{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T04:28:17Z","timestamp":1786508897018,"version":"build-2736575974"},"posted":{"date-parts":[[2026,8,10]]},"group-title":"Biochemistry","reference-count":63,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,8,10]],"date-time":"2026-08-10T00:00:00Z","timestamp":1786320000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"award":["ASAP-000350"],"award-info":[{"award-number":["ASAP-000350"]}],"id":[{"id":"https:\/\/ror.org\/03zj4c476","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R01 NS134598"],"award-info":[{"award-number":["R01 NS134598"]}],"id":[{"id":"https:\/\/ror.org\/01s5ya894","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,8,10]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Rubicon is a negative regulator of autophagy and the endolysosomal network (ELN) and an antagonist of the class III phosphatidylinositol 3-kinase complex II (PI3KC3-C2). Inhibition of Rubicon is considered a potential means to therapeutically upregulate autophagy and the ELN to treat Parkinson\u2019s disease and other conditions characterized by autophagic and ELN dysfunction. Rubicon is specific for the UVRAG-containing PI3KC3-C2 over the purely autophagic ATG14- containing PI3KC3-C1 complex. Here, we determined the high-resolution cryo-electron microscopy structure of PI3KC3-C2 in complex with the PI3KC3-binding domain (PIKBD) of Rubicon and compared it to cryo-EM structures of unbound PI3KC3-C2 and PI3KC3-C1. Rubicon binds directly to PI3KC3-C2 only via the BARA domain of the BECN1 subunit, which is common to both C1 and C2. The selectivity of Rubicon for the PI3KC3-C2 complex over the PI3KC3-C1 complex is attributed to a conformation of the BECN1\n                  <jats:sup>BARA<\/jats:sup>\n                  domain induced by UVRAG, rather than to direct contact with UVRAG or direct antagonism by the ATG14 subunit of PI3KC3-C1. Targeted disruption of the Rubicon:PI3K3-C2 structural interface by site-directed mutations enhances mitophagic activity in human epithelial cells to levels comparable to those observed in Rubicon knockout (KO) cells. Similarly, disruption of the interaction in Rubicon-overexpressing hippocampal neurons restored lysosomal flux to wild-type levels. These data show that suppressing the function of PI3K3- C2 can fully account for the negative regulatory effects of Rubicon in the autophagy and ELN pathways.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Significance Statement<\/jats:title>\n                  <jats:p>Endolysosome maturation and autophagosome-lysosome fusion require the production of phosphatidylinositol 3-phosphate (PI(3)P) by the class III phosphatidylinositol 3-kinase complex II (PI3KC3-C2). Rubicon is a key negative regulator of endolysosomes and autophagy that suppresses PI3KC3-C2 activity. Here, we reveal in atomistic detail how Rubicon selectively recognizes PI3KC3-C2. Disrupting the Rubicon\u2013PI3KC3-C2 interaction restores mitophagy and enhances lysosomal activity to the same extent as Rubicon gene deletion, establishing that PI3KC3-C2 inhibition fully accounts for the biological regulatory effects of Rubicon in the autophagy and lysosome pathways.<\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.08.07.743589","type":"posted-content","created":{"date-parts":[[2026,8,10]],"date-time":"2026-08-10T12:30:13Z","timestamp":1786365013000},"source":"Crossref","is-referenced-by-count":0,"title":["Structural basis for the selective inhibition of the PI3KC3-C2 complex by Rubicon in endolysosome maturation and mitophagy"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7668-3974","authenticated-orcid":false,"given":"Minghao","family":"Chen","sequence":"first","affiliation":[{"name":"Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA"},{"name":"California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA"},{"name":"Aligning Science Across Parkinson\u2019s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-9374-4584","authenticated-orcid":false,"given":"Aniketh","family":"Bishnu","sequence":"additional","affiliation":[{"name":"Aligning Science Across Parkinson\u2019s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA"},{"name":"MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-7553-0124","authenticated-orcid":false,"given":"Yongjia","family":"Duan","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA"},{"name":"California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA"},{"name":"Aligning Science Across Parkinson\u2019s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8518-9786","authenticated-orcid":false,"given":"Julia F.","family":"Riley","sequence":"additional","affiliation":[{"name":"Aligning Science Across Parkinson\u2019s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA"},{"name":"Physiology Department, University of Pennsylvania Perelman School of Medicine, and the Neuroscience Graduate Group, University of Pennsylvania, Philadelphia, PA 19104, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-7727-4080","authenticated-orcid":false,"given":"Qingyang","family":"Ni","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA"},{"name":"California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9049-2384","authenticated-orcid":false,"given":"Aaron","family":"Joiner","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA"},{"name":"California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA"},{"name":"Aligning Science Across Parkinson\u2019s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-2723-2017","authenticated-orcid":false,"given":"Isaac J.","family":"Allen","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5389-4114","authenticated-orcid":false,"given":"Erika L. 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