Tom40 Functions as a Channel for Protein Retrotranslocation in the Mitochondria-Associated Degradation (MAD) Pathway

Tom40 Functions as a Channel for Protein Retrotranslocation in the Mitochondria-Associated Degradation (MAD) Pathway | 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 Tom40 Functions as a Channel for Protein Retrotranslocation in the Mitochondria-Associated Degradation (MAD) Pathway Liza Pon, Pin-Chao Liao, Catherine Tsang, Tzu-Ying Lin This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3446714/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Jul, 2025 Read the published version in Communications Biology → Version 1 posted You are reading this latest preprint version Abstract The mitochondria-associated degradation pathway (MAD) mediates removal and elimination of unfolded mitochondrial proteins by the ubiquitin-proteasome system (UPS). Here, we reconstituted retrotranslocation of MAD substrates from the mitochondrial matrix across mitochondrial inner and outer membranes in cell-free systems. This retrotranslocation is ATP- dependent but membrane potential-independent. We also identified a role for the TOM complex, the protein import channel in the mitochondrial outer membrane (MOM), in this process. Inhibition of protein translocation across the Tom40p channel inhibits retrotranslocation of MAD substrates in vitro. Deletion of the TOM5 subunit of the TOM complex inhibits retrotranslocation in vitro, and results in increased sensitivity to oxidative stress and decreased association of a MAD substrate with Cdc48p, a subunit of the segregase complex that removes proteins from mitochondria and targets them for proteasomal degradation in MAD, in vivo. Our studies indicate that unfolded proteins in the mitochondrial matrix are retrotranslocated across both mitochondrial membranes and the TOM complex is the retrotranslocation channel in the MOM in MAD. Biological sciences/Cell biology Biological sciences/Cell biology/Proteolysis/Protein quality control Full Text Additional Declarations There is NO Competing Interest. Supplementary Files Liaoetal.SupplementalFigures2023.pdf Cite Share Download PDF Status: Published Journal Publication published 29 Jul, 2025 Read the published version in Communications Biology → 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-3446714","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":276721491,"identity":"2d5957a4-6f7f-4aa6-bae8-c1d31231508a","order_by":0,"name":"Liza Pon","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYBACPgYG9g8fYLwHDAwJbCAGDx4tQAVsjDNgvARitTDDFSSAESEt7GePPbapORzN38D88EFi27Y8PvYDjA/etuHRwpOXbpxz7HDujANsxgaJbbeL2XgSmA3n4tPCkGMgndtwOHcDAw+bBFBLYhvQO9K8+LTwvzGQtkTRwv+A/TdeLRI5ZtKMKFokEtiY8Wt5l2zYcyw9d8ZhoF8SzgH9IvGwWXLOOdxa+PlzDz74UWOd29/e/PDBh7LbefL9yQc/vCnDrQURBcxwEcYGfOoZ8MfaKBgFo2AUjAIQAAC3lUytdhMtyAAAAABJRU5ErkJggg==","orcid":"","institution":"Columbia University","correspondingAuthor":true,"prefix":"","firstName":"Liza","middleName":"","lastName":"Pon","suffix":""},{"id":276721492,"identity":"b5dc6f31-9c65-4b71-a520-cc30c1908faf","order_by":1,"name":"Pin-Chao Liao","email":"","orcid":"https://orcid.org/0000-0002-1969-5565","institution":"Dept. of Life Science, National Tsing Hua University","correspondingAuthor":false,"prefix":"","firstName":"Pin-Chao","middleName":"","lastName":"Liao","suffix":""},{"id":276721493,"identity":"112ea45d-0bc5-4479-89b2-5004386cd50c","order_by":2,"name":"Catherine Tsang","email":"","orcid":"","institution":"Columbia University","correspondingAuthor":false,"prefix":"","firstName":"Catherine","middleName":"","lastName":"Tsang","suffix":""},{"id":276721494,"identity":"f49f3912-d999-4ded-802c-9d3402de5d7b","order_by":3,"name":"Tzu-Ying Lin","email":"","orcid":"","institution":"Dept. of Life Science, National Tsing Hua University","correspondingAuthor":false,"prefix":"","firstName":"Tzu-Ying","middleName":"","lastName":"Lin","suffix":""}],"badges":[],"createdAt":"2023-10-14 19:45:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3446714/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3446714/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s42003-025-08549-z","type":"published","date":"2025-07-29T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88506178,"identity":"b1f01ad5-3ee2-4c7c-884e-34b6f421991e","added_by":"auto","created_at":"2025-08-07 07:32:16","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1109296,"visible":true,"origin":"","legend":"","description":"","filename":"Liaoetal.2023.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3446714/v1_covered_0be66cc5-0dae-4b26-9194-2b0f53e92821.pdf"},{"id":52144175,"identity":"3e100d39-59d7-43e4-87b1-72220e20b037","added_by":"auto","created_at":"2024-03-07 11:59:24","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1851915,"visible":true,"origin":"","legend":"","description":"","filename":"Liaoetal.SupplementalFigures2023.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3446714/v1/9c5bb6eae54cb272d3bf8dc0.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Tom40 Functions as a Channel for Protein Retrotranslocation in the Mitochondria-Associated Degradation (MAD) Pathway","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3446714/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3446714/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"The mitochondria-associated degradation pathway (MAD) mediates removal and elimination of unfolded mitochondrial proteins by the ubiquitin-proteasome system (UPS). 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