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

Abstract

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.

Vaccinia Virus Infection Requires Maturation of Macropinosomes

“…The first conversion that macropinosomes undergo after fission from the plasma membrane is the recruitment of EE factors such as Rab5 and its effector EEA1 as well as the accumulation of PtdIns(3)P 17, 18, 20. We observed MVs in Rab5a‐positive, and to a lesser extent in EEA1‐positive, vacuoles already 5 min after warming.…”

“…After detachment from the plasma membrane, the PtdIns(3)P on newly formed macropinosomes is replaced by PtdIns(3,4,5)P 3 17. Rab5 accumulates together with its GEF, Rabex5 and effector Rabankyrin 5 17, 18. Macropinosomes may also acquire Rab34 and early endosome antigen 1 (EEA1) needed for pinosome formation and fusion 19, 20.…”

Differential phosphorylation of the phosphoinositide 3-phosphatase MTMR2 regulates its association with early endosomal subtypes

“…We postulated that the partial colocalization with Rab5 may be due to the phosphatase activity of MTMR2 since many Rab5 effectors require PI(3)P for localizing to Rab5-positive endosomes Merithew et al, 2003;Schnatwinkel et al, 2004;Zerial and McBride, 2001). Therefore we created a MTMR2 phosphatase inactive mutant in the S58A/S631E background and reassessed the ability to colocalize to Rab5-positive and APPL1-positive endosomes.…”

“…Furthermore, Rab5 collaborates with numerous peripheral endosome binding proteins to facilitate fusion of endosome membranes including Rab5 effectors whose endosome targeting requires association with endosomal PI(3)P Merithew et al, 2003;Schnatwinkel et al, 2004;Zerial and McBride, 2001). This property reveals that the endosomal activities of Rab5 can be dramatically affected by PI(3)P levels.…”

“…This property reveals that the endosomal activities of Rab5 can be dramatically affected by PI(3)P levels. For example, the critical endosomal effectors Rabenosyn-5 and Early Endosomal Antigen 1 (EEA1) are essential tethering factors important for endosome fusion events (Merithew et al, 2003;Schnatwinkel et al, 2004;Zerial and McBride, 2001). These effectors require association with Rab5 and PI(3)P simultaneously to localize to early endosomes.…”