Department of Physiology and Biophysics, University of Arkansas for Medical Sciences Little Rock, AR, USA.
Front Cell Dev Biol. 2016 Mar 1;4:13. doi: 10.3389/fcell.2016.00013. eCollection 2016.
Unexpectedly, members of the Rab VI subfamily exhibit considerable variation in their effects on Golgi organization and trafficking. By fluorescence microscopy, neither depletion nor overexpression of the GDP-locked form of Rab6a/a', the first trans Golgi-associated Rab protein discovered, affects Golgi ribbon organization while, on the other hand, both Rab41/6d depletion and overexpression of GDP-locked form cause Golgi fragmentation into a cluster of punctate elements, suggesting that Rab41/6d has an active role in maintenance of Golgi ribbon organization. To establish a molecular basis for these differences, we screened for Rab41/6d interacting proteins by yeast two-hybrid assay. 155 non-repetitive hits were isolated and sequenced, and after searching in NCBI database, 102 different proteins and protein fragments were identified. None of these hits overlapped with any published Rab6a/a' effector. Eight putative Rab41 interactors involved in membrane trafficking were found. Significantly, these exhibited a preferential interaction with GTP- vs. GDP-locked Rab41/6d. Of the 8 hits, the dynactin 6, syntaxin 8, and Kif18A plasmids were the only ones expressing the full-length protein. Hence, these 3 proteins were selected for further study. We found that depletion of dynactin 6 or syntaxin 8, but not Kif18A, resulted in a fragmented Golgi apparatus that displayed a Rab41/6d knockdown phenotype, i.e., the Golgi apparatus was disrupted into a cluster of punctate Golgi elements. Co-immunoprecipation experiments verified that the interaction of dynactin 6 and syntaxin 8 with GTP-locked Rab41/6d was stronger than that with wild type Rab41/6d and least with the GDP-locked form. In contrast, co-immunoprecipitation interaction with Rab6a was greatest with the GDP-locked Rab6a, suggestive of a non-physiological interaction. In conclusion, we suggest that dynactin 6, a subunit of dynactin complex, the minus-end-directed, dynein motor, provides a sufficient molecular basis to explain the active role of Rab41/6d in maintaining Golgi ribbon organization while syntaxin 8 contributes more indirectly to Golgi positioning.
出人意料的是，Rab VI 亚家族的成员在其对高尔基体组织和运输的影响方面表现出相当大的差异。通过荧光显微镜观察，第一个被发现的跨高尔基体相关 Rab 蛋白 Rab6a/a'的 GDP 锁定形式的耗尽或过表达均不影响高尔基体带的组织，而另一方面，Rab41/6d 的耗尽和 GDP 锁定形式的过表达均导致高尔基体碎裂成一群点状元件，这表明 Rab41/6d 在维持高尔基体带的组织中具有活跃的作用。为了为这些差异建立分子基础，我们通过酵母双杂交测定筛选 Rab41/6d 的相互作用蛋白。分离并测序了 155 个非重复的阳性克隆，在 NCBI 数据库中搜索后，鉴定出 102 种不同的蛋白质和蛋白质片段。这些阳性克隆中没有一个与任何已发表的 Rab6a/a'效应子重叠。发现了 8 种涉及膜运输的推定 Rab41 相互作用蛋白。值得注意的是，这些蛋白与 GTP-而非 GDP-锁定的 Rab41/6d 优先相互作用。在 8 个阳性克隆中，dynactin 6、syntaxin 8 和 Kif18A 质粒是唯一表达全长蛋白的质粒。因此，选择这 3 种蛋白进行进一步研究。我们发现，dynactin 6 或 syntaxin 8 的耗竭而不是 Kif18A 的耗竭导致高尔基体碎裂，显示出 Rab41/6d 敲低表型，即高尔基体被破坏成一群点状高尔基体元件。免疫共沉淀实验验证了 dynactin 6 和 syntaxin 8 与 GTP-锁定 Rab41/6d 的相互作用强于与野生型 Rab41/6d 的相互作用，与 GDP-锁定形式的相互作用最弱。相比之下，与 Rab6a 的免疫共沉淀相互作用以 GDP 锁定的 Rab6a 最强，提示存在非生理相互作用。总之，我们认为 dynactin 6，dynactin 复合物的一个亚基，是负向指向的，动力蛋白，为解释 Rab41/6d 在维持高尔基体带的组织中的活跃作用提供了充分的分子基础，而 syntaxin 8 对高尔基体定位的贡献更间接。
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