Many receptor-ligand complexes are internalized and delivered to the endosomal pathway, allowing the receptors to be recycled and the ligands degraded. The lumenal pH of various endosomal compartments is acidic; this acidic pH, which is generated by a proton-pumping vacuolar ATPase (V-ATPase), is required for formation of an endosomal protein coat and transport along the endocytic pathway. This depends on the pH-sensitive recruitment of the small GTPase Arf6 and the guanine nucleotide exchange factor ARNO; however, the mechanisms governing their recruitment have been unclear (see Recchi and Chavrier). The V-ATPase is a multisubunit complex with a number of transmembrane and cytosolic subunits; Hurtado-Lorenzo et al. found that the a2-isoform of the transmembrane a-subunit was specifically targeted to early endosomes of mouse renal proximal tubule cells. Co-immunoprecipitation, pull-down, and mammalian two-hybrid analyses indicated that a2 interacted with ARNO; this interaction involved the a2 N-terminal cytosolic tail. Arf6, on the other hand, interacted with the V-ATPase transmembrane c-subunit. These interactions were found in HeLa cells, human embryonic kidney (HEK) cells, and mouse tubular cells (MTCs), indicating that they were widespread. Pharmacological blockade of endosomal acidification inhibited interaction between ARNO and a2, as well as inhibited albumin endocytosis. This specifically involved disruption of trafficking from early to late endosomes in the degradative pathway. Disruption of the a2-ARNO interaction by overexpression of a fluorescently labeled soluble a2 tail, which sequestered ARNO, also inhibited endocytosis. Endocytosis was rescued by expression of an interaction-competent but not an interaction-deficient ARNO mutant. Thus, the authors propose that the V-ATPase not only is responsible for endosomal acidification but also acts as the pH sensor that enables recruitment of cytosolic proteins and the subsequent trafficking of endosomes.
A. Hurtado-Lorenzo, M. Skinner, J. El Annan, M. Futai, G.-H. Sun-Wada, S. Bourgoin, J. Casanova, A. Wildeman, S. Bechoua, D. A. Ausiello, D. Brown, V. Marshansky, V-ATPase interacts with ARNO and Arf6 in early endosomes and regulates the protein degradative pathway. Nat. Cell Biol. 8, 124-136 (2006). [PubMed]
C. Recchi, P. Chavrier, V-ATPase: A potential pH sensor. Nat. Cell Biol. 8, 107-109 (2006). [PubMed]