Department of Biochemistry and Molecular Biology, Peggy and Charles Stephenson Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
Department of Biochemistry and Molecular Biology, Peggy and Charles Stephenson Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
J Biol Chem. 2022 Sep;298(9):102281. doi: 10.1016/j.jbc.2022.102281. Epub 2022 Jul 19.
Rab22 and Rab31 belong to the Rab5 subfamily of GTPases that regulates endocytic traffic and endosomal sorting. Rab22 and Rab31 (a.k.a. Rab22b) are closely related and share 87% amino acid sequence similarity, but they show distinct intracellular localization and function in the cell. Rab22 is localized to early endosomes and regulates early endosomal recycling, while Rab31 is mostly localized to the Golgi complex with only a small fraction in the endosomes at steady state. The specific determinants that affect this differential localization, however, are unclear. In this study, we identify a novel membrane targeting domain (MTD) consisting of the C-terminal hypervariable domain (HVD), interswitch loop (ISL), and N-terminal domain as a major determinant of endosomal localization for Rab22 and Rab31, as well as Rab5. Rab22 and Rab31 share the same N-terminal domain, but we find Rab22 chimeras with Rab31 HVD exhibit phenotypic Rab31 localization to the Golgi complex, while Rab31 chimeras with the Rab22 HVD localize to early endosomes, similar to wildtype Rab22. We also find that the Rab22 HVD favors interaction with the early endosomal effector protein Rabenosyn-5, which may stabilize the Rab localization to the endosomes. The importance of effector interaction in endosomal localization is further demonstrated by the disruption of Rab22 endosomal localization in Rabenosyn-5 knockout cells and by the shift of Rab31 to the endosomes in Rabenosyn-5-overexpressing cells. Taken together, we have identified a novel MTD that mediates localization of Rab5 subfamily members to early endosomes via interaction with an effector such as Rabenosyn-5.
Rab22 和 Rab31 属于 Rab5 亚家族 GTP 酶，调节内吞运输和内体分拣。Rab22 和 Rab31（又名 Rab22b）密切相关，氨基酸序列相似度为 87%，但它们在细胞内的定位和功能明显不同。Rab22 定位于早期内体，调节早期内体再循环，而 Rab31 主要定位于高尔基体复合物，在稳定状态下只有一小部分位于内体中。然而，影响这种差异定位的特定决定因素尚不清楚。在这项研究中，我们确定了一个新的膜靶向结构域（MTD），由 C 端高变区（HVD）、开关环（ISL）和 N 端结构域组成，该结构域是 Rab22 和 Rab31 以及 Rab5 内体定位的主要决定因素。Rab22 和 Rab31 共享相同的 N 端结构域，但我们发现 Rab22 嵌合体具有 Rab31 HVD 表现出 Rab31 定位于高尔基体复合物的表型，而 Rab31 嵌合体具有 Rab22 HVD 则定位于早期内体，类似于野生型 Rab22。我们还发现，Rab22 HVD 有利于与早期内体效应蛋白 Rabenosyn-5 相互作用，这可能稳定 Rab 向内体的定位。效应物相互作用在内体定位中的重要性进一步通过在 Rabenosyn-5 敲除细胞中破坏 Rab22 内体定位和在 Rabenosyn-5 过表达细胞中 Rab31 向内体转移得到证明。总之，我们已经确定了一个新的 MTD，它通过与 Rabenosyn-5 等效应物相互作用，介导 Rab5 亚家族成员向早期内体的定位。
