Laboratory of Membrane Trafficking Mechanisms, Department of Integrative Life Sciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi 980-8578, Japan.
J Cell Sci. 2023 Mar 15;136(6). doi: 10.1242/jcs.260522. Epub 2023 Mar 8.
The small GTPase Rab22A is an important regulator of the formation of tubular endosomes, which are one of the types of recycling endosome compartments of the clathrin-independent endocytosis pathway. In order to regulate tubular endosome formation, Rab22A must be activated by a specific guanine-nucleotide-exchange factor (GEF); however, all of the GEFs that have been reported to exhibit Rab22A-GEF activity in vitro also activate Rab5A, an essential regulator of the clathrin-mediated endocytosis pathway, and no Rab22A-specific GEF has ever been identified. Here, we identified Vps9d1, a previously uncharacterized vacuolar protein sorting 9 (VPS9) domain-containing protein, as a novel Rab22A-GEF. The formation of tubular endosome structures was found to be severely impaired in Vps9d1-depleted HeLa cells, but Rab5A localization was unaffected. Expression of a constitutively active Rab22A mutant in Vps9d1-depleted HeLa cells restored tubular endosomes, but expression of a GEF-activity-deficient Vps9d1 mutant did not. Moreover, Vps9d1 depletion altered the distribution of clathrin-independent endocytosed cargos and impaired their recycling. Our findings indicate that Vps9d1 promotes tubular endosome formation by specifically activating Rab22A.
小分子 GTPase Rab22A 是管状内体形成的重要调节剂，管状内体是网格蛋白非依赖型内吞作用途径的再循环内体隔室之一。为了调节管状内体的形成，Rab22A 必须被特定的鸟嘌呤核苷酸交换因子（GEF）激活；然而，所有在体外表现出 Rab22A-GEF 活性的 GEFs也激活 Rab5A，Rab5A 是网格蛋白介导的内吞作用途径的关键调节剂，并且从未鉴定出 Rab22A 特异性的 GEF。在这里，我们鉴定了 Vps9d1，一种以前未被表征的液泡蛋白分选 9（VPS9）结构域蛋白，为一种新的 Rab22A-GEF。发现 Vps9d1 耗尽的 HeLa 细胞中管状内体结构的形成严重受损，但 Rab5A 定位不受影响。在 Vps9d1 耗尽的 HeLa 细胞中表达组成型活性 Rab22A 突变体恢复了管状内体，但表达 GEF 活性缺陷的 Vps9d1 突变体则没有。此外，Vps9d1 耗尽改变了无网格蛋白内吞作用载体的分布，并损害了它们的回收。我们的研究结果表明，Vps9d1 通过特异性激活 Rab22A 促进管状内体的形成。
