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1.
    
Niemann-Pick disease type C (NPC), caused by mutations in the NPC1 gene or the NPC2 gene, is characterized by the accumulation of unesterified cholesterol and other lipids in endo/lysosomal compartments. NPC2 is a small, soluble, lysosomal protein that is targeted to this compartment via a mannose 6-phosphate-inhibitable pathway. To obtain insight into the roles of mannose 6-phosphate receptors (MPRs) in NPC2 targeting, we here examine the trafficking and function of NPC2 in fibroblast lines deficient in one or both of the two MPRs, MPR46 and MPR300. We demonstrate that either MPR alone is sufficient to transport NPC2 to the endo/lysosomal compartment, although MPR300 seems to be more efficient than MPR46. In the absence of both MPRs, NPC2 is secreted into the culture medium, and only a small amount of intracellular NPC2 can be detected, mainly in the endoplasmic reticulum. This leads to massive accumulation of unesterified cholesterol in the endo/lysosomal compartment of the MPR46/300-deficient fibroblasts, a phenotype similar to that of the NPC patient fibroblasts. In addition, we observed an upregulation of NPC1 protein and mRNA in the MPR-double-deficient cells. Taken together, our results suggest that the lysosomal targeting of NPC2 is strictly dependent on MPRs in fibroblasts.  相似文献   

2.
    
Mammalian cells acquire most exogenous cholesterol through receptor‐mediated endocytosis of low‐density lipoproteins (LDLs). After internalization, LDL cholesteryl esters are hydrolyzed to release free cholesterol, which then translocates to late endosomes (LEs)/lysosomes (LYs) and incorporates into the membranes by co‐ordinated actions of Niemann‐Pick type C (NPC) 1 and NPC2 proteins. However, how cholesterol exits LEs/LYs and moves to other organelles remain largely unclear. Growing evidence has suggested that nonvesicular transport is critically involved in the post‐endosomal cholesterol trafficking. Numerous sterol‐transfer proteins (STPs) have been identified to mediate directional cholesterol transfer at membrane contact sites (MCSs) formed between 2 closely apposed organelles. In addition, a recent study reveals that lysosome‐peroxisome membrane contact (LPMC) established by a non‐STP synaptotagmin VII and a specific phospholipid phosphatidylinositol 4,5‐bisphosphate also serves as a novel and important path for LDL‐cholesterol trafficking. These findings highlight an essential role of MCSs in intracellular cholesterol transport, and further work is needed to unveil how various routes are regulated and integrated to maintain proper cholesterol distribution and homeostasis in eukaryotic cells.   相似文献   

3.
    
Niemann‐Pick type C (NPC) disease is a fatal neurodegenerative disorder characterized by over‐accumulation of low‐density lipoprotein‐derived cholesterol and glycosphingolipids in late endosomes/lysosomes (LE/L) throughout the body. Human mutations in either NPC1 or NPC2 genes have been directly associated with impaired cholesterol efflux from LE/L. Independent from its role in cholesterol homeostasis and its NPC2 partner, NPC1 was unexpectedly identified as a critical player controlling intracellular entry of filoviruses such as Ebola. In this study, a yeast three‐hybrid system revealed that the NPC1 cytoplasmic tail directly interacts with the clathrin adaptor protein AP‐1 via its acidic/di‐leucine motif. Consequently, a nonfunctional AP‐1A cytosolic complex resulted in a typical NPC‐like phenotype mainly due to a direct impairment of NPC1 trafficking to LE/L and a partial secretion of NPC2. Furthermore, the mislocalization of NPC1 was not due to cholesterol accumulation in LE/L, as it was not rescued upon treatment with Mβ‐cyclodextrin, which almost completely eliminated intracellular free cholesterol. Our cumulative data demonstrate that the cytosolic clathrin adaptor AP‐1A is essential for the lysosomal targeting and function of NPC1 and NPC2.  相似文献   

4.
    
The present nomenclature of the splice variants of the lysosome-associated membrane protein type 2 (LAMP-2) is confusing. The LAMP-2a isoform is uniformly named in human, chicken, and mouse, but the LAMP-2b and LAMP-2c isoforms are switched in human as compared with mouse and chicken. We propose to change the nomenclature of the chicken and mouse b and c isoforms to agree with that currently used for the human isoforms. To avoid confusion in the literature, we further propose to adopt the use of capital letters for the updated nomenclature of all the isoforms in all three species: LAMP-2A, LAMP-2B, and LAMP-2C.  相似文献   

5.
    
Cholesterol homeostasis is critical for cell function and human health. Cholesterol is heterogeneously distributed among cellular membranes, with the redistribution of endocytosed dietary cholesterol playing a pivotal role in the regulation of cholesterol homeostasis. While gaps remain in our understanding of intracellular dietary cholesterol transport, a highly complex network of pathways is starting to emerge, often involving inter‐dependent vesicular and non‐vesicular transport mechanisms. The last decade has seen a surge in interest in non‐vesicular transport and inter‐organellar communication at membrane contact sites. By providing platforms for protein interactions, signalling events, lipid exchange and calcium flux, membrane contact sites (MCS) are now appreciated as controlling the fate of large amounts of lipid and play central roles in the regulation and co‐ordination of endocytic trafficking. Here, we review the role of MCS in multiple pathways for cholesterol export from the endocytic pathway and highlight the intriguing interplay between vesicular and non‐vesicular transport mechanisms and relationship with neurodegenerative disease.  相似文献   

6.
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7.
The Niemann–Pick C protein (NPC1) is required for cholesterol transport from late endosomes and lysosomes to other cellular membranes. Mutations in NPC1 cause lysosomal lipid storage and progressive neurological degeneration. Cloning of the NPC1 gene has given us tools with which to investigate the function of this putative cholesterol transporter. Here, we discuss recent studies indicating that NPC1 is not a cholesterol-specific transport molecule. Instead, NPC1 appears to be required for the vesicular shuttling of both lipids and fluid-phase constituents from multivesicular late endosomes to destinations such as the trans -Golgi network.  相似文献   

8.
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9.
    
