Active recycling of yeast Golgi mannosyltransferase complexes through the endoplasmic reticulum.
Active recycling of yeast Golgi mannosyltransferase complexes through the endoplasmic reticulum.
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DOI：
10.1073/pnas.250472397
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发表时间：
2000-12
影响因子：
11.1
通讯作者：
Zlatko Todorow;A. Spang;E. Carmack;John R. Yates;Randy W. Schekman
中科院分区：
文献类型：
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作者：
Zlatko Todorow;A. Spang;E. Carmack;John R. Yates;Randy W. Schekman
Mnn9p是酿酒酵母顺式高尔基酵母中两种不同的多蛋白复合物的组成部分，这两种复合物在体外均具有α -1,6-甘露糖基转移酶活性。在其中一个复合体中，Mnn9p与其他四种膜蛋白Anp1p、Mnn10p、Mnn11p和Hoc1p结合，而另一个复合体由Mnn9p和Van1p组成。含mnn9p复合物的成员被纳入体外COPII囊泡中，这些囊泡是由环己亚胺处理的细胞分离的内质网（ER）膜制成的。这种行为与活跃的高尔基体到ER的回收过程是一致的。为了在体内检查这一途径，我们监测了在内质网逆行运输中受阻的细胞中复合物亚基的逆行运输。在这种情况下，在没有新蛋白质合成的情况下，观察到蛋白质从高尔基体特异性重新定位到内质网。相反，当体内的逆行转运被阻断时，甘露糖基转移酶复合物的亚基在液泡中积累。利用涂层依赖的囊泡出芽试验，研究了纯化高尔基膜的copi包被囊泡中Mnn9p的包装。梯度分选实验表明Mnn9p和逆行v-SNARE Sec22p被纳入copi包被的囊泡中。这些观察结果表明，含有mnn9p的甘露糖基转移酶复合物在内质网和高尔基体之间来回循环。
Mnn9p is a component of two distinct multiprotein complexes in the Saccharomyces cerevisiae cis-Golgi that have both been shown to have alpha-1,6-mannosyltransferase activity in vitro. In one of these complexes, Mnn9p associates with four other membrane proteins, Anp1p, Mnn10p, Mnn11p, and Hoc1p, whereas the other complex consists of Mnn9p and Van1p. Members of the Mnn9p-containing complexes were incorporated into COPII vesicles made in vitro from endoplasmic reticulum (ER) membranes isolated from cycloheximide-treated cells. This behavior is consistent with an active Golgi to ER recycling process. To examine this path in vivo, we monitored retrograde transport of subunits of the complex in cells blocked in anterograde transport from the ER. In this situation, specific relocation of the proteins from the Golgi to the ER was observed in the absence of new protein synthesis. Conversely, when retrograde transport was blocked in vivo, subunits of the mannosyltransferase complex accumulated in the vacuole. Packaging of Mnn9p in COPI-coated vesicles from purified Golgi membranes was also investigated using a coatomer-dependent vesicle budding assay. Gradient fractionation experiments showed that Mnn9p and the retrograde v-SNARE, Sec22p, were incorporated into COPI-coated vesicles. These observations indicate that the Mnn9p-containing mannosyltransferase complexes cycle back and forth between the ER and Golgi.