Identification and characterization of GCP16, a novel acylated Golgi protein that interacts with GCP170
Identification and characterization of GCP16, a novel acylated Golgi protein that interacts with GCP170
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DOI：
10.1074/jbc.m310014200
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发表时间：
2003-12-19
影响因子：
4.8
通讯作者：
Ikehara, Y
中科院分区：
文献类型：
--
作者：
Ohta, E;Misumi, Y;Ikehara, Y
GCP 170是与高尔基体膜的胞质面相关的高尔基体蛋白家族的成员，发现其在NH 2-末端区域（位置137 - 237）具有高尔基体定位信号。在酵母双杂交筛选系统中，利用该结构域作为诱饵，我们鉴定了一个与GCP 170相互作用的新蛋白。编码16 kDa的137个氨基酸的蛋白质的2.0-脱氢酶mRNA被广泛表达，命名为GCP 16。免疫荧光显微镜显示，GCP 16与GCP 170和Giantin共定位于高尔基体区域。尽管缺乏足以参与膜定位的疏水结构域，但发现GCP 16与膜紧密相关，就像一个完整的膜蛋白。用[H-3]棕榈酸标记实验和突变分析表明，GCP 16在Cys（69）和Cys（72）处被酰化，这解释了其与膜的紧密结合。一个没有潜在酰化位点的突变体（C69 A/C72 A）不再定位于高尔基体，表明酰化是GCP 16高尔基体定位的先决条件。尽管突变体GCP 16即使过表达也对蛋白质转运没有影响，但野生型GCP 16的过表达对蛋白质从高尔基体到细胞表面的转运产生了抑制作用。总之，这些结果表明，GCP 16是酰化的膜蛋白，与GCP 170，并可能参与从高尔基体到细胞表面的囊泡运输。
GCP170, a member of the golgin family associated with the cytoplasmic face of the Golgi membrane, was found to have a Golgi localization signal at the NH2-terminal region ( positions 137 - 237). Using this domain as bait in the yeast two-hybrid screening system, we identified a novel protein that interacted with GCP170. The 2.0-kilobase mRNA encoding a 137-amino acid protein of 16 kDa designated GCP16 was ubiquitously expressed. Immunofluorescence microscopy showed that GCP16 was co-localized with GCP170 and giantin in the Golgi region. Despite the absence of a hydrophobic domain sufficient for participating in membrane localization, GCP16 was found to be tightly associated with membranes like an integral membrane protein. Labeling experiments with [H-3] palmitic acid and mutational analysis demonstrated that GCP16 was acylated at Cys(69) and Cys(72), accounting for its tight association with the membrane. A mutant without potential acylation sites (C69A/C72A) was no longer localized to the Golgi, indicating that the acylation is prerequisite for the Golgi localization of GCP16. Although the mutant GCP16, even when overexpressed, had no effect on protein transport, overexpression of the wild type GCP16 caused an inhibitory effect on protein transport from the Golgi to the cell surface. Taken together, these results indicate that GCP16 is the acylated membrane protein, associated with GCP170, and possibly involved in vesicular transport from the Golgi to the cell surface.