Annexin A6 depletion rescues cholesterol egress in NPC1 mutant cells via formation of endosome–ER membrane contact sites

Grewal T1, Meneses-Salas E2, Garcia-Melero A2, Bianco-Muñoz P2, Egert A1, Beevi SS1, Rentero C2 and Enrich C2

  1. School of Pharmacy, University of Sydney, Sydney, NSW 2006, Australia.
  2. Departament de Biomedicina, Unitat de Biologia Cel·lular, Facultat de Medicina i Ciències de la Salut, Universitat de Barcelona, 08036-Barcelona, Spain.

The accumulation of cholesterol in late endosomes (LE)/lysosomes of Niemann-Pick type C1 (NPC1) mutant cells critically disturbs intracellular cholesterol transport. Besides NPC1, oxysterol-binding proteins (ORP1L, ORP5), StAR related lipid transfer domain (STARD) proteins, and several Rab proteins also facilitate cholesterol export from LE. Some of these proteins are engaged in the formation of membrane contact sites (MCS), defined by the apposition of two cellular membranes, providing opportunity for non-vesicular transport of lipids, including cholesterol. Hence, yet unidentified players or gatekeepers in LE may control activation or fine-tune alternative LE-cholesterol transport routes other than NPC1. Annexin A6 (AnxA6), a member of the annexin family, has been implicated in endo- and exocytic pathways and cholesterol homeostasis. We previously demonstrated that AnxA6 is recruited to Low Density Lipoprotein (LDL) -cholesterol enriched LE. Moreover, similar to the loss of NPC1, AnxA6 overexpression led to LE-cholesterol accumulation. Here, we demonstrate that this cellular cholesterol imbalance is due to AnxA6 recruiting TBC1D15, a Rab7-GTPase activating protein (Rab7-GAP), to cholesterol-rich LE to inhibit Rab7 GTPase activity. In contrast, AnxA6 depletion elevated Rab7 activity, rescuing cholesterol export from LE in NPC1 mutant cells. This was associated with peripheral distribution and increased mobility of LE and enhanced lipid accumulation in lipid droplets (LDs) in an acetyl-coenzyme A acetyltransferase (ACAT)-dependent manner. Moreover, StARD3 depletion compromised LE-Chol export and lipid deposition in LD in AnxA6-deficient NPC1 mutant cells. Electron microscopy revealed a significant increase of MCS between LE and the endoplasmic reticulum (ER) in NPC1 mutant cells lacking AnxA6, suggesting LE-cholesterol transfer to the ER via StARD3-dependent MCS formation. Taken together, this study identifies an annexin, AnxA6, as a novel gatekeeper that controls cellular distribution of LE-Chol via two critical biological activities: regulation of a Rab7-GAP and MCS functioning.