Kim Hon-Song, Takahashi Mikiko, Matsuo Kazuhiko, Ono Yoshitaka
Graduate School of Science and Technology, Kobe University, Kobe 657-8501, Japan.
Genes Cells. 2007 Mar;12(3):421-34. doi: 10.1111/j.1365-2443.2007.01055.x.
The structural organization and position of the Golgi apparatus are highly regulated by microtubule cytoskeleton and microtubule motor proteins. The mechanisms linking these proteins to the Golgi apparatus remain elusive. Here, we found that centrosome and Golgi-localized PKN associated protein (CG-NAP) was localized to the Golgi apparatus in a microtubule-dependent manner. Microtubule-binding experiments revealed that CG-NAP possessed two microtubule-binding domains. We also found that CG-NAP was well co-localized with cytoplasmic dynein subunits during recovery from the on-ice treatment of cells that induced dissociation of CG-NAP from the Golgi. Similar co-localization was observed during recovery from the acetate treatment, which has been reported to inhibit the dynein-mediated transport. CG-NAP was co-immunoprecipitated with a dynactin subunit p150(Glued). Expressing the p150(Glued)-binding region of CG-NAP fused with mitochondria-targeting sequence induced recruitment of mitochondria to the pericentriolar area, suggesting that this region interacts with functional cytoplasmic dynein in vivo. Moreover, over-expression of this region caused fragmentation of the Golgi similar to that of dynamitin. These results suggest that CG-NAP is recruited to the minus ends of microtubules by interacting with cytoplasmic dynein, thereby localizes to the Golgi apparatus in a microtubule-dependent manner and possibly involved in the formation of the Golgi near the centrosomes.
高尔基体的结构组织和位置受到微管细胞骨架和微管运动蛋白的高度调控。将这些蛋白与高尔基体联系起来的机制仍然不清楚。在这里，我们发现中心体和高尔基体定位的PKN相关蛋白（CG-NAP）以微管依赖的方式定位于高尔基体。微管结合实验表明，CG-NAP具有两个微管结合结构域。我们还发现，在诱导CG-NAP从高尔基体解离的细胞冰上处理恢复过程中，CG-NAP与细胞质动力蛋白亚基共定位良好。在乙酸处理恢复过程中也观察到类似的共定位，据报道乙酸处理会抑制动力蛋白介导的运输。CG-NAP与动力蛋白激活蛋白亚基p150（Glued）共免疫沉淀。表达与线粒体靶向序列融合的CG-NAP的p150（Glued）结合区域会诱导线粒体向中心粒周围区域募集，这表明该区域在体内与功能性细胞质动力蛋白相互作用。此外，该区域的过表达导致高尔基体碎片化，类似于动力蛋白抑制蛋白的作用。这些结果表明，CG-NAP通过与细胞质动力蛋白相互作用被募集到微管的负端，从而以微管依赖的方式定位于高尔基体，并可能参与中心体附近高尔基体的形成。
