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The exit of low-density lipoprotein derived cholesterol (LDL-C) from late endosomes (LE)/lysosomes (Ly) is mediated by Niemann-Pick C1 (NPC1), a multipass integral membrane protein on the limiting membranes of LE/Ly, and by NPC2, a cholesterol-binding protein in the lumen of LE/Ly. NPC2 delivers cholesterol to the N-terminal domain of NPC1, which is believed to insert cholesterol into the limiting membrane for subsequent transport to other subcellular organelles. Few cytoplasmic factors have been identified to govern cholesterol efflux from LE/Ly, and much less is known about the underlying molecular mechanisms. Here we establish VPS4, an AAA ATPase that has a well-established role in disassembling the ESCRT (endosomal sorting complex required for transport)-III polymer, as an important regulator of endosomal cholesterol transport. Knocking down VPS4 in HeLa cells resulted in prominent accumulation of LDL-C in LE/Ly, and disrupted cholesterol homeostatic responses at the endoplasmic reticulum. The level and localization of NPC1 and NPC2 appeared to be normal in VPS4 knockdown cells. Importantly, depleting any of the ESCRT-III components did not exert a significant effect on endosomal cholesterol transport. Our results thus identify an important cytoplasmic regulator of endosomal cholesterol trafficking and represent the first functional separation of VPS4 from ESCRT-III. [ABSTRACT FROM AUTHOR]

Phosphatidylinositol phosphates (PIPs) and cholesterol are known to regulate the function of late endosomes and lysosomes (LELs), and ORP1L specifically localizes to LELs. Here, we show in vitro that ORP1 is a PI(4,5)P2- or PI(3,4)P2-dependent cholesterol transporter, but cannot transport any PIPs. In cells, both ORP1L and PI(3,4)P2 are required for the efficient removal of cholesterol from LELs. Structures of the lipid-binding domain of ORP1 (ORP1-ORD) in complex with cholesterol or PI(4,5)P2 display open conformations essential for ORP function. PI(4,5)P2/PI(3,4)P2 can facilitate ORP1-mediated cholesterol transport by promoting membrane targeting and cholesterol extraction. Thus, our work unveils a distinct mechanism by which PIPs may allosterically enhance OSBP/ORPs-mediated transport of major lipid species such as cholesterol. Phosphatidylinositol phosphates (PIPs) and cholesterol are known to regulate the function of late endosomes and lysosomes (LELs). Here authors unveil a mechanism by which PI(4,5)P2/PI(3,4)P2 allosterically enhances ORP1-mediated cholesterol exit from LELs. [ABSTRACT FROM AUTHOR]

The integrity of the plasma membrane is critical to cell function and survival. Cells have developed multiple mechanisms to repair damaged plasma membranes. A key process during plasma membrane repair is to limit the size of the damage, which is facilitated by the presence of tetraspanin-enriched rings surrounding damage sites. Here, we identify phosphatidylserine-enriched rings surrounding damaged sites of the plasma membrane, resembling tetraspanin-enriched rings. Importantly, the formation of both the phosphatidylserine- and tetraspanin-enriched rings requires phosphatidylserine and its transfer proteins ORP5 and ORP9. Interestingly, ORP9, but not ORP5, is recruited to the damage sites, suggesting cells acquire phosphatidylserine from multiple sources upon plasma membrane damage. We further demonstrate that ORP9 contributes to efficient plasma membrane repair. Our results thus unveil a role for phosphatidylserine and its transfer proteins in facilitating the formation of tetraspanin-enriched macrodomains and plasma membrane repair., (© 2024 Li et al.)

Nuclear lipid droplets (nLDs) are poorly characterized outside of the liver. In this issue, Sołtysik et al. (2020. J. Cell Biol.https://doi.org/10.1083/jcb.202005026) show that seipin is absent from the nucleus but seipin deficiency promotes nLD formation by increasing nuclear phosphatidic acid., (© 2020 Du and Yang.)

Lipid droplets (LDs) are evolutionarily conserved organelles that play important roles in cellular metabolism. Each LD is enclosed by a monolayer of phospholipids, distinct from bilayer membranes. During LD biogenesis and growth, this monolayer of lipids expands by acquiring phospholipids from the endoplasmic reticulum (ER) through nonvesicular mechanisms. Here, in a mini-screen, we find that ORP5, an integral membrane protein of the ER, can localize to ER-LD contact sites upon oleate loading. ORP5 interacts with LDs through its ligand-binding domain, and ORP5 deficiency enhances neutral lipid synthesis and increases the size of LDs. Importantly, there is significantly more phosphatidylinositol-4-phosphate (PI(4)P) and less phosphatidylserine (PS) on LDs in ORP5-deficient cells than in normal cells. The increased presence of PI(4)P on LDs in ORP5-deficient cells requires phosphatidylinositol 4-kinase 2-α. Our results thus demonstrate the existence of PI(4)P on LDs and suggest that LD-associated PI(4)P may be primarily used by ORP5 to deliver PS to LDs., (© 2019 Du et al.)

Generating a cholesterol storage phenotype of Niemann-Pick Type C (NPC) disease is important for investigating the mechanisms of intracellular cholesterol trafficking, as well as screening drugs for potential treatment of NPC disease. The use of the CRISPR/Cas9 technology to knockout specific genes within the genome of mammals has become routine in the past few years. Here, we describe a protocol for producing a cellular NPC cholesterol storage phenotype in HeLa cells using the CRISPR-Cas9 system to disrupt the NPC1 gene. The protocol details the steps for single guide RNA oligo cloning, cell colony selection, and cell line verification by filipin staining and immunoblotting.

Null mutations of the Niemann-Pick type C1 (NPC1) gene cause NPC disease, a lysosomal storage disorder characterized by cholesterol accumulation in late endosomes (LE) and lysosomes (Ly). Nascent or mutated NPC1 is degraded through the ubiquitin-proteasome pathway, but how NPC1 degradation is regulated remains currently unknown. In the present study, we demonstrated a link between NPC1 degradation and the Akt (protein kinase B)/mTOR [mammalian (or mechanistic) target of rapamycin] signalling pathway in cervical cancer cell lines. We provided evidence that activated Akt/mTOR pathway increased NPC1 degradation by ∼50% in C33A cells when compared with SiHa or HeLa cells. NPC1 degradation in C33A cells was reversed when Akt/mTOR activation was blocked by specific inhibitors or when mTORC1 (mTOR complex 1) was disrupted by regulatory associated protein of mTOR (Raptor) knockdown. Importantly, inhibition of the Akt/mTOR pathway led to decreased NPC1 ubiquitination in C33A cells, pointing to a role of Akt/mTOR in the proteasomal degradation of NPC1. Moreover, we found that NPC1 depletion in several cancer cell lines inhibited cell proliferation and migration. Our results uncover Akt as a key regulator of NPC1 degradation and link NPC1 to cancer cell proliferation and migration., (© 2015 Authors; published by Portland Press Limited.)