McConville Malcolm J, Ilgoutz Steven C, Teasdale Rohan D, Foth Bernardo J, Matthews Antony, Mullin Kylie A, Gleeson Paul A
Russell Grimwade School of Biochemistry and Molecular Biology, University of Melbourne, Victoria, Australia.
Eur J Cell Biol. 2002 Sep;81(9):485-95. doi: 10.1078/0171-9335-00268.
The GRIP domain, found in a family of coiled-coil peripheral membrane Golgi proteins, is a specific targeting sequence for the trans-Golgi network of animal cells. In this study we show that a coiled-coil protein with a GRIP domain occurs in the primitive eukaryote, Trypanosoma brucei, and that reporter proteins containing this domain can be used as a marker for the poorly characterized trans Golgi/trans-Golgi network of trypanosomatid parasites. The T. brucei GRIP domain, when fused to the carboxyl terminus of the green fluorescent protein (GFP-TbGRIP), was efficiently localized to the Golgi apparatus of transfected COS cells. Overexpression of GFP-TbGRIP in COS cells displaced the endogenous GRIP protein, GCC1p, from the Golgi apparatus indicating that the trypanosomatid and mammalian GRIP sequences interact with similar membrane determinants. GFP fusion proteins containing either the T. brucei GRIP domain or the human p230 GRIP (p230GRIP) domain were also expressed in the trypanosomatid parasite, Leishmania mexicana, and localized by fluorescence and immuno-electron microscopy to the trans face of the single Golgi apparatus and a short tubule that extended from the Golgi apparatus. Binding of GFP-p230GRIP to Golgi membranes in L. mexicana was abrogated by mutation of a critical tyrosine residue in the p230 GRIP domain. The levels of GFP-GRIP fusion proteins were dramatically reduced in stationary-phase L. mexicana promastigotes, suggesting that specific Golgi trafficking steps may be down-regulated as the promastigotes cease dividing. This study provides a protein marker for the trans-Golgi network of trypanosomatid parasites and suggests that the GRIP domain binds to a membrane component that has been highly conserved in eukaryotic evolution.
GRIP结构域存在于一类卷曲螺旋外周膜高尔基体蛋白家族中，是动物细胞反式高尔基体网络的特定靶向序列。在本研究中，我们发现一种带有GRIP结构域的卷曲螺旋蛋白存在于原始真核生物布氏锥虫中，并且含有该结构域的报告蛋白可作为锥虫寄生虫中特征不明确的反式高尔基体/反式高尔基体网络的标记物。当布氏锥虫GRIP结构域与绿色荧光蛋白（GFP-TbGRIP）的羧基末端融合时，能有效地定位于转染的COS细胞的高尔基体。在COS细胞中过表达GFP-TbGRIP会使内源性GRIP蛋白GCC1p从高尔基体移位，这表明锥虫和哺乳动物的GRIP序列与相似的膜决定簇相互作用。含有布氏锥虫GRIP结构域或人p230 GRIP（p230GRIP）结构域的GFP融合蛋白也在锥虫寄生虫墨西哥利什曼原虫中表达，并通过荧光和免疫电子显微镜定位于单个高尔基体的反面以及从高尔基体延伸出的短小管。p230 GRIP结构域中一个关键酪氨酸残基的突变消除了GFP-p230GRIP与墨西哥利什曼原虫高尔基体膜的结合。在静止期的墨西哥利什曼原虫前鞭毛体中，GFP-GRIP融合蛋白的水平显著降低，这表明随着前鞭毛体停止分裂，特定的高尔基体运输步骤可能会下调。本研究为锥虫寄生虫的反式高尔基体网络提供了一种蛋白质标记物，并表明GRIP结构域与真核生物进化中高度保守的膜成分结合。
