Derby Merran C, Lieu Zi Zhao, Brown Darren, Stow Jennifer L, Goud Bruno, Gleeson Paul A
Department of Biochemistry and Molecular Biology, University of Melbourne, Melbourne, Victoria 3010, Australia, and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Melbourne, Victoria 3010, Australia.
Traffic. 2007 Jun;8(6):758-73. doi: 10.1111/j.1600-0854.2007.00563.x. Epub 2007 May 4.
Four mammalian golgins are specifically targeted to the trans-Golgi network (TGN) membranes via their C-terminal GRIP domains. The TGN golgins, p230/golgin-245 and golgin-97, are recruited via the GTPase Arl1, whereas the TGN golgin GCC185 is recruited independently of Arl1. Here we show that GCC185 is localized to a region of the TGN distinct from Arl1 and plays an essential role in maintaining the organization of the Golgi apparatus. Using both small interfering RNA (siRNA) and microRNA (miRNA), we show that depletion of GCC185 in HeLa cells frequently resulted in fragmentation of the Golgi apparatus. Golgi apparatus fragments were dispersed throughout the cytoplasm and contained both cis and trans markers. Trafficking of anterograde and retrograde cargo was analysed over an extended period following GCC185 depletion. Early effects of GCC185 depletion included a perturbation in the distribution of the mannose-6-phosphate receptor and a block in shiga toxin trafficking to the Golgi apparatus, which occurred in parallel with the fragmentation of the Golgi ribbon. Internalized shiga toxin accumulated in Rab11-positive endosomes, indicating GCC185 is essential for transport between the recycling endosome and the TGN. In contrast, the plasma membrane-TGN recycling protein TGN38 was efficiently transported into GCC185-depleted Golgi apparatus fragments throughout a 96-h period, and anterograde transport of E-cadherin was functional until a late stage of GCC185 depletion. This study demonstrated (i) a more effective long-term depletion of GCC185 using miRNA than siRNA and (ii) a dual role for the GCC185 golgin in the regulation of endosome-to-TGN membrane transport and in the organization of the Golgi apparatus.
四种哺乳动物的高尔基体蛋白通过其C端的GRIP结构域特异性定位于反式高尔基体网络（TGN）膜。TGN高尔基体蛋白p230/高尔基体蛋白-245和高尔基体蛋白-97通过GTP酶Arl1募集，而TGN高尔基体蛋白GCC185的募集独立于Arl1。在此我们表明，GCC185定位于TGN中与Arl1不同的区域，并在维持高尔基体的组织结构中起关键作用。使用小干扰RNA（siRNA）和微小RNA（miRNA），我们发现HeLa细胞中GCC185的缺失经常导致高尔基体的碎片化。高尔基体片段分散在整个细胞质中，同时包含顺式和反式标记物。在GCC185缺失后的较长时间内分析了顺行和逆行货物的运输。GCC185缺失的早期影响包括甘露糖-6-磷酸受体分布的紊乱以及志贺毒素向高尔基体运输的阻断，这与高尔基体带的碎片化同时发生。内化的志贺毒素积聚在Rab11阳性的内体中，表明GCC185对于回收内体和TGN之间的运输至关重要。相反，质膜-TGN循环蛋白TGN38在96小时内有效地转运到GCC185缺失的高尔基体片段中，并且E-钙粘蛋白的顺行运输在GCC185缺失的后期仍保持功能。这项研究表明：（i）使用miRNA比siRNA能更有效地长期耗尽GCC185；（ii）GCC185高尔基体蛋白在调节内体到TGN的膜运输以及高尔基体的组织中具有双重作用。
