Misumi Y, Sohda M, Tashiro A, Sato H, Ikehara Y
Department of Biochemistry, Fukuoka University School of Medicine, Jonan-ku, Fukuoka 814-0180, Japan.
J Biol Chem. 2001 Mar 2;276(9):6867-73. doi: 10.1074/jbc.M010121200. Epub 2000 Dec 11.
Giantin is a resident Golgi protein that has an extremely long cytoplasmic domain (about 370 kDa) and is anchored to the Golgi membrane by the COOH-terminal membrane-anchoring domain (CMD) with no luminal extension. We examined the essential domain of giantin required for Golgi localization by mutational analysis. The Golgi localization of giantin was not affected by the deletion of its CMD or by substitution with the CMD of syntaxin-2, a plasma membrane protein. The giantin CMD fused to the cytoplasmic domain of syntaxin-2 could not retain the chimera in the Golgi apparatus. Sequential deletion analysis showed that the COOH-terminal sequence (positions 3059--3161) adjacent to the CMD was the essential domain required for the Golgi localization of giantin. We also examined two other Golgi-resident proteins, golgin-84 and syntaxin-5, with a similar membrane topology as giantin. It was confirmed that the cytoplasmic domain of about 100 residues adjacent to the CMD was required for their Golgi localization. Taken together, these results suggest that the COOH-terminally anchored Golgi proteins with long cytoplasmic extensions have the Golgi localization signal(s) in the cytoplasmic sequence adjacent to the CMD. This is in contrast to previous observations that a transmembrane domain is required for Golgi localization by other Golgi proteins transported from the endoplasmic reticulum.
巨蛋白是一种驻留高尔基体蛋白，其胞质结构域极长（约370 kDa），并通过COOH末端膜锚定结构域（CMD）锚定在高尔基体膜上，无腔延伸。我们通过突变分析研究了巨蛋白高尔基体定位所需的关键结构域。巨蛋白的高尔基体定位不受其CMD缺失或被质膜蛋白Syntaxin-2的CMD替代的影响。与Syntaxin-2胞质结构域融合的巨蛋白CMD无法将嵌合体保留在高尔基体中。序列缺失分析表明，与CMD相邻的COOH末端序列（第3059 - 3161位）是巨蛋白高尔基体定位所需的关键结构域。我们还研究了另外两种与巨蛋白具有相似膜拓扑结构的高尔基体驻留蛋白，golgin-84和Syntaxin-5。证实了与CMD相邻的约100个残基的胞质结构域是它们高尔基体定位所必需的。综上所述，这些结果表明，具有长胞质延伸的COOH末端锚定高尔基体蛋白在与CMD相邻的胞质序列中具有高尔基体定位信号。这与之前观察到的从内质网转运的其他高尔基体蛋白需要跨膜结构域进行高尔基体定位的情况形成对比。
