Localization of a yeast early Golgi mannosyltransferase, Och1p, involves retrograde transport.
Localization of a yeast early Golgi mannosyltransferase, Och1p, involves retrograde transport.
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DOI：
10.1083/jcb.132.6.985
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发表时间：
1996-03
期刊：
影响因子：
--
通讯作者：
Waters MG
中科院分区：
文献类型：
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作者：
Harris SL;Waters MG
为了分析酿酒酵母早期高尔基体中整合膜蛋白定位的机制，我们使用了 Och1p，一种顺式高尔基体甘露糖基转移酶。构建了一系列流感病毒血凝素 (HA) 表位标记的融合蛋白，其中转化酶附加到全长 Och1p 的高尔基体腔羧基末端。一些构建体包括 Och1p 和转化酶部分之间的 Kex2p 切割位点，以监测向含有 Kex2p 的 TGN 的转运。表达 Och1p-转化酶融合蛋白的细胞不分泌转化酶，但表达 Och1p-Kex2p 位点转化酶融合蛋白的细胞以 Kex2p 依赖性方式分泌高水平的转化酶。 Och1p-Kex2p 位点-转化酶融合蛋白在 5 分钟的半衰期内被切割，并且该过程继续完成。在裂解之前，蛋白质接受糖基修饰，表明通过内侧和反面高尔基体，因此裂解发生在通过高尔基体有序顺行运输至 TGN 后。转化酶部分不会诱导向远端区室的转运，因为非转化酶融合构建体也会遇到相同的糖基转移酶和 Kex2p。 Och1p-HA 部分，无论是通过 TGN 中的融合蛋白裂解产生还是从头合成，都会在约 60 分钟的半衰期内降解。因此，降解半衰期比达到 TGN 所需的时间长 12 倍。在稳定状态下，从头合成和 TGN 生成的带有 HA 表位标记的 Och1p 驻留在一个隔室中，其浮力密度与野生型 Och1p 相同，但与液泡或 TGN 的浮力密度不同。最后，表达 Ochlp 融合构建体的 och1 无效细胞已知能够以与野生型细胞相同的程度快速遇到 TGN 糖基化转化酶，表明它们具有表型野生型 Och1p 活性。这些结果使我们提出了一种 Och1p-HA 定位模型，该模型涉及移动到远端隔室，至少远至 TGN，然后通过囊泡运输恢复到顺式隔室。
To analyze the mechanism of integral membrane protein localization in the early Golgi apparatus of Saccharomyces cerevisiae, we have used Och1p, a cis-Golgi mannosyltransferase. A series of influenza virus hemagglutinin (HA) epitope-tagged fusion proteins was constructed in which invertase is appended to the Golgi-luminal carboxy terminus of full-length Och1p. Several constructs included a Kex2p cleavage site between the Och1p and invertase moieties to monitor transit to the Kex2p-containing TGN. Cells expressing an Och1p-invertase fusion do not secrete invertase, but those expressing an Och1p-Kex2p site-invertase fusion protein secrete high levels of invertase in a Kex2p-dependent manner. The Och1p-Kex2p site-invertase fusion protein is cleaved with a half-time of 5 min, and the process proceeds to completion. Before cleavage the protein receives glycosyl modifications indicative of passage through the medial- and trans-Golgi, therefore cleavage occurs after ordered anterograde transport through the Golgi to the TGN. Transit to distal compartments is not induced by the invertase moiety, since noninvertase fusion constructs encounter the same glycosyltransferases and Kex2p as well. The Och1p-HA moiety, irrespective of whether it is generated by cleavage of the fusion protein in the TGN or synthesized de novo, is degraded with a half-time of about 60 min. Thus, the half-time of degradation is 12-fold longer than the time required to reach the TGN. At steady state, de novo- synthesized and TGN-generated HA epitope-tagged Och1p reside in a compartment with a buoyant density identical to that of wild-type Och1p and distinct from that of the vacuole or the TGN. Finally, och1 null cells that express an Ochlp fusion construct known to rapidly encounter the TGN glycosylate invertase to the same extent as wild-type cells, indicating that they have phenotypically wild-type Och1p activity. These results lead us to propose a model for Och1p-HA localization that involves movement to distal compartments, at least as far as the TGN, followed by retrieval to the cis compartment, presumably by vesicular transport.