V-ATPase interacts with ARNO and Arf6 in early endosomes and regulates the protein degradative pathway

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The recruitment of the small GTPase Arf6 and ARNO from cytosol to endosomal membranes is driven by V-ATPase-dependent intra-endosomal acidification. The molecular mechanism that mediates this pH-sensitive recruitment and its role are unknown. Here, we demonstrate that Arf6 interacts with the c-subunit, and ARNO with the a2-isoform of V-ATPase. The a2-isoform is targeted to early endosomes, interacts with ARNO in an intra-endosomal acidification-dependent manner, and disruption of this interaction results in reversible inhibition of endocytosis. Inhibition of endosomal acidification abrogates protein trafficking between early and late endosomal compartments. These data demonstrate the crucial role of early endosomal acidification and V-ATPase/ARNO/Arf6 interactions in the regulation of the endocytic degradative pathway. They also indicate that V-ATPase could modulate membrane trafficking by recruiting and interacting with ARNO and Arf6; characteristics that are consistent with the role of V-ATPase as an essential component of the endosomal pH-sensing machinery.

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Figure 1: Expression, distribution and targeting of a-isoforms of V-ATPase in kidney proximal tubule epithelial cells in situ.
Figure 2: The a2-isoform of V-ATPase is targeted to early endosomes.
Figure 3: Endosomal V-ATPase directly interacts with cytosolic ARNO and Arf6.
Figure 4: Vesicular trafficking in proximal tubule cells is an acidification-dependent process that correlates with intra-endosomal acidification-dependent V-ATPase–ARNO interaction.
Figure 5: Overexpression of soluble cytosolic tail of the a2-isoform (a2N) inhibits receptor-mediated endocytosis in MTC cells.
Figure 6: An interaction-competent mutant of ARNO rescues MTC cells from the inhibitory effect of a2N on receptor-mediated protein endocytosis.
Figure 7: Differential role of V-ATPase-driven endosomal acidification on vesicular trafficking via recycling and degradative pathways during endocytosis.
Figure 8: Model of the novel role of V-ATPase as an essential component of the endosomal pH-sensing machinery.


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We would like to thank C. Reinecker for generously providing Rab7–EGFP and Rab11–EGFP constructs and for advice on Volocity software. We would like to thank J. Donaldson for generously providing the Arf6–HA construct and M.A. Billeter for kindly providing the HEK cell line expressing T7-polymerase. We also wish to thank H. Huang for expert technical assistance on FACS analysis. We are grateful to V. Hsu for critical discussion and reading of the manuscript. M.F. and G.-H.S.-W. were supported by CREST, the Japan Science and Technology Agency. This work was supported by a National Institutes of Health grants (DK38452 and DK42956). The Microscopy Core facility of the MGH Program in Membrane Biology receives additional support from the Boston Area Diabetes and Endocrinology Research Center (DK57521) and the Center for the Study of Inflammatory Bowel Disease (DK43341).

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Correspondence to Vladimir Marshansky.

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