NPC1 regulates ER contacts with endocytic organelles to mediate cholesterol egress

Abstract Transport of dietary cholesterol from endocytic organelles to the endoplasmic reticulum (ER) is essential for cholesterol homoeostasis, but the mechanism and regulation of this transport remains poorly defined. Membrane contact sites (MCS), microdomains of close membrane apposition, are gaining attention as important platforms for non-vesicular, inter-organellar communication. Here we investigate the impact of ER-endocytic organelle MCS on cholesterol transport. We report a role for Niemann-Pick type C protein 1 (NPC1) in tethering ER-endocytic organelle MCS where it interacts with the ER-localised sterol transport protein Gramd1b to regulate cholesterol egress. We show that artificially tethering MCS rescues the cholesterol accumulation that characterises NPC1-deficient cells, consistent with direct lysosome to ER cholesterol transport across MCS. Finally, we identify an expanded population of lysosome-mitochondria MCS in cells depleted of NPC1 or Gramd1b that is dependent on the late endosomal sterol-binding protein STARD3, likely underlying the mitochondrial cholesterol accumulation in NPC1-deficient cells.

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Annexin A6 modulates TBC1D15/Rab7/StARD3 axis to control endosomal cholesterol export in NPC1 cells

Cellular and Molecular Life Sciences ◽

10.1007/s00018-019-03330-y ◽

2019 ◽

Vol 77 (14) ◽

pp. 2839-2857 ◽

Cited By ~ 4

Author(s):

Elsa Meneses-Salas ◽

Ana García-Melero ◽

Kristiina Kanerva ◽

Patricia Blanco-Muñoz ◽

Frederic Morales-Paytuvi ◽

...

Keyword(s):

Late Endosome ◽

Dependent Manner ◽

Lipid Transfer ◽

Cholesterol Accumulation ◽

Membrane Contact Sites ◽

Late Endosomes ◽

Membrane Contact ◽

Domain 3 ◽

Niemann Pick ◽

Mutant Cells

Abstract Cholesterol accumulation in late endosomes is a prevailing phenotype of Niemann-Pick type C1 (NPC1) mutant cells. Likewise, annexin A6 (AnxA6) overexpression induces a phenotype reminiscent of NPC1 mutant cells. Here, we demonstrate that this cellular cholesterol imbalance is due to AnxA6 promoting Rab7 inactivation via TBC1D15, a Rab7-GAP. In NPC1 mutant cells, AnxA6 depletion and eventual Rab7 activation was associated with peripheral distribution and increased mobility of late endosomes. This was accompanied by an enhanced lipid accumulation in lipid droplets in an acyl-CoA:cholesterol acyltransferase (ACAT)-dependent manner. Moreover, in AnxA6-deficient NPC1 mutant cells, Rab7-mediated rescue of late endosome-cholesterol export required the StAR-related lipid transfer domain-3 (StARD3) protein. Electron microscopy revealed a significant increase of membrane contact sites (MCS) between late endosomes and ER in NPC1 mutant cells lacking AnxA6, suggesting late endosome-cholesterol transfer to the ER via Rab7 and StARD3-dependent MCS formation. This study identifies AnxA6 as a novel gatekeeper that controls cellular distribution of late endosome-cholesterol via regulation of a Rab7-GAP and MCS formation.

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Cholesterol Overload: Contact Sites to the Rescue!

Contact ◽

10.1177/2515256419893507 ◽

2019 ◽

Vol 2 ◽

pp. 251525641989350 ◽

Cited By ~ 1

Author(s):

Carlos Enrich ◽

Carles Rentero ◽

Thomas Grewal ◽

Clare E. Futter ◽

Emily R. Eden

Keyword(s):

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Cholesterol Homeostasis ◽

Low Density ◽

Cholesterol Accumulation ◽

Membrane Contact Sites ◽

Contact Sites ◽

Late Endosomes ◽

Membrane Contact ◽

Niemann Pick

Delivery of low-density lipoprotein-derived cholesterol to the endoplasmic reticulum (ER) is essential for cholesterol homeostasis, yet the mechanism of this transport has largely remained elusive. Two recent reports shed some light on this process, uncovering a role for Niemann Pick type-C1 protein (NPC1) in the formation of membrane contact sites (MCS) between late endosomes (LE)/lysosomes (Lys) and the ER. Both studies identified a loss of MCS in cells lacking functional NPC1, where cholesterol accumulates in late endocytic organelles. Remarkably, and taking different approaches, both studies have made a striking observation that expansion of LE/Lys-ER MCS can rescue the cholesterol accumulation phenotype in NPC1 mutant or deficient cells. In both cases, the cholesterol was shown to be transported to the ER, demonstrating the importance of ER-LE/Lys contact sites in the direct transport of low-density lipoprotein-derived cholesterol to the ER.

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Quantitative imaging of membrane contact sites for sterol transfer between endo-lysosomes and mitochondria in living cells

Scientific Reports ◽

10.1038/s41598-021-87876-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Alice Dupont Juhl ◽

Christian W. Heegaard ◽

Stephan Werner ◽

Gerd Schneider ◽

Kathiresan Krishnan ◽

...

