Secretion in yeast: in vitro analysis of the sec53 mutant.
Secretion in yeast: in vitro analysis of the sec53 mutant.
复制标题
酵母中的分泌：sec53 突变体的体外分析。
DOI：
10.1002/j.1460-2075.1988.tb03062.x
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发表时间：
1988
期刊：
影响因子：
--
通讯作者：
Meyer,DI
中科院分区：
文献类型：
--
作者：
Hibbs,AR;Meyer,DI
通过遗传和生物化学相结合的方法研究蛋白质易位的核心重要性是酵母条件致死分泌突变体的体外分析。sec 53的分析提供了一个机会，不仅可以看到，如果突变体可以在最近开发的酵母体外易位系统进行检查，但也进一步表征这种突变体的性质，最初假设是有缺陷的蛋白质易位。来自sec 53的膜能够在不允许突变细胞生长的温度下在sec 53和野生型裂解物中在体外易位和糖基化新生前原-α-因子。这些结果表明，Sec 53蛋白不直接在前原-α-因子的易位和糖基化中起作用。为了进一步研究这一点，我们从在破坏之前已经在非允许温度下生长的sec 53细胞中分离膜。在这种情况下，无论测定温度如何，来自sec 53细胞的膜有效地易位，但未能在体外糖基化前原-α-因子。Sec 53的体外表型可以通过从在衣霉素存在下生长1小时的野生型细胞中分离粗糙微粒体来模仿。总之，这些结果表明，sec 53是没有缺陷的易位，而是在组装的N-连接的糖基化所需的长醇-寡糖底物。图像
Of central importance to studying protein translocation via a combined genetic and biochemical approach is the in vitro analysis of yeast conditionally-lethal secretory mutants. Analysis of sec53 presented an opportunity not only to see if mutants could be examined in recently developed yeast in vitro translocation systems, but also to characterize further the nature of this mutant originally postulated to be defective in protein translocation. Membranes from sec53 were capable of translocating and glycosylating nascent prepro-alpha-factor in vitro in both sec53 and wild-type lysates at temperatures that were non-permissive for growth of the mutant cells. These results suggested that the Sec53 protein does not function directly in the translocation and glycosylation of prepro-alpha-factor. To examine this point further, we isolated membranes from sec53 cells that had been grown at the non-permissive temperature prior to disruption. In such cases, regardless of assay temperature, membranes from sec53 cells efficiently translocated but failed to glycosylate prepro-alpha-factor in vitro. The in vitro phenotype of sec53 could be mimicked by isolating rough microsomes from wild-type cells that had been grown for 1 h in the presence of tunicamycin. Together, these results demonstrate that sec53 is not defective in translocation, rather in assembly of the dolichol-oligosaccharide substrate needed for N-linked glycosylation. Images