Complexes of syndapin II with dynamin II promote vesicle formation at the trans-Golgi network
Complexes of syndapin II with dynamin II promote vesicle formation at the trans-Golgi network
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DOI：
10.1242/jcs.02877
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发表时间：
2006-04-15
影响因子：
4
通讯作者：
Qualmann, B
中科院分区：
文献类型：
--
作者：
Kessels, MM;Dong, JX;Qualmann, B
动力素和所谓的辅助蛋白在内吞作用中的作用已经得到了充分的证实。然而，动力素II在高尔基体中的功能(S)的分子细节在很大程度上还不清楚。我们证明了普遍表达的Syndapin II亚型通过其Src同源3(SH3)结构域与Dynamin II的富含脯氨酸的结构域(PRD)相互作用。内源性Syndapin II和Dynamin II的免疫共沉淀以及此类复合体在COS-7细胞膜上的成功重组，表明了这种相互作用在体内的相关性。Syndapin II可与高尔基体膜结合，这种结合在高尔基体出口阻断时增强。Brefeldin A处理清楚地表明，观察到的Syndapin II与Synaxin 6共存的核周定位反映了高尔基复合体，这需要高尔基体的功能完整性。突触素对高尔基体小泡的形成至关重要，因为用于体外重组或活细胞中的抗突触素抗体抑制了这一过程。这两种检测还揭示了Syndapin II SH3与Dynamin II PRD相互作用在囊泡形成中的重要作用。过量的Syndapin SH3结构域在体外强烈抑制高尔基体膜的萌发。同样，过表达Syndapin SH3结构域或不能与Syndapin II(Dynamin II Delta PRD)结合的Dynamin II变异体，会削弱水泡性口炎病毒糖蛋白(VSVG)-GFP在体内的转运。相反，全长Syndapin II-L没有负面影响，反而促进了VSVG-GFP从高尔基体输出。重要的是，含有内源性突触素-动力蛋白复合体的胞质组分足以促进高尔基体膜以突触不依赖的方式形成囊泡。因此，Syndapin-Dynamin复合体对于促进跨高尔基体网络的囊泡形成至关重要。
The role of dynamin and so-called accessory proteins in endocytosis is well established. However, molecular details of the function(s) of dynamin II at the Golgi are largely unclear. We demonstrate that the ubiquitously expressed syndapin II isoform interacts with the proline-rich domain (PRD) of dynamin II through its Src-homology 3 (SH3) domain. Co-immunoprecipitation of endogenous syndapin II and dynamin II, and successful reconstitutions of such complexes at membranes in COS-7 cells, show the in vivo relevance of the interaction. Syndapin II can associate with Golgi membranes and this association increases upon Golgi exit block. Brefeldin A treatment clearly shows that the observed perinuclear localization of syndapin II colocalizing with syntaxin 6 reflects the Golgi complex and that it requires functional integrity of the Golgi. Syndapins are crucial for Golgi vesicle formation because anti-syndapin antibodies, used either in in vitro reconstitutions or in living cells, inhibited this process. Both types of assays additionally revealed the essential role of syndapin II SH3 interactions with the dynamin II PRD in vesicle formation. An excess of the syndapin SH3 domain strongly inhibited budding from Golgi membranes in vitro. Likewise, overexpression of the syndapin SH3 domain or of a dynamin II variant incapable of associating with syndapin II (dynamin II Delta PRD) impaired trafficking of vesicular stomatitis virus glycoprotein (VSVG)-GFP in vivo. By contrast, full-length syndapin II-l had no negative effect, and instead promoted VSVG-GFP export from the Golgi. Importantly, a cytosolic fraction containing endogenous syndapin-dynamin complexes was sufficient to promote vesicle formation from Golgi membranes in a syndapindependent manner. Thus, syndapin-dynamin complexes are crucial and sufficient to promote vesicle formation from the trans-Golgi network.