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Natl. Acad. Sci. U.S.A."],"published-print":{"date-parts":[[2025,12,30]]},"abstract":"<jats:p>\n                    The Niemann-Pick type 1 and 2 proteins (NPC1 and NPC2) coordinate cholesterol egress from late endosomes-lysosomes (LE\/LY). Proper folding, trafficking, and localization of both NPC proteins are essential for normal LE\/LY cholesterol handling. Accordingly, mutations in\n                    <jats:italic toggle=\"yes\">NPC<\/jats:italic>\n                    genes cause Niemann\u2013Pick type C (NPC) disease, a progressive neurodegenerative lysosomal cholesterol storage disorder. The routes by which NPC1 reaches the LE\/LY compartment in mammalian cells are not fully elucidated. Therefore, to interrogate NPC1 trafficking, we developed genome-engineered HeLa cells expressing endogenous NPC1\n                    <jats:sup>mNeon<\/jats:sup>\n                    . We demonstrate that endogenous NPC1 localizes to the LE\/LY compartment and by using protein proximity-based approaches that NPC1 resides in the same membranes as Vacuolar Protein Sorting-associated protein 41 (VPS41), one of the two unique subunits of the homotypic fusion and vacuole protein sorting complex. Loss of VPS41 increases NPC1 and Lysosomal Associated Membrane Protein 1 (LAMP1) abundance. Paradoxically, this results in marked accumulation of lysosomal cholesterol and induction of sterol regulatory element-binding protein signaling. Mechanistically, using immuno-fluorescence and electron microscopy imaging in combination with a VPS41-dependent ectopic recruitment assay, we demonstrate that this is due to a shift in the localization of NPC1 and LAMP1 from LE\/LY to biosynthetic vesicles called LAMP carriers. These vesicles have been recently described to transport lysosomal-destined cargo directly from the\n                    <jats:italic toggle=\"yes\">trans-<\/jats:italic>\n                    Golgi (TGN) network to LE\/LY. In conclusion, we identify NPC1 as a cargo for VPS41-dependent LAMP carriers that are instrumental for the delivery of NPC1 to LE\/LY and maintaining cellular cholesterol homeostasis.\n                  <\/jats:p>","DOI":"10.1073\/pnas.2521979122","type":"journal-article","created":{"date-parts":[[2025,12,26]],"date-time":"2025-12-26T19:14:15Z","timestamp":1766776455000},"update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":3,"title":["NPC1 trafficking via VPS41-dependent LAMP carriers regulates endosomal cholesterol homeostasis"],"prefix":"10.1073","volume":"122","author":[{"ORCID":"https:\/\/orcid.org\/0009-0002-5902-7673","authenticated-orcid":false,"given":"Klevis","family":"Ndoj","sequence":"first","affiliation":[{"name":"Department of Medical Biochemistry, Amsterdam University Medical Centers location AMC, University of Amsterdam","place":["Amsterdam, The Netherlands"]},{"name":"Amsterdam Gastroenterology, Endocrinology, and Metabolism Institute, Amsterdam University Medical Centers, University of Amsterdam","place":["Amsterdam, The Netherlands"]},{"name":"Amsterdam Cardiovascular Sciences Institute, Amsterdam University Medical Centers, University of Amsterdam","place":["Amsterdam, The Netherlands"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matteo","family":"Tantucci","sequence":"additional","affiliation":[{"name":"Center for Molecular Medicine\u2014Cell Biology, University Medical Center Utrecht, University of Utrecht","place":["Utrecht, The Netherlands"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-5934-7457","authenticated-orcid":false,"given":"Paolo","family":"Sanza","sequence":"additional","affiliation":[{"name":"Center for Molecular Medicine\u2014Cell Biology, University Medical Center Utrecht, University of Utrecht","place":["Utrecht, The 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