The multivesicular body (MVB) is a specialized Rab7+ late endosome (LE) containing multiple intralumenal vesicles that function in targeting ubiquitinylated cell surface proteins to the lysosome for degradation. African trypanosomes lack a morphologically well‐defined MVB, but contain orthologs of the ESCRT (Endosomal Sorting Complex Required for Transport) machinery that mediates MVB formation. We investigate the role of TbVps23, an early ESCRT component, and TbVps4, the terminal ESCRT ATPase, in lysosomal trafficking in bloodstream form trypanosomes. Both localize to the TbRab7+ LE and RNAi silencing of each rapidly blocks growth. TbVps4 silencing results in approximately threefold accumulation of TbVps23 at the LE, consistent with blocking terminal ESCRT disassembly. Trafficking of endocytic and biosynthetic cargo, but not default lysosomal reporters, is also negatively affected. Others reported that TbVps23 mediates ubiquitin‐dependent lysosomal degradation of invariant surface glycoproteins (ISG65) (Leung et al., Traffic 2008;9:1698–1716). In contrast, we find that TbVps23 ablation does not affect ISG65 turnover, while TbVps4 silencing markedly enhances lysosomal degradation. We propose several models to accommodate these results, including that the ESCRT machinery actually retrieves ISG65 from the LE to earlier endocytic compartments, and in its absence ISG65 traffics more efficiently to the lysosome. Overall, these results confirm that the ESCRT machinery is essential in Trypanosoma brucei and plays important and novel role(s) in LE function in trypanosomes .  相似文献   

10.
    
The role of the endoplasmic reticulum (ER) in phagocytosis has been the subject of debate for over a decade. Proteomic determinations and dynamic microscopy of live cells led to conflicting conclusions. Recent insights into the existence of a variety of membrane contact sites (MCS) may help reconcile the seemingly disparate views. Specifically, earlier results can be rationalized considering that the ER forms specialized MCS with nascent and maturing phagosomes, without undergoing fusion. The composition and function of documented ER‐to‐phagosome contact sites is described. In addition, we speculate about the possible existence of additional phagosomal contact sites, based on available knowledge of interactions between the ER and other endocytic compartments. The interaction between phagosomes and the ER has been the subject of debate. Earlier observations that led to the suggestion that the ER fuses with the phagosomal membrane can now be explained in the light of recent evidence that intimate contacts form between the two organelles.  相似文献   

11.
Cholesterol is an important precursor for numerous biologically active molecules, and it plays a major role in membrane structure and function. Cholesterol can be endogenously synthesized or exogenously taken up via the endocytic vesicle system and subsequently delivered to post-endo/lysosomal sites including the plasma membrane and the endoplasmic reticulum. Niemann–Pick C (NPC) disease results in the accumulation of exogenously-derived cholesterol, as well as other lipids, in late endosomes and lysosomes (LE/LY). Identification of the two genes that underlie NPC disease, NPC1 and NPC2, has focused attention on the mechanisms by which lipids, in particular cholesterol, are transported out of the LE/LY compartment. This review discusses the role of the NPC2 protein in cholesterol transport, and the potential for concerted action of NPC1 and NPC2 in regulating normal intracellular cholesterol homeostasis.  相似文献   

12.
    
Many studies have demonstrated a role for ubiquitin (Ub) in the down-regulation of cell surface proteins. In yeast, down-regulation is marked by the internalization of proteins, followed by their delivery to the lumen of the vacuole where both the cytosolic and lumenal domains are degraded. It is generally believed that the regulatory step of this process is internalization from the plasma membrane and that protein delivery to the lysosome or vacuole is by default. By separating the process of internalization from degradation, we demonstrate that incorporation of proteins into intralumenal vesicles represents a distinct sorting step along the endocytic pathway that is controlled by recognition of ubiquitin. We show that attachment of a single ubiquitin can serve as a specific sorting signal for the degradative pathway by redirecting recycling Golgi proteins and resident vacuolar proteins into intralumenal vesicles of the yeast vacuole. This pathway is independent of PtdIns(3,5) P2 and does not rely on the specific composition of transmembrane domain segments. These data provide a physiological basis for how ubiquitination of cell surface proteins guides their degradation and removal from the recycling pathway.  相似文献   

13.
    
Bis(monoacylglycero)phosphate (BMP) is a unique phospholipid (PL) preferentially found in late endosomal membranes, where it forms specialized lipid domains. Recently, using cultured macrophages treated with anti-BMP antibody, we showed that BMP-rich domains are involved in cholesterol homeostasis. We had previously stressed the high propensity of BMP to accumulate docosahexaenoic acid (DHA), compared with other