Coupled ER to Golgi transport reconstituted with purified cytosolic proteins.
Coupled ER to Golgi transport reconstituted with purified cytosolic proteins.
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DOI：
10.1083/jcb.139.5.1097
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发表时间：
1997-12-01
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影响因子：
--
通讯作者：
Barlowe C
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文献类型：
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作者：
Barlowe C
无细胞囊泡融合试验再现了α-因子原从内质网转运到高尔基复合体的亚反应，该试验已用于破碎酵母胞质溶胶。在ATP和GTP存在下纯化的Sec 18 p、Uso 1 p和LMA 1满足ER衍生的囊泡与高尔基体膜融合对胞质溶胶的要求。虽然这些纯化的因子足以用于囊泡对接和融合，但酵母半完整细胞中的总体ER到高尔基体的运输取决于COPII蛋白（驱动囊泡从ER出芽的膜包衣的组分）。因此，即使在纯化的融合因子的饱和水平的存在下，膜融合也与ER到高尔基体运输中的囊泡形成相耦合。半完整细胞试验的操作用于区分含前α因子的自由扩散ER衍生囊泡与对接囊泡和融合囊泡。Uso 1 p介导囊泡对接并产生耐稀释中间体。Sec 18 p和LMA 1不是对接阶段所需的，但却是ER衍生的囊泡与高尔基体复合体有效融合所需的。令人惊讶的是，升高水平的Sec 23 p复合物（COPII外壳的亚基）以可逆的方式防止囊泡融合，但不干扰囊泡对接。使用耐稀释中间体和可逆Sec 23 p复合物抑制的排序实验表明，在LMA 1发挥功能之前需要Sec 18 p作用。
A cell-free vesicle fusion assay that reproduces a subreaction in transport of pro-α-factor from the ER to the Golgi complex has been used to fractionate yeast cytosol. Purified Sec18p, Uso1p, and LMA1 in the presence of ATP and GTP satisfies the requirement for cytosol in fusion of ER-derived vesicles with Golgi membranes. Although these purified factors are sufficient for vesicle docking and fusion, overall ER to Golgi transport in yeast semi-intact cells depends on COPII proteins (components of a membrane coat that drive vesicle budding from the ER). Thus, membrane fusion is coupled to vesicle formation in ER to Golgi transport even in the presence of saturating levels of purified fusion factors. Manipulation of the semi-intact cell assay is used to distinguish freely diffusible ER- derived vesicles containing pro-α-factor from docked vesicles and from fused vesicles. Uso1p mediates vesicle docking and produces a dilution resistant intermediate. Sec18p and LMA1 are not required for the docking phase, but are required for efficient fusion of ER- derived vesicles with the Golgi complex. Surprisingly, elevated levels of Sec23p complex (a subunit of the COPII coat) prevent vesicle fusion in a reversible manner, but do not interfere with vesicle docking. Ordering experiments using the dilution resistant intermediate and reversible Sec23p complex inhibition indicate Sec18p action is required before LMA1 function.