RECONSTITUTION OF THE TRANSPORT OF PROTEIN BETWEEN SUCCESSIVE COMPARTMENTS OF THE GOLGI MEASURED BY THE COUPLED INCORPORATION OF N-ACETYLGLUCOSAMINE
RECONSTITUTION OF THE TRANSPORT OF PROTEIN BETWEEN SUCCESSIVE COMPARTMENTS OF THE GOLGI MEASURED BY THE COUPLED INCORPORATION OF N-ACETYLGLUCOSAMINE
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DOI：
10.1016/0092-8674(84)90019-9
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发表时间：
1984-01-01
期刊：
影响因子：
64.5
通讯作者：
ROTHMAN, JE
中科院分区：
文献类型：
--
作者：
BALCH, WE;DUNPHY, WG;ROTHMAN, JE
VSV[水泡性口炎病毒]编码的糖蛋白（G蛋白）在高尔基体的连续隔室之间的转运在无细胞系统中重建，并在一种快速灵敏的新测定中通过偶联掺入3 H-N-乙酰葡糖胺（GlcNAc）进行测定。当G蛋白在混合孵育期间从来自VSV感染的CHO克隆15 B细胞的高尔基体中的供体区室（缺少关键的高尔基体GlcNAc转移酶）转移到来自野生型CHO细胞的高尔基体中的下一个连续的受体区室（含有GlcNAc转移酶）时，发生这种糖基化。在该测定中使用的高尔基体级分被广泛纯化，并解释了细胞中的所有供体和受体活性。连同其他几个证据线，这表明，无细胞系统是高度特异性的，仅测量高尔基体堆栈中连续隔室之间的运输。体外运输几乎与细胞中一样有效，除了高尔基体膜细胞质表面的蛋白质成分外，还需要ATP和细胞质组分。
Transport of the VSV[vesicular stomatitis virus]-encoded glycoprotein (G protein) between successive compartments of the Golgi was reconstituted in a cell-free system and is measured, in a rapid and sensitive new assay, by the coupled incorporation of 3H-N-acetylglucosamine (GlcNAc). This glycosylation occurs when G protein is transproted during mixed incubations from the donor compartment in Golgi from VSV-infected CHO clone 15B cells (missing a key Golgi GlcNAc transferase) to the next, successive acceptor compartment (containing the GlcNAc transferase) in Golgi from wild-type CHO cells. Golgi fractions used in this assay were extensively purified and account for all of the donor and acceptor activity in the cells. Together with several other lines of evidence, this indicates that the cell-free system is highly specific, measuring only transport between sequential compartments in the Golgi stack. Transport in vitro is almost as efficient as in the cell and requires ATP and the cytosol fraction in addition to protein components on the cytoplasmic surface of the Golgi membranes.