Nelson D S, Alvarez C, Gao Y S, García-Mata R, Fialkowski E, Sztul E
Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
J Cell Biol. 1998 Oct 19;143(2):319-31. doi: 10.1083/jcb.143.2.319.
The mammalian protein TAP/p115 and its yeast homologue Uso1p have an essential role in membrane traffic (Nakajima et al., 1991; Waters et al., 1992; Sztul et al., 1993; Rabouille et al.; 1995). To inquire into the site and mechanism of TAP/p115 action, we aimed to localize it and to identify domains required for its function. We show that in interphase cells, TAP/p115 localizes predominantly to the Golgi and to peripheral structures that represent vesicular tubular clusters (VTCs) involved in ER to Golgi transport. Using BFA/ nocodazole treatments we confirm that TAP/p115 is present on ER to Golgi transport intermediates. TAP/ p115 redistributes to peripheral structures containing ERGIC-53 during a 15 degreesC treatment, suggesting that it is a cycling protein. Within the Golgi, TAP/p115 is associated with pleiomorphic structures on the cis side of the cis-Golgi cisterna and the cis-most cisterna, but is not detected in more distal compartments of the Golgi. TAP/p115 binds the cis-Golgi protein GM130, and the COOH-terminal acidic domain of TAP/p115 is required for this interaction. TAP/p115 interaction with GM130 occurs only in the Golgi and is not required for TAP/p115 association with peripheral VTCs. To examine whether interaction with GM130 is required to recruit TAP/p115 to the Golgi, TAP/p115 mutants lacking the acidic domain were expressed and localized in transfected cells. Mutants lacking the GM130-binding domain showed normal Golgi localization, indicating that TAP/p115 is recruited to the Golgi independently of its ability to bind GM130. Such mutants were also able to associate with peripheral VTCs. Interestingly, TAP/p115 mutants containing the GM130-binding domain but lacking portions of the NH2-terminal region were restricted from the Golgi and localized to the ER. The COOH-terminal domain required for GM130 binding and the NH2-terminal region required for Golgi localization appear functionally relevant since expression of TAP/p115 mutants lacking either of these domains leads to loss of normal Golgi morphology.
哺乳动物蛋白TAP/p115及其酵母同源物Uso1p在膜泡运输中起关键作用（中岛等人，1991年；沃特斯等人，1992年；斯图尔等人，1993年；拉布耶等人，1995年）。为了探究TAP/p115的作用位点和机制，我们旨在对其进行定位并鉴定其功能所需的结构域。我们发现，在间期细胞中，TAP/p115主要定位于高尔基体以及代表参与内质网到高尔基体运输的囊泡管状簇（VTCs）的周边结构。通过BFA/诺考达唑处理，我们证实TAP/p115存在于内质网到高尔基体的运输中间体上。在15℃处理期间，TAP/p115重新分布到含有ERGIC-53的周边结构，这表明它是一种循环蛋白。在高尔基体中，TAP/p115与顺面高尔基体潴泡和顺面最外侧潴泡顺面的多形性结构相关，但在高尔基体更靠后的区室中未检测到。TAP/p115与顺面高尔基体蛋白GM130结合，这种相互作用需要TAP/p115的COOH末端酸性结构域。TAP/p115与GM130的相互作用仅发生在高尔基体中，且对于TAP/p115与周边VTCs的结合并非必需。为了研究与GM130的相互作用是否是将TAP/p115招募到高尔基体所必需的，我们表达了缺乏酸性结构域的TAP/p115突变体并将其定位在转染细胞中。缺乏GM130结合结构域的突变体显示出正常的高尔基体定位，这表明TAP/p115被招募到高尔基体与其结合GM130的能力无关。此类突变体也能够与周边VTCs结合。有趣的是，含有GM130结合结构域但缺乏部分NH2末端区域的TAP/p115突变体被限制在高尔基体之外并定位于内质网。GM130结合所需的COOH末端结构域和高尔基体定位所需的NH2末端区域似乎在功能上相关，因为缺乏这些结构域之一的TAP/p115突变体的表达会导致正常高尔基体形态的丧失。
