Analysis of intracellular translocation of glycoproteins using inhibitors as probes

特异性抑制剂有望成为分析糖蛋白细胞内易位机制的有用探针。目前这类抑制剂非常有限。通过一种新建立的筛选方法来寻找新的抑制剂。本研究中分离的许多化合物被发现在H^+易位atp酶中特异性抑制v型atp酶。在这种化合物的存在下，病毒糖蛋白的细胞表面表达受到极大抑制，并在细胞内积累。通过分析n -糖苷结合的低聚糖片段的结构，表明阻滞位点在高尔基体之前或在高尔基体上。相反，尽管在v型atp酶抑制剂的存在下，这些糖蛋白的糖部分被不成熟地加工，但酿酒酵母的转化酶向外周质分泌和羧肽酶Y向液泡的靶向不受影响。已经报道的brefeldin的新阻断被诺卡唑部分逆转，提示诱导从高尔基体到内质网的逆行运输是阻断病毒糖蛋白细胞表面表达的原因。

Specific inhibitors are expected to be useful probes in analysis of the mechanism of intracellular translocation of glycoproteins. Very restricted are such inhibitors at present. Novel inhibitors have been searched for by a newly established screening method.Many of the compounds isolated in this study were found to inhibit specifically the V-type ATPase among H^+-translocating ATPases. In the presence of such a compounds, cell surface expression of virus glycoprotein was greatly suppressed and accumulated intracellularly. The site(s) of blockade was indicated to be before or at the Golgi by analysing the structure of the N-glycosidically bound oligosaccharide moiety. On the contrary, invertase secretion to the periplasm and targetting of carboxypeptidase Y to the vacuole in Saccharomyces cerevisiae were not affected in spite of the fact the saccharide moiety of these glycoproteins was immaturely processed in the presence of inhibitor of V-type ATPase.The already reported novel blockade by brefeldin was partly reversed by nocadazole, suggesting that induction of the retrograde trafficking from the Golgi to endoplasmic reticulum is the cause of the blockade of cell surface expression of virus glycoprotein.