The Rab5 Effector Rabankyrin-5 Regulates and Coordinates Different Endocytic Mechanisms

The small GTPase Rab5 is a key regulator of clathrin-mediated endocytosis. On early endosomes, within a spatially restricted domain enriched in phosphatydilinositol-3-phosphate [PI(3)P], Rab5 coordinates a complex network of effectors that functionally cooperate in membrane tethering, fusion, and organelle motility. Here we discovered a novel PI(3)P-binding Rab5 effector, Rabankyrin-5, which localises to early endosomes and stimulates their fusion activity. In addition to early endosomes, however, Rabankyrin-5 localises to large vacuolar structures that correspond to macropinosomes in epithelial cells and fibroblasts. Overexpression of Rabankyrin-5 increases the number of macropinosomes and stimulates fluid-phase uptake, whereas its downregulation inhibits these processes. In polarised epithelial cells, this function is primarily restricted to the apical membrane. Rabankyrin-5 localises to large pinocytic structures underneath the apical surface of kidney proximal tubule cells, and its overexpression in polarised Madin-Darby canine kidney cells stimulates apical but not basolateral, non-clathrin-mediated pinocytosis. In demonstrating a regulatory role in endosome fusion and (macro)pinocytosis, our studies suggest that Rab5 regulates and coordinates different endocytic mechanisms through its effector Rabankyrin-5. Furthermore, its active role in apical pinocytosis in epithelial cells suggests an important function of Rabankyrin-5 in the physiology of polarised cells.

小GTP酶Rab5（small GTPase Rab5）是网格蛋白介导的内吞作用（clathrin-mediated endocytosis）的关键调控因子。在早期内体（early endosomes）上，于富含磷脂酰肌醇-3-磷酸[PI(3)P]（phosphatydilinositol-3-phosphate [PI(3)P]）的空间受限结构域内，Rab5可协调形成一套复杂的效应因子网络，这些效应因子在膜拴连（membrane tethering）、膜融合（fusion）及细胞器运动（organelle motility）中发挥功能性协同作用。本研究发现了一种新型PI(3)P结合型Rab5效应因子——Rabankyrin-5，该因子定位于早期内体并可促进其融合活性。然而除早期内体外，Rabankyrin-5还定位于上皮细胞（epithelial cells）与成纤维细胞（fibroblasts）中对应于巨胞饮体（macropinosomes）的大型液泡结构。过表达（Overexpression）Rabankyrin-5可增加巨胞饮体的数量并促进液相胞吞（fluid-phase uptake）过程，而下调（downregulation）该因子则会抑制上述进程。在极化上皮细胞（polarised epithelial cells）中，该功能主要局限于顶膜（apical membrane）。Rabankyrin-5定位于肾近端小管细胞（kidney proximal tubule cells）顶膜下方的大型胞饮结构中，在极化的马-达二氏犬肾（Madin-Darby canine kidney）细胞中过表达该因子，可促进顶膜而非基底外侧膜的非网格蛋白介导胞吞作用（non-clathrin-mediated pinocytosis）。本研究证实Rabankyrin-5在内体融合及（巨）胞饮作用中发挥调控作用，表明Rab5可通过其效应因子Rabankyrin-5调控并协调不同的内吞机制。此外，该因子在上皮细胞顶膜胞吞过程中的主动作用，提示Rabankyrin-5在极化细胞的生理过程中具有重要功能。