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

Received December The Rab5 Effector Rabankyrin-5 Regulates and Coordinates Different Endocytic Mechanisms Carsten Schnatwinkel 0 Savvas Christoforidis 0 Margaret R. Lindsay 0 Sandrine Uttenweiler-Joseph 0 Matthias Wilm 0 Robert G. Parton 0 Marino Zerial 0 Peter Walter, University of California at San Francisco 0 1 Max-Planck Institute of Molecular Cell Biology and Genetics , Dresden, Germany, 2 Laboratory of Biological Chemistry , Medical School, University of Ioannina, Ioannina, Greece, 3 Institute for Molecular Bioscience, Centre for Microscopy and Microanalysis, School of Biomedical Sciences, University of Queensland , Brisbane, Queensland , Australia , 4 European Molecular Biology Laboratory , Heidelberg , Germany 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 MadinDarby 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. - In mammalian cells multiple mechanisms of endocytosis operate within a single cell to perform nutrient uptake, cellular homeostasis, neurotransmission, signal transduction, antigen presentation, and defence against pathogens. Endocytosis comprises two major categories, phagocytosis and pinocytosis, depending on the uptake of particles or fluid, respectively (reviewed in Conner and Schmid 2003). Pinocytosis encompasses various membrane entry routes and mechanisms. Clathrin-mediated endocytosis, the best-studied route at the molecular level to date, primarily serves receptor-mediated uptake. Caveolae assemble on sphingolipid and cholesterol rafts and may internalise molecules partitioning in these lipid microdomains. To a certain extent, ligand-receptor complexes and raft components can follow both entry routes but can also be internalised via clathrinand caveolae-independent endocytosis. Large volumes of fluid are engulfed by the closure of plasma membrane protrusions in a process termed macropinocytosis. Although it was the first mode of pinocytosis reported (Lewis 1931), little is known on the molecular mechanisms underlying this physiologically very important endocytic process. Constitutively active in immature dendritic cells, macropinocytosis favors antigen sampling (Steinman and Swanson 1995). However, it can be transiently induced in most cells by growth factors (Haigler et al. 1979; Shao et al. 2002), tumorpromoting chemicals such as phorbol 12-myristate 13-acetate (PMA), or oncogenes like H-Ras or v-Src (Bar-Sagi and Feramisco 1986; Veithen et al. 1996), and it has been proposed to downregulate signalling molecules from the cell surface. Since macropinosomes arise from membrane ruffles, regions of intense actin remodelling which are also a trait of motile cells, they have been implicated in directed cell locomotion (Carpentier et al. 1991). Macropinocytosis is exploited by several invasive pathogens as entry route (Francis et al. 1993; Sansonetti 1997) but differs from phagocytosis with respect to regulation (e.g., receptor mediated) and cargo (e.g., uptake and degradation of opsonised particles) (Galan and Zhou 2000). Macropinosomes are distinct from early endosomes, morphologically and Figure 1. A Protein of 130 kDa Is a New Rab5 Effector (A) GST-Rab5-GDP and GST-Rab-GTPcS were loaded on beads and incubated with bovine brain cytosol. Bound proteins were eluted and analysed by SDS-PAGE followed by Coommasie Blue staining. The positions of the already known Rab5 effectors (EEA1, Rabaptin-5, hVps34, p110b, and Rabenosyn-5) and of the new Rab5 effector are indicated. (B) Schematic representation of the domain organisation in Rabankyrin-5. ANK, ankyrin repeats. (C) Bovine brain cytosol or HeLa cell cytosol was incubated with GST-Rab5-GDP or GST-Rab5-GTPcSloaded beads. Subsequently the beads were washed, and bound proteins were eluted and analysed by Western blotting using antiRabankyrin-5 antibodies. (D) GST-Rab4, -5, -7, and -11 fusion proteins were preloaded with GDP or GTPcS and incubated with in vitro-translated 35S-methioninelabelled Rabankyrin-5 full-length protein. As a control, bound and unbound material was analysed by SDS-PAGE followed by phosphoimager analysis. (E) Rabankyrin-5 binds most strongly to PI(3)P. Recombinant full-length Rabankyrin-5 was incubated with liposomes containing 2% of the indicated phosphoinositide. Bound Rabankyrin-5 was detected by Western blotting. DOI: 10.1371/journal.pbio.0020261.g001 biochemically. In epidermal growth factor (EGF)-stimulated A431 cells, whereas macropinosomes can fuse homotypically, they seldom fuse with early endosomes (Hewlett et al. 1994). However, this partition is not absolute, since in dendritic cells macropinosomes do fuse with endosomes (Racoosin and Swanson 1993). The intracellular trafficking properties of macropinosomes may therefore be governed by cell typespecific mechanisms and fulfil specialised functions. Finally, macropinocytosis shares mechanistic features with apical fluid-phase endocytosis in polarised cells (Gottlieb et al. 1993; Jackman et al. 1994; Holm et al. 1995; Amyere et al. 2002). In kidney, apical pinocytosis contributes an essential function to the physiology of the renal system by contributing to vectorial fluid-phase transport across the membranes (Goligorsky and Hruska 1986). Given the dependence on actin remodelling, it is no surprise that Rho GTPases (West et al. 2000), ARF6 (Radhakrishna et al. 1996), and type 1 phosphatidylinositol3 kinases (PI3-Ks) (Hooshmand-Rad et al. 1997) are involved in macropinocytosis, presumably through their role in membrane ruffling. Much less clear is the function of two Rab GTPases, Rab5 and Rab34/Rah, both of which are implicated in the formation of macro (...truncated)