Non-muscle myosin IIA transports a Golgi glycosyltransferase to the endoplasmic reticulum by binding to its cytoplasmic tail.
Non-muscle myosin IIA transports a Golgi glycosyltransferase to the endoplasmic reticulum by binding to its cytoplasmic tail.
复制标题
非肌肉肌球蛋白IIA通过与其细胞质尾巴结合，将高尔基糖基转移酶转运到内质网。
DOI：
10.1016/j.biocel.2012.04.004
复制
发表时间：
2012-07
期刊：
影响因子：
--
通讯作者：
Cheng PW
中科院分区：
文献类型：
--
作者：
Petrosyan A;Ali MF;Verma SK;Cheng H;Cheng PW
高尔基体糖基转移酶的高尔基体-内质网转运机制尚不清楚。我们利用表达c-Myc标记的核心2 N-乙酰葡糖胺转移酶-M（C2 GnT M）的细胞系来探索这种机制。通过使用抗c-Myc抗体的免疫沉淀结合蛋白质组学分析，我们已经鉴定了免疫沉淀物中的几种蛋白质，包括非肌肉肌球蛋白IIA（NMIIA）、热休克蛋白（HSP）-70和泛素激活酶E1。采用酵母双杂交分析和下拉实验，我们表明，NMIIA重链的C-末端区域结合C2 GnT-M的胞质尾中的1-6个氨基酸。我们已经发现，NMIIA与C2 GnT-M共定位在高尔基体的外围。此外，NMIIA的抑制或敲低防止布雷菲德菌素A诱导的高尔基体塌陷，如通过抑制Giantin（一种高尔基体基质蛋白）和C2 GnT-M（一种高尔基体非基质蛋白）向ER的迁移所示。相比之下，HSP-70的敲除将Giantin保留在高尔基体中，但将C2 GnT-M移动到ER，该过程也被NMIIA的抑制或敲除阻断。此外，C2 GnT-M的细胞内分布不受β-外被体蛋白的敲低（有或没有HSP抑制）的影响，表明C2 GnT-M的高尔基体至ER运输不依赖于外被蛋白复合物-I。此外，蛋白酶体的抑制导致泛素化C2 GnT-M的积累，表明其被蛋白酶体降解。因此，NMIIA而不是外壳蛋白复合物-I负责将高尔基体糖基转移酶转运到ER进行蛋白酶体降解。这些数据表明NMIIA参与了高尔基体的重塑。
The mechanism of the Golgi-to-ER transport of Golgi glycosyltransferases is not clear. We utilize a cell line expressing the core 2 N-acetylglucosaminyltransferase-M (C2GnT M) tagged with c-Myc to explore this mechanism. By immunoprecipitation using anti-c-Myc antibodies coupled with proteomics analysis, we have identified several proteins including non-muscle myosin IIA (NMIIA), heat shock protein (HSP)-70 and ubiquitin activating enzyme E1 in the immunoprecipitate. Employing yeast-two-hybrid analysis and pulldown experiments, we show that the C-terminal region of the NMIIA heavy chain binds to the 1-6 amino acids in the cytoplasmic tail of C2GnT-M. We have found that NMIIA colocalizes with C2GnT-M at the periphery of the Golgi. In addition, inhibition or knockdown of NMIIA prevents the Brefeldin A-induced collapse of the Golgi as shown by the inhibition of the migration of both Giantin, a Golgi matrix protein, and C2GnT-M, a Golgi non-matrix protein, to the ER. In contrast, knockdown of HSP-70 retains Giantin in the Golgi but moves C2GnT-M to the ER, a process also blocked by inhibition or knockdown of NMIIA. Also, the intracellular distribution of C2GnT-M is not affected by knockdown of β-coatomer protein with or without inhibition of HSPs, suggesting that the Golgi-to-ER trafficking of C2GnT-M does not depend on coat protein complex-I. Further, inhibition of proteasome results in accumulation of ubiquitinated C2GnT-M, suggesting its degradation by proteasome. Therefore, NMIIA and not coat protein complex-I is responsible for transporting the Golgi glycosyltransferase to the ER for proteasomal degradation. The data suggest that NMIIA is involved in the Golgi remodeling.