Ohta Eiji, Misumi Yoshio, Sohda Miwa, Fujiwara Toshiyuki, Yano Akiko, Ikehara Yukio
Department of Cell Biology, Fukuoka University School of Medicine, Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan.
J Biol Chem. 2003 Dec 19;278(51):51957-67. doi: 10.1074/jbc.M310014200. Epub 2003 Oct 1.
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 [3H]palmitic acid and mutational analysis demonstrated that GCP16 was acylated at Cys69 and Cys72, 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.
GCP170是一种与高尔基体膜细胞质面相关的golgin家族成员，发现在其NH2末端区域（第137 - 237位）有一个高尔基体定位信号。利用该结构域作为酵母双杂交筛选系统中的诱饵，我们鉴定出一种与GCP170相互作用的新蛋白。编码16 kDa的137个氨基酸蛋白（命名为GCP16）的2.0千碱基mRNA在各处均有表达。免疫荧光显微镜显示GCP16与GCP170和巨蛋白在高尔基体区域共定位。尽管缺乏足以参与膜定位的疏水结构域，但GCP16被发现像整合膜蛋白一样与膜紧密结合。用[3H]棕榈酸进行的标记实验和突变分析表明，GCP16在Cys69和Cys72处被酰化，这解释了它与膜的紧密结合。一个没有潜在酰化位点的突变体（C69A/C72A）不再定位于高尔基体，表明酰化是GCP16高尔基体定位的先决条件。尽管突变型GCP16即使过表达也对蛋白质转运没有影响，但野生型GCP16的过表达对从高尔基体到细胞表面的蛋白质转运产生抑制作用。综上所述，这些结果表明GCP16是一种酰化膜蛋白，与GCP170相关，可能参与从高尔基体到细胞表面的囊泡运输。
