEST-like sequences located at the interface between the myosin Va- and actin-binding domains (DeltaPEST; amino acids 399-405) is more stable than the wild-type protein, both in vitro and in melanocytes. Expression of the mutant Slac2-a-DeltaPEST with an N-terminal green fluorescence protein tag often induced perinuclear aggregation of melanosomes (similar to40% of the transfected cells) compared with the wild-type Slac2-a. Our findings suggest that protein degradation of Slac2-a is an essential process for proper melanosome distribution in melanocytes.