Endosome maturation is orchestrated by inside-out proton signaling through a Na+/H+ exchanger and pH-dependent Rab GTPase cycling

Endosome maturation is orchestrated by inside-out proton signaling through a Na+/H+ exchanger and pH-dependent Rab GTPase cycling | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Endosome maturation is orchestrated by inside-out proton signaling through a Na+/H+ exchanger and pH-dependent Rab GTPase cycling Eric Morrow, YouJin Lee, Qing Ouyang, Li Ma, Morgan Fleishman, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5515864/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Endosome maturation requires lumen acidification. Is progressive lumen acidification sensed by cytosolic-side molecules driving maturation? We show here that proton efflux through the endosomal Na⁺/H⁺ Exchanger (NHE6) activates the late endosome master regulator Rab7. Importantly, NHE6 is mutated in the childhood neurologic disorder Christianson Syndrome. We demonstrate that NHE6 interacts with the Rab7 GTPase-activating protein (GAP) TBC1D5 in a complex with Rab7 on the late endosome. This interaction and proton efflux are both required for Rab7 activation. TBC1D5 is potently inactivated with decreasing pH. A conserved histidine in the TBC1D5 GAP domain mediates pH-dependence. Furthermore, we show that neurons from mice engineered with a selective defect in NHE6 proton efflux exhibit blocked endosome maturation and disrupted Rab7 GTP-GDP cycling. In addition, knock-down of TBC1D5, thereby reducing Rab7 GAP activity, in NHE6 mutant neurons rescues Rab7 GTP-GDP cycling and endosome maturation. Finally, we present a biophysical model of proton signaling through acidic pH microdomains within the NHE6-TBC1D5-Rab7 protein complex upon endosome acidification. In conclusion, our studies provide evidence supporting a mechanism involving “inside-out” proton signaling, whereby lumen acidification drives endosome maturation through pH-dependent Rab GTPase cycling. Failure in this mechanism may have broad impact in neurodegenerative disease. Biological sciences/Neuroscience/Cellular neuroscience Biological sciences/Cell biology/Mechanisms of disease Full Text Additional Declarations There is NO Competing Interest. Supplementary Files LeeetalExtendedFiguresLegends.pdf Extended Data Figure Legends LeeetalSupplementaryEquation.pdf Supplementary Equation Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5515864","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":584927646,"identity":"592c83f4-d5c8-4a61-b531-11f00de7fce3","order_by":0,"name":"Eric 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Is progressive lumen acidification sensed by cytosolic-side molecules driving maturation? We show here that proton efflux through the endosomal Na⁺/H⁺ Exchanger (NHE6) activates the late endosome master regulator Rab7. Importantly, NHE6 is mutated in the childhood neurologic disorder Christianson Syndrome. We demonstrate that NHE6 interacts with the Rab7 GTPase-activating protein (GAP) TBC1D5 in a complex with Rab7 on the late endosome. This interaction and proton efflux are both required for Rab7 activation. TBC1D5 is potently inactivated with decreasing pH. A conserved histidine in the TBC1D5 GAP domain mediates pH-dependence. Furthermore, we show that neurons from mice engineered with a selective defect in NHE6 proton efflux exhibit blocked endosome maturation and disrupted Rab7 GTP-GDP cycling. In addition, knock-down of TBC1D5, thereby reducing Rab7 GAP activity, in NHE6 mutant neurons rescues Rab7 GTP-GDP cycling and endosome maturation. Finally, we present a biophysical model of proton signaling through acidic pH microdomains within the NHE6-TBC1D5-Rab7 protein complex upon endosome acidification. In conclusion, our studies provide evidence supporting a mechanism involving “inside-out” proton signaling, whereby lumen acidification drives endosome maturation through pH-dependent Rab GTPase cycling. 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