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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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).
The authors declare no competing financial interests.
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Hurtado-Lorenzo, A., Skinner, M., Annan, J. et al. V-ATPase interacts with ARNO and Arf6 in early endosomes and regulates the protein degradative pathway. Nat Cell Biol 8, 124–136 (2006). https://doi.org/10.1038/ncb1348
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