Spatiotemporal dynamics of the COPI vesicle machinery
Spatiotemporal dynamics of the COPI vesicle machinery
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DOI：
10.1038/sj.embor.embor942
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发表时间：
2003-10-01
期刊：
影响因子：
7.7
通讯作者：
Weiss, M
中科院分区：
文献类型：
--
作者：
Elsner, M;Hashimoto, H;Weiss, M
外壳蛋白I（COPI）囊泡外壳的组装由小GTP酶ADP核糖基化因子1（ARF 1）及其GTP酶激活蛋白ARFGAP 1控制。在这里，我们调查的扩散行为的coatomer，涂层的主要成分，也是那些ARF 1和ARFGAP 1。使用荧光相关光谱，我们发现大多数ARF 1和ARFGAP 1分子在胞质溶胶中是高度移动的（扩散常数D约为15 μ m（2）s（-1）），而涂层分子的扩散比预期慢5 - 10倍（D约为1 μ m（2）s（-1））。这种缓慢的扩散导致高尔基体膜的扩散限制结合动力学，在FRAP（光漂白后的荧光恢复）实验中，转化为两倍慢的结合速率。氟化铝的加入将coatomer锁定在高尔基体膜上，并且还降低了ARF 1和ARFGAP 1的结合动力学，这表明这些蛋白质协同作用以介导分选和囊泡形成。
Assembly of the coat protein I ( COPI) vesicle coat is controlled by the small GTPase ADP ribosylation factor 1 (ARF1) and its GTPase-activating protein, ARFGAP1. Here, we investigate the diffusional behaviours of coatomer, the main component of the coat, and also those of ARF1 and ARFGAP1. Using fluorescence-correlation spectroscopy, we found that most ARF1 and ARFGAP1 molecules are highly mobile in the cytosol ( diffusion constant D approximate to 15 mum(2) s(-1)), whereas coatomer diffuses 5 - 10 times more slowly than expected (D approximate to 1 mum(2) s(-1)). This slow diffusion causes diffusion-limited binding kinetics to Golgi membranes, which, in FRAP ( fluorescence recovery after photobleaching) experiments, translates into a twofold slower binding rate. The addition of aluminium fluoride locks coatomer onto Golgi membranes and also decreases the binding kinetics of both ARF1 and ARFGAP1, suggesting that these proteins function in concert to mediate sorting and vesicle formation.