Hara Shigeo, Kiyokawa Etsuko, Iemura Shun-ichiro, Natsume Tohru, Wassmer Thomas, Cullen Peter J, Hiai Hiroshi, Matsuda Michiyuki
Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.
Mol Biol Cell. 2008 Sep;19(9):3823-35. doi: 10.1091/mbc.e08-03-0314. Epub 2008 Jul 2.
DOCK180 is the archetype of the DOCK180-family guanine nucleotide exchange factor for small GTPases Rac1 and Cdc42. DOCK180-family proteins share two conserved domains, called DOCK homology region (DHR)-1 and -2. Although the function of DHR2 is to activate Rac1, DHR1 is required for binding to phosphoinositides. To better understand the function of DHR1, we searched for its binding partners by direct nanoflow liquid chromatography/tandem mass spectrometry, and we identified sorting nexins (SNX) 1, 2, 5, and 6, which make up a multimeric protein complex mediating endosome-to-trans-Golgi-network (TGN) retrograde transport of the cation-independent mannose 6-phosphate receptor (CI-MPR). Among these SNX proteins, SNX5 was coimmunoprecipitated with DOCK180 most efficiently. In agreement with this observation, DOCK180 colocalized with SNX5 at endosomes. The RNA interference-mediated knockdowns of SNX5 and DOCK180, but not Rac1, resulted in the redistribution of CI-MPR from TGN to endosomes. Furthermore, expression of the DOCK180 DHR1 domain was sufficient to restore the perturbed CI-MPR distribution in DOCK180 knockdown cells. These data suggest that DOCK180 regulates CI-MPR trafficking via SNX5 and that this function is independent of its guanine nucleotide exchange factor activity toward Rac1.
DOCK180是小GTP酶Rac1和Cdc42的DOCK180家族鸟嘌呤核苷酸交换因子的原型。DOCK180家族蛋白共享两个保守结构域，称为DOCK同源区域（DHR）-1和-2。虽然DHR2的功能是激活Rac1，但DHR1是与磷酸肌醇结合所必需的。为了更好地理解DHR1的功能，我们通过直接纳流液相色谱/串联质谱法寻找其结合伙伴，并鉴定出分选连接蛋白（SNX）1、2、5和6，它们组成了一个多聚体蛋白复合物，介导阳离子非依赖性甘露糖6-磷酸受体（CI-MPR）从内体到反式高尔基体网络（TGN）的逆行运输。在这些SNX蛋白中，SNX5与DOCK180的共免疫沉淀效率最高。与这一观察结果一致，DOCK180与SNX5在内体中共定位。RNA干扰介导的SNX5和DOCK180而非Rac1的敲低导致CI-MPR从TGN重新分布到内体。此外，DOCK180 DHR1结构域的表达足以恢复DOCK180敲低细胞中受干扰的CI-MPR分布。这些数据表明，DOCK180通过SNX5调节CI-MPR的运输，并且该功能独立于其对Rac1的鸟嘌呤核苷酸交换因子活性。
