ATG8ylation facilitates an ESCRT-independent vacuolar membrane invagination in plants

ATG8ylation facilitates an ESCRT-independent vacuolar membrane invagination in plants | 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 Letter ATG8ylation facilitates an ESCRT-independent vacuolar membrane invagination in plants Xuanang Zheng, Juncai Ma, Jing Li, Siyu Chen, Jun Luo, Jianxiong Wu, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3878363/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Jul, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract ATG8-family proteins have been found to be incorporated into single-membrane structures, a process referred to as non-canonical autophagy. While several physiological roles of non-canonical conjugation of ATG8 (ATG8ylation) have been established, the specific functions of ATG8 on single membrane remain largely elusive. Here, we demonstrate that ionophores induce conjugation of ATG8 to vacuolar membrane to promote invagination in Arabidopsis. Upon monensin treatment, ATG8 is rapidly translocated to the vacuolar membrane, which is reliant on the ATG conjugation system rather than upstream autophagic regulators such as ATG1, ATG9, and phosphoinositide 3-kinase (PI3K). Moreover, inhibiting reactive oxygen species (ROS) generation or V-ATPase activity greatly impedes the targeting of ATG8 to the vacuolar membrane. Intriguingly, the recruitment of ATG8 to the tonoplast exhibits a pronounced enhancement of invagination and fosters the formation of intraluminal vesicles within the vacuoles. Further analyses elucidate that the topological remodeling of the vacuolar membrane is achieved in a ESCRT machinery-independent manner. Collectively, this study reveals a previously unrecognized role of ATG8ylation in driving the topological transformation of vacuolar membranes in plants. Biological sciences/Plant sciences/Plant cell biology Biological sciences/Plant sciences/Plant physiology Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryVideo1.mov Supplementary Video 1 SupplementaryVideo2.avi Supplementary Video 2 SupplementaryVideo3.avi Supplementary Video 3 Cite Share Download PDF Status: Published Journal Publication published 17 Jul, 2025 Read the published version in Nature Communications → 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. 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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-3878363","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Letter","associatedPublications":[],"authors":[{"id":270518598,"identity":"85430d7b-c471-4c1d-894d-919ebbd11f56","order_by":0,"name":"Xuanang Zheng","email":"","orcid":"","institution":"Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, South China Normal University","correspondingAuthor":false,"prefix":"","firstName":"Xuanang","middleName":"","lastName":"Zheng","suffix":""},{"id":270518599,"identity":"8494fd44-b8f8-4fd9-b78f-2203db4af8c1","order_by":1,"name":"Juncai 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