The TRAPP complex is a nucleotide exchanger for Ypt1 and Ypt31/32
The TRAPP complex is a nucleotide exchanger for Ypt1 and Ypt31/32
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DOI：
10.1091/mbc.11.12.4403
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发表时间：
2000-12-01
影响因子：
3.3
通讯作者：
Segev, N
中科院分区：
文献类型：
--
作者：
Jones, S;Newman, C;Segev, N
在酵母中，Ypt1 GTPase是er -to-顺式高尔基蛋白和顺式-to-medial高尔基蛋白转运所必需的，而Ypt31/32是从反式高尔基蛋白转运所必需的一对GTPase。我们之前已经确定了一个Ypt1鸟嘌呤核苷酸交换因子（GEF）活性，并将其描述为一个大的膜相关蛋白复合物，定位于高尔基体，可以通过盐从膜中提取，但不能通过洗涤剂提取。TRAPP是ER-to-Golgi转运所需的大型蛋白质复合物，具有与Ypt1 GEF相似的特性。这里我们显示TRAPP具有Ypt1 GEF活性。gst标记的Bet3p或Bet5p是TRAPP的两个亚基，在酵母细胞中表达，并被谷胱甘肽琼脂糖（GA）珠沉淀。由此产生的沉淀可以刺激Ypt1p释放GDP和吸收GTP。与GST-Bet3p沉淀物相关的大部分Ypt1 GEF活性的表观分子量为bb0 670 kDa，表明GEF活性存在于TRAPP复合物中。令人惊讶的是，TRAPP还可以刺激Ypt31/32 GTPase上的核苷酸交换，但不能刺激Sec4p，这是ypt家族GTPase在胞外通路的最后一步所必需的。与先前表征的Ypt1 GEF一样，TRAPP的Ypt1-GEF活性可以被无核苷酸的Ypt1- d124n突变蛋白抑制。该突变蛋白也抑制TRAPP的Ypt32 GEF活性。共沉淀和过表达研究表明，TRAPP在体内可作为Ypt1和Ypt31/32的GEF。这些数据表明一种令人兴奋的可能性，即Ypt1和Ypt31/32共有的GEF复合体可能协调这些gtpase进入和退出高尔基体的功能。
Ln yeast, the Ypt1 GTPase is required for ER-to-cis-Golgi and cis-to-medial-Golgi protein transport, while Ypt31/32 are a functional pair of GTPases essential for exit from the trans-Golgi. We have previously identified a Ypt1 guanine nucleotide exchange factor (GEF) activity and characterized it as a large membrane-associated protein complex that localizes to the Golgi and can be extracted from the membrane by salt, but not by detergent. TRAPP is a large protein complex that is required for ER-to-Golgi transport and that has properties similar to those of Ypt1 GEF. Here we show that TRAPP has Ypt1 GEF activity. GST-tagged Bet3p or Bet5p, two of the TRAPP subunits, were expressed in yeast cells and were precipitated by glutathione-agarose (GA) beads. The resulting precipitates can stimulate both GDP release and GTP uptake by Ypt1p. The majority of the Ypt1 GEF activity associated with the GST-Bet3p precipitate has an apparent molecular weight of > 670 kDa, indicating that the GEF activity resides in the TRAPP complex. Surprisingly TRAPP can also stimulate nucleotide exchange on the Ypt31/32 GTPases, but not on Sec4p, a Ypt-family GTPase required for the last step of the exocytic pathway. Like the previously characterized Ypt1 GEF, the TRAPP Ypt1-GEF activity can be inhibited by the nucleotide-free Ypt1-D124N mutant protein. This mutant protein also inhibits the Ypt32 GEF activity of TRAPP. Coprecipitation and overexpression studies suggest that TRAPP can act as a GEF for Ypt1 and Ypt31/32 in vivo. These data suggest the exciting possibility that a GEF complex common to Ypt1 and Ypt31/32 might coordinate the function of these GTPases in entry into and exit from the Golgi.