Annexin II is a major component of fusogenic endosomal vesicles.
Annexin II is a major component of fusogenic endosomal vesicles.
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DOI：
10.1083/jcb.120.6.1357
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发表时间：
1993-03
期刊：
影响因子：
--
通讯作者：
Gruenberg J
中科院分区：
文献类型：
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作者：
Emans N;Gorvel JP;Walter C;Gerke V;Kellner R;Griffiths G;Gruenberg J
我们已经使用体外测定来跟踪早期内体融合后从供体转移到受体的蛋白质。受体是在珠上免疫分离的纯化的早期内体部分，供体是悬浮液中的代谢标记的早期内体部分。在测定中，在未标记的胞质溶胶的存在下混合两种级分，然后回收并洗涤珠。通过二维凝胶电泳和放射自显影对转移到受体的供体蛋白进行鉴定。转移了大约50种主要蛋白质，这种转移满足了为体外内体融合建立的所有标准。然而，如果在测定前对供体内体进行简单超声处理以产生小（0.1微米直径）囊泡，则仅一小部分蛋白质被有效转移。这些包括两个酸性膜蛋白，和三个碱性外周蛋白暴露在细胞质面的膜。部分测序和蛋白质印迹表明，后者的组成部分之一是膜联蛋白II，一种已知介导膜-膜相互作用的蛋白质。免疫金标记的冷冻切片证实，膜联蛋白II是存在于体内早期内涵体。这些数据表明，膜联蛋白II，连同其他四种蛋白质，我们已经确定，是一个融合的内体囊泡的主要组成部分，这表明这些蛋白质参与的结合和/或融合过程。
We have used an in vitro assay to follow the proteins transferred from a donor to an acceptor upon fusion of early endosomes. The acceptor was a purified early endosomal fraction immunoisolated on beads and the donor was a metabolically-labeled early endosomal fraction in suspension. In the assay, both fractions were mixed in the presence of unlabeled cytosol, and then the beads were retrieved and washed. The donor proteins transferred to the acceptor were identified by two- dimensional gel electrophoresis and autoradiography. Approximately 50 major proteins were transferred and this transfer fulfilled all criteria established for endosome fusion in vitro. However, only a small subset of proteins was efficiently transferred, if donor endosomes were briefly sonicated to generate small (0.1 micron diam) vesicles before the assay. These include two acidic membrane proteins, and three alkaline peripheral proteins exposed on the cytoplasmic face of the membrane. Partial sequencing and Western blotting indicated that one of the latter components is annexin II, a protein known to mediate membrane-membrane interactions. Immunogold labeling of cryosections confirmed that annexin II is present on early endosomes in vivo. These data demonstrate that annexin II, together with the other four proteins we have identified, is a major component of fusogenic endosomal vesicles, suggesting that these proteins are involved in the binding and/or fusion process.