CHARACTERIZATION OF PROTEIN-TRANSPORT BETWEEN SUCCESSIVE COMPARTMENTS OF THE GOLGI-APPARATUS - ASYMMETRIC PROPERTIES OF DONOR AND ACCEPTOR ACTIVITIES IN A CELL-FREE SYSTEM
CHARACTERIZATION OF PROTEIN-TRANSPORT BETWEEN SUCCESSIVE COMPARTMENTS OF THE GOLGI-APPARATUS - ASYMMETRIC PROPERTIES OF DONOR AND ACCEPTOR ACTIVITIES IN A CELL-FREE SYSTEM
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DOI：
10.1016/0003-9861(85)90046-3
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发表时间：
1985-01-01
影响因子：
3.9
通讯作者：
ROTHMAN, JE
中科院分区：
文献类型：
--
作者：
BALCH, WE;ROTHMAN, JE
在无细胞系统中，通过N-[3H]乙酰氨基葡萄糖（GlcNAc）的偶联结合，测量了水疱性口炎病毒（VSV）编码的糖蛋白（G蛋白）在高尔基体连续隔室之间的运输。当G蛋白从缺乏GlcNAc转移酶I的高尔基体膜的供体区室（来自vsv感染的CHO克隆15B细胞）从野生型CHO细胞（含有GlcNAc转移酶但不含G蛋白）运输到高尔基体群体中的下一个受体区室时，发生糖基化。提出了实现体外转运所需条件的详细特征。供体和受体的活性在某些性质上明显不同。施体活性被n -乙基马来酰亚胺抑制，而受体活性则具有抗性。在没有ATP或细胞质组分的情况下，供体活性是不稳定的；受体活性要稳定得多。这种不对称可能反映了蛋白质运输的潜在生物化学载体性质。供体和受体都对胰蛋白酶敏感，这意味着需要细胞质导向的膜蛋白。转运只发生在接近生理条件的有限范围内。ATP是绝对必需的，尽管只有0.1 μ。M是充分的。转运受到ATP- γ的抑制。-硫酸盐和钒酸盐，表明ATP需要水解。相比之下，耗散膜电位和质子梯度的离子载体不抑制运输。莫能菌素在无细胞系统中也没有作用。
Transport of the vesicular stomatitis virus (VSV)-encoded glycoprotein (G protein) between successive compartments of the Golgi in a cell-free system is measured by the coupled incorporation of N-[3H]acetylglucosamine (GlcNAc). This glycosylation occurs when G protein is transported from a donor compartment in Golgi membranes that lack GlcNAc transferase I (from VSV-infected CHO clone 15B cells) to the next acceptor compartment in a Golgi population from wild-type CHO cells (containing the GlcNAc transferase but not G protein). A detailed characterization of the conditions required to achieve transport in vitro is presented. Donor and acceptor activities differ markedly in certain of their properties. The donor activity is inhibited by N-ethylmaleimide but the acceptor activity is resistant. Donor activity is unstable in the absence of ATP or the cytosol fraction; acceptor activity is much more stable. This asymmetry may reflect the vectorial nature of the underlying biochemistry of protein transport. Both donor and acceptor are trypsin-sensitive, implying a need for cytoplasmically oriented membrane proteins. Transport occurs only in a restricted range of close to physiological conditions. ATP is absolutely required, although as little as 1 .mu.M is sufficient. Transport is inhibited by ATP-.gamma.-sulfate and vanadate, suggesting the ATP hydrolysis is needed. By contrast, ionophores that dissipate membrane potentials and proton gradients do not inhibit transport. Monensin was also without effect in the cell-free system.