Keyword(s):

Close Contact ◽

Quantitative Imaging ◽

Human Fibroblasts ◽

Time Lapse ◽

Living Cells ◽

Membrane Contact Sites ◽

Contact Sites ◽

Sterol Transport ◽

Membrane Contact ◽

Niemann Pick

AbstractMitochondria receive cholesterol from late endosomes and lysosomes (LE/LYSs) or from the plasma membrane for production of oxysterols and steroid hormones. This process depends on the endo-lysosomal sterol transfer protein Niemann Pick C2 (NPC2). Using the intrinsically fluorescent cholesterol analog, cholestatrienol, we directly observe sterol transport to mitochondria in fibroblasts upon treating NPC2 deficient human fibroblasts with NPC2 protein. Soft X-ray tomography reveals the ultrastructure of mitochondria and discloses close contact to endosome-like organelles. Using fluorescence microscopy, we localize endo-lysosomes containing NPC2 relative to mitochondria based on the Euclidian distance transform and use statistical inference to show that about 30% of such LE/LYSs are in contact to mitochondria in human fibroblasts. Using Markov Chain Monte Carlo image simulations, we show that interaction between both organelle types, a defining feature of membrane contact sites (MCSs) can give rise to the observed spatial organelle distribution. We devise a protocol to determine the surface fraction of endo-lysosomes in contact with mitochondria and show that this fraction does not depend on functional NPC1 or NPC2 proteins. Finally, we localize MCSs between LE/LYSs containing NPC2 and mitochondria in time-lapse image sequences and show that they either form transiently or remain stable for tens of seconds. Lasting MCSs between endo-lysosomes containing NPC2 and mitochondria move by slow anomalous sub-diffusion, providing location and time for sterol transport between both organelles. Our quantitative imaging strategy will be of high value for characterizing the dynamics and function of MCSs between various organelles in living cells.

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A role for NPC1 and NPC2 in intestinal cholesterol absorption – the hypothesis gutted

Biochemical Journal ◽

10.1042/bj20071340 ◽

2007 ◽

Vol 408 (1) ◽

Cited By ~ 5

Author(s):

Laura Liscum

Keyword(s):

Endoplasmic Reticulum ◽

Dietary Cholesterol ◽

Cholesterol Absorption ◽

Cholesterol Transport ◽

Intestinal Epithelial ◽

Intestinal Cholesterol Absorption ◽

Biochemical Journal ◽

Good Target ◽

Type C ◽

Niemann Pick

Dietary and biliary cholesterol are taken up by intestinal epithelial cells and transported to the endoplasmic reticulum. At the endoplasmic reticulum, cholesterol is esterified, packaged into chylomicrons and secreted into the lymph for delivery to the bloodstream. NPC1L1 (Niemann–Pick C1-like 1) is a protein on the enterocyte brush-border membrane that facilitates cholesterol absorption. Cholesterol's itinerary as it moves to the endoplasmic reticulum is unknown, as is the identity of any cellular proteins that facilitate the movement. Two proteins that play an important role in intracellular cholesterol transport and could potentially influence NPC1L1-mediated cholesterol uptake are NPC1 and NPC2 (Niemann–Pick type C disease proteins 1 and 2). In this issue of the Biochemical Journal, Dixit and colleagues show that the absence or presence of NPC1 and NPC2 has no effect on intestinal cholesterol absorption in the mouse. Thus neither protein fills the gap in our knowledge of intra-enterocyte cholesterol transport. Furthermore, the NPC1/NPC2 pathway would not be a good target for limiting the uptake of dietary cholesterol.

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Intracellular Cholesterol Transport by Sterol Transfer Proteins at Membrane Contact Sites

Trends in Biochemical Sciences ◽

10.1016/j.tibs.2018.10.001 ◽

2019 ◽

Vol 44 (3) ◽

pp. 273-292 ◽

Cited By ~ 35

Author(s):

Jie Luo ◽

Lu-Yi Jiang ◽

Hongyuan Yang ◽

Bao-Liang Song

Keyword(s):

Cholesterol Transport ◽

Membrane Contact Sites ◽

Contact Sites ◽

Membrane Contact ◽

Intracellular Cholesterol

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Cholesterol sensor ORP1L contacts the ER protein VAP to control Rab7–RILP–p150Glued and late endosome positioning

The Journal of Cell Biology ◽

10.1083/jcb.200811005 ◽

2009 ◽

Vol 185 (7) ◽

pp. 1209-1225 ◽

Cited By ~ 379

Author(s):

Nuno Rocha ◽

Coenraad Kuijl ◽

Rik van der Kant ◽

Lennert Janssen ◽

Diane Houben ◽

...

Keyword(s):

Motor Proteins ◽

Oxysterol Binding Protein ◽

Membrane Contact Sites ◽

Cholesterol Levels ◽

Dynein Motor ◽

Late Endosomes ◽

In Trans ◽

Membrane Contact ◽

Cholesterol Control ◽

Niemann Pick

Late endosomes (LEs) have characteristic intracellular distributions determined by their interactions with various motor proteins. Motor proteins associated to the dynactin subunit p150Glued bind to LEs via the Rab7 effector Rab7-interacting lysosomal protein (RILP) in association with the oxysterol-binding protein ORP1L. We found that cholesterol levels in LEs are sensed by ORP1L and are lower in peripheral vesicles. Under low cholesterol conditions, ORP1L conformation induces the formation of endoplasmic reticulum (ER)–LE membrane contact sites. At these sites, the ER protein VAP (VAMP [vesicle-associated membrane protein]-associated ER protein) can interact in trans with the Rab7–RILP complex to remove p150Glued and associated motors. LEs then move to the microtubule plus end. Under high cholesterol conditions, as in Niemann-Pick type C disease, this process is prevented, and LEs accumulate at the microtubule minus end as the result of dynein motor activity. These data explain how the ER and cholesterol control the association of LEs with motor proteins and their positioning in cells.

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