Sec16p potentiates the action of COPII proteins to bud transport vesicles.
Sec16p potentiates the action of COPII proteins to bud transport vesicles.
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Sec16p增强了Copii蛋白对芽囊泡的作用。
DOI：
10.1083/jcb.200207053
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发表时间：
2002-09-16
期刊：
影响因子：
--
通讯作者：
Schekman R
中科院分区：
文献类型：
--
作者：
Supek F;Madden DT;Hamamoto S;Orci L;Schekman R
SEC16编码一个240-kD的亲水蛋白，它是从酿酒酵母的内质网运输囊泡萌发所必需的。Sec16p与内质网细胞膜紧密结合，因此它不是在无细胞反应中重建运输囊泡发芽所需的胞浆蛋白之一。然而，Sec16p是通过盐洗从膜上去除的，使用这种膜，我们重新构建了依赖于COPII蛋白和纯Sec16p的囊泡萌发反应。虽然GTP或非水解性类似物鸟苷二磷酸(GMP-PNP)可促进COPII囊泡萌发，但Sec16p的刺激作用依赖于GTP。合成脂质体包括酸性磷脂(主要-次要混合物)或简单的磷脂酰胆碱(PC)和磷脂酰乙醇胺(PE)二元混合物，用于研究外壳蛋白组装和Sec16P刺激的囊泡萌发的细节。Sec16p与主要-次要混合脂质体结合，在Sar1p和GMP-PNP刺激的反应中促进COPII蛋白的募集和囊泡萌发。薄片电子显微镜证实了脂质体与COPII和Sec16P孵育产生的萌发轮廓的刺激作用。而主要-次要混合物中的酸性磷脂需要将纯Sec16p聚集到脂质体中，而PC/PE脂质体结合Sar1p-GTP，从而刺激Sec16p和Sec23/24p的结合。我们认为，Sec16p核化了依赖Sar1-GTP的COPII组装的启动，并在Sar1p水解GTP后稳定外壳使其过早解体。
SEC16 encodes a 240-kD hydrophilic protein that is required for transport vesicle budding from the ER in Saccharomyces cerevisiae. Sec16p is tightly and peripherally bound to ER membranes, hence it is not one of the cytosolic proteins required to reconstitute transport vesicle budding in a cell-free reaction. However, Sec16p is removed from the membrane by salt washes, and using such membranes we have reconstituted a vesicle budding reaction dependent on the addition of COPII proteins and pure Sec16p. Although COPII vesicle budding is promoted by GTP or a nonhydrolyzable analogue, guanylimide diphosphate (GMP-PNP), Sec16p stimulation is dependent on GTP in the reaction. Details of coat protein assembly and Sec16p-stimulated vesicle budding were explored with synthetic liposomes composed of a mixture of lipids, including acidic phospholipids (major–minor mix), or a simple binary mixture of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Sec16p binds to major–minor mix liposomes and facilitates the recruitment of COPII proteins and vesicle budding in a reaction that is stimulated by Sar1p and GMP-PNP. Thin-section electron microscopy confirms a stimulation of budding profiles produced by incubation of liposomes with COPII and Sec16p. Whereas acidic phospholipids in the major–minor mix are required to recruit pure Sec16p to liposomes, PC/PE liposomes bind Sar1p-GTP, which stimulates the association of Sec16p and Sec23/24p. We propose that Sec16p nucleates a Sar1-GTP–dependent initiation of COPII assembly and serves to stabilize the coat to premature disassembly after Sar1p hydrolyzes GTP.
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DOI：
10.1006/meth.2000.0955
发表时间：
2000-04-01
期刊：
METHODS-A COMPANION TO METHODS IN ENZYMOLOGY
影响因子：
--
作者：
Matsuoka, K;Schekman, R
通讯作者：
Schekman, R
影响因子：
3.3
作者：
Gimeno, RE;Espenshade, P;Kaiser, CA
通讯作者：
Kaiser, CA
影响因子：
7.8
作者：
ESPENSHADE, P;GIMENO, RE;KAISER, CA
通讯作者：
KAISER, CA
影响因子：
64.8
作者：
Kuehn, MJ;Herrmann, JM;Schekman, R
通讯作者：
Schekman, R
影响因子：
64.5
作者：
LATTERICH, M;SCHEKMAN, R
通讯作者：
SCHEKMAN, R