Low-density lipoprotein (LDL)-derived cholesterol must leave late endosomes/lysosomes to reach the endoplasmic reticulum (ER) and other organelles, and Niemann-Pick type C1 protein (NPC1) loss blocks this egress, causing the fatal lysosomal storage disorder Niemann-Pick type C (NPC). Höglinger et al. (Nature Communications, 2019) showed that NPC1 tethers ER-lysosome membrane contact sites (MCS) where it interacts with the ER sterol-transfer protein Gramd1b, that artificially expanding ER-lysosome MCS with a sterol-transport-deficient ORP1L tether rescues cholesterol accumulation in NPC1-deficient cells, and that NPC1 or Gramd1b depletion expands a STARD3-dependent lysosome-mitochondria MCS population. These findings left three linked questions open: (1) whether sterol traverses the ER-lysosome interface via a dedicated carrier (Gramd1b, ORP family members), passively down a concentration gradient across expanded contacts, or both; (2) how STARD3 anchors late endocytic membranes to mitochondria — the mitochondrial binding partner and attachment mode are unidentified; (3) what upstream signals (e.g., Rab7 GTPase-cycle status, sterol-sensitive switches) coordinate assembly and disassembly of the competing NPC1-Gramd1b and STARD3 contact populations.

Background

Most mammalian cells acquire cholesterol through receptor-mediated endocytosis of LDL (low-density lipoprotein). LDL is delivered to late endosomes and lysosomes, where its cholesteryl-ester core is hydrolyzed and the released cholesterol is handed off from the soluble luminal protein NPC2 to the membrane protein NPC1 for egress; cholesterol then moves to the ER, where the enzyme ACAT (acyl-CoA:cholesterol acyltransferase) re-esterifies it and the sterol-regulatory feedback loop is tuned. In Niemann-Pick type C (NPC) disease, loss-of-function mutations in NPC1 (or NPC2) trap unesterified cholesterol in late endosomes/lysosomes, causing neurovisceral degeneration. A substantial fraction of LDL-derived cholesterol is thought to reach the ER without passing through the plasma membrane, by a mechanism that remained undefined. Membrane contact sites (MCS) — regions where two organelles are apposed within roughly 10-30 nm — are established platforms for non-vesicular lipid exchange. Several protein families tether ER-endocytic organelle contacts and can transfer sterol: ORP1L (an oxysterol-binding-protein-related protein, a lipid-transfer protein that can carry cholesterol between organelles) binds the ER protein VAP (vesicle-associated membrane protein-associated protein) through its FFAT motif (a short two-phenylalanines-in-an-acidic-tract peptide that docks onto VAP) in a sterol-sensitive manner and regulates late-endosome positioning and cholesterol egress; STARD3 (also called MLN64), a late-endosomal protein whose START domain (StAR-related lipid-transfer domain, a hydrophobic pocket that binds single sterol molecules) can transfer cholesterol, tethers ER-endosome contacts with VAP; and Gramd1b (also called Aster-B), an ER-resident protein with a StART-like lipid-transfer domain that moves accessible cholesterol from the plasma membrane to the ER. Höglinger et al. (2019) added NPC1 itself to this picture: NPC1 interacts with Gramd1b at ER-lysosome MCS when endosomal cholesterol is replete, and NPC1 or Gramd1b depletion both reduces ER-lysosome contacts and impairs cholesterol esterification. Strikingly, forcing ER-lysosome contact expansion with a sterol-transport-deficient, constitutively VAP-bound ORP1L mutant restored cholesterol egress in NPC1-deficient cells — implying that when the interface is enlarged, cholesterol can still leave the lysosome, either because a residual carrier (endogenous ORP1L or Gramd1b) suffices, or because sterol diffuses passively down its concentration gradient across the widened apposition. The same study found that NPC1 or Gramd1b loss expands a sterol-responsive population of lysosome-mitochondria MCS that depends on STARD3 — plausibly explaining the mitochondrial cholesterol accumulation and dysfunction seen in NPC — while noting that Rab7 (a small GTPase master regulator of late-endosome identity whose GTP hydrolysis is inhibited by cholesterol loading and is required for mitochondria-lysosome contact disassembly) may couple sterol status to contact dynamics. Rab7 activity is turned off by GTPase-activating proteins (GAPs) such as TBC1D15. The paper's discussion explicitly leaves unresolved: the precise transport mechanism across ER-lysosome MCS, the mitochondrial anchoring mechanism of STARD3, and the regulation of the competing sterol-responsive contact populations.

Problem Statement

The common objective is a complete mechanistic account of how LDL-derived cholesterol leaves the late endocytic compartment across membrane contact sites — which proteins carry it and how those contact populations are regulated. Specifically, determine: (1) the molecular mechanism by which cholesterol traverses the ER-lysosome interface at these membrane contact sites — whether endogenous sterol transfer requires a dedicated carrier protein (e.g., the ER sterol-transfer protein Gramd1b or ORP-family lipid carriers such as ORP1L), can occur passively down a concentration gradient across expanded contacts without a dedicated carrier, or proceeds by a context-dependent combination of both, accounting for the observation that artificial expansion of ER-lysosome contacts with a sterol-transport-deficient ORP1L tether restores cholesterol egress in cells lacking NPC1 (the lysosomal membrane protein whose loss causes Niemann-Pick type C disease); (2) the molecular basis by which STARD3 (a late-endosomal START-domain cholesterol-transfer protein) anchors late endocytic membranes to mitochondria — the identity of the mitochondrial binding partner(s) and the structural mode of attachment at lysosome-mitochondria contact sites; and (3) the upstream regulatory signals that sterol-sensitively switch contact-site populations between NPC1-Gramd1b-mediated lysosome-ER contacts and STARD3-mediated contacts (including how the GTPase cycle of Rab7, a small GTPase controlling late-endosome identity, and sterol-induced changes in small-GTPase cycles coordinate contact assembly and disassembly).

References

  1. Höglinger D, Burgoyne T, Sanchez-Heras E, Hartwig P, Colaco A, Newton J, Futter CE, Spiegel S, Platt FM, Eden ER. NPC1 regulates ER contacts with endocytic organelles to mediate cholesterol egress. Nature Communications 10:4276 (2019). doi:10.1038/s41467-019-12152-2, https://doi.org/10.1038/s41467-019-12152-2
  2. Wilhelm LP, Wendling C, Védie B, Kobayashi T, Chenard M-P, Tomasetto C, Drin G, Alpy F. STARD3 mediates endoplasmic reticulum-to-endosome cholesterol transport at membrane contact sites. The EMBO Journal 36:1412-1433 (2017). doi:10.15252/embj.201695917, https://doi.org/10.15252/embj.201695917
  3. Zhao K, Ridgway ND. Oxysterol-Binding Protein-Related Protein 1L Regulates Cholesterol Egress from the Endo-Lysosomal System. Cell Reports 19:1807-1818 (2017). doi:10.1016/j.celrep.2017.05.028, https://doi.org/10.1016/j.celrep.2017.05.028
  4. Rocha N, Kuijl C, van der Kant R, Janssen L, Houben D, Janssen H, Zwart W, Neefjes J. Cholesterol sensor ORP1L contacts the ER protein VAP to control Rab7-RILP-p150Glued and late endosome positioning. Journal of Cell Biology 185:1209-1225 (2009). doi:10.1083/jcb.200811005, https://doi.org/10.1083/jcb.200811005
  5. Dong J, Du X, Wang H, Wang J, Lu C, Chen X, Zhu Z, Luo Z, Yu L, Brown AJ, Yang H, Wu J-W. Allosteric enhancement of ORP1-mediated cholesterol transport by PI(4,5)P2/PI(3,4)P2. Nature Communications 10:829 (2019). doi:10.1038/s41467-019-08791-0, https://doi.org/10.1038/s41467-019-08791-0
  6. Lim C-Y, Davis OB, Shin HR, Zhang J, Berdan CA, Jiang X, Counihan JL, Ory DS, Nomura DK, Zoncu R. ER-lysosome contacts enable cholesterol sensing by mTORC1 and drive aberrant growth signalling in Niemann-Pick type C. Nature Cell Biology 21:1206-1218 (2019). doi:10.1038/s41556-019-0391-5, https://doi.org/10.1038/s41556-019-0391-5
  7. Wijdeven RH, Janssen H, Nahidiazar L, Janssen L, Jalink K, Berlin I, Neefjes J. Cholesterol and ORP1L-mediated ER contact sites control autophagosome transport and fusion with the endocytic pathway. Nature Communications 7:11808 (2016). doi:10.1038/ncomms11808, https://doi.org/10.1038/ncomms11808
  8. Trinh MN, Brown MS, Goldstein JL, Han J, Vale G, McDonald JG, Seemann J, Mendell JT, Lu F. Last step in the path of LDL cholesterol from lysosome to plasma membrane to ER is governed by phosphatidylserine. Proceedings of the National Academy of Sciences USA 117:18521-18529 (2020). doi:10.1073/pnas.2010682117, https://doi.org/10.1073/pnas.2010682117
  9. Lu A, Hsieh F, Sharma BR, Vaughn SR, Enrich C, Pfeffer SR. CRISPR screens for lipid regulators reveal a role for ER-bound SNX13 in lysosomal cholesterol export. Journal of Cell Biology 221:e202105060 (2022). doi:10.1083/jcb.202105060, https://doi.org/10.1083/jcb.202105060
  10. Lu A. Endolysosomal cholesterol export: More than just NPC1. BioEssays 44:e2200111 (2022). doi:10.1002/bies.202200111, https://doi.org/10.1002/bies.202200111
  11. Fang X, Wei P, Zhao K, Sheng Z, Song B-L, Yin L, Luo J. Fatty acid-binding proteins 3, 7, and 8 bind cholesterol and facilitate its egress from lysosomes. Journal of Cell Biology 223:e202211062 (2024). doi:10.1083/jcb.202211062, https://doi.org/10.1083/jcb.202211062
  12. Meneses-Salas E, García-Melero A, Kanerva K, Blanco-Muñoz P, Morales-Paytuvi F, Bonjoch J, Casas J, Egert A, Beevi SS, Jose J, Llorente-Cortés V, Rye K-A, Heeren J, Lu A, Pol A, Tebar F, Ikonen E, Grewal T, Enrich C, Rentero C. Annexin A6 modulates TBC1D15/Rab7/StARD3 axis to control endosomal cholesterol export in NPC1 cells. Cell and Molecular Life Sciences 77:2839-2857 (2020). doi:10.1007/s00018-019-03330-y, https://doi.org/10.1007/s00018-019-03330-y
  13. Palladino END, Bernas T, Green CD, Weigel C, Singh SK, Senkal CE, Martello A, Kennelly JP, Bieberich E, Tontonoz P, Ford DA, Milstien S, Eden ER, Spiegel S. Sphingosine kinases regulate ER contacts with late endocytic organelles and cholesterol trafficking. Proceedings of the National Academy of Sciences USA 119:e2204396119 (2022). doi:10.1073/pnas.2204396119, https://doi.org/10.1073/pnas.2204396119
  14. Juhl AD, Heegaard CW, Werner S, Schneider G, Krishnan K, Covey DF, Wüstner D. Quantitative imaging of membrane contact sites for sterol transfer between endo-lysosomes and mitochondria in living cells. Scientific Reports 11:8927 (2021). doi:10.1038/s41598-021-87876-7, https://doi.org/10.1038/s41598-021-87876-7
  15. Nara A, Inoue A, Aoyama Y, Yazawa T. The ultrastructural function of MLN64 in the late endosome-mitochondria membrane contact sites in placental cells. Experimental Cell Research 429:113668 (2023). doi:10.1016/j.yexcr.2023.113668, https://doi.org/10.1016/j.yexcr.2023.113668
  16. Bernaus-Esqué M, Liu Y, Prats E, Estanyol JM, Martin G, Calvo M, Gigourtsi PA, Nguyen MKL, Álvarez AR, Zanlungo S, Agell N, Lu A, Tebar F, Enrich C, Grewal T, Rentero C. Annexin A6 controls multi-organelle contact site formation and endolysosomal positioning, and remodels the STARD3 interactome. iScience 29:116387 (2026). doi:10.1016/j.isci.2026.116387, https://doi.org/10.1016/j.isci.2026.116387
  17. Pastore R, Stanley J, Kiraly S, Dubois C, Monfregola J, De Luca A, Guerra G, Skehel M, Ballabio A, Platt FM, Eden ER. HKDC1 contributes to aberrant lysosome-mitochondria contact in Niemann-Pick disease type C. bioRxiv preprint (2026). doi:10.64898/2026.06.01.729255, https://doi.org/10.64898/2026.06.01.729255
  18. Eichler J, Wendling C, Huver S, Zouiouich M, Hanss V, Cardinal A, Fimbel V, Birck C, McEwen AG, Knorr C, Fromental-Ramain C, Boutry M, Chenard M-P, Drin G, Tomasetto C, Alpy F. STARD3 regulates lysosome positioning and contacts via a GSK3-controlled phosphorylation switch. The EMBO Journal 45:2239-2277 (2026). doi:10.1038/s44318-026-00705-3, https://doi.org/10.1038/s44318-026-00705-3
  19. Battista B, Sambrailo A, Biglione FA, Lombardo VA, Mansilla MC, Albanesi D, Lisa MN, de Mendoza D, Binolfi A. STARD3 mediates non-vesicular cholesterol transport in Caenorhabditis elegans. bioRxiv preprint (2026). doi:10.64898/2026.01.09.698688, https://doi.org/10.64898/2026.01.09.698688
  20. Nguyen MKL, Pinkenburg C, Du JJ, Bernaus-Esqué M, Enrich C, Rentero C, Grewal T. The multiple facets of Rab proteins modulating the cellular distribution of cholesterol from the late endosomal compartment. Biochimica et Biophysica Acta - Molecular Cell Research 1872:119896 (2025). doi:10.1016/j.bbamcr.2025.119896, https://doi.org/10.1016/j.bbamcr.2025.119896
  21. Nguyen MKL, Nikenich MR, Seifert K, Pinkenburg C, Lai F, Walther HL, Hartmann M, Szulc A, Sparkes E, Chen S, Wikramanayake R, Bernaus-Esqué M, Liu Y, Tebar F, Serwetnyk M, Wenninger A, Patzke C, Blagg BSJ, Ashfeld BL, Groundwater PW, Hibbs DE, Hoy AJ, Rentero C, Enrich C, Cho A-N, Du JJ, Grewal T. Small Molecules to Elevate Rab7-GTPase Activity and Lower Cholesterol Accumulation in Niemann-Pick Type C Disease. Pharmaceutical Research 43:1009-1031 (2026). doi:10.1007/s11095-026-04058-8, https://doi.org/10.1007/s11095-026-04058-8
  22. Pastore R, Yao L, Hatcher N, Helley M, Brownlees J, Desai R. Deficiency in NPC2 results in disruption of mitochondria-late endosome/lysosomes contact sites and endo-lysosomal lipid dyshomeostasis. Scientific Reports 15:325 (2025). doi:10.1038/s41598-024-83460-x, https://doi.org/10.1038/s41598-024-83460-x
  23. Boutry M, Kim PK. ORP1L mediated PI(4)P signaling at ER-lysosome-mitochondrion three-way contact contributes to mitochondrial division. Nature Communications 12:5354 (2021). doi:10.1038/s41467-021-25621-4, https://doi.org/10.1038/s41467-021-25621-4
  24. Olkkonen VM, Ikonen E. Getting to Grips with the Oxysterol-Binding Protein Family - a Forty Year Perspective. Contact 7:25152564241273598 (2024). doi:10.1177/25152564241273598, https://doi.org/10.1177/25152564241273598
  25. Loughran RM, Arora GK, Sun J, Llorente A, Crabtree S, Zhang CY, Ly K, Huynh R-L, Cho W, Emerling BM. Noncanonical PI(4,5)P2 coordinates lysosome positioning through cholesterol trafficking. Science Advances 12:eaeb8658 (2026). doi:10.1126/sciadv.aeb8658, https://doi.org/10.1126/sciadv.aeb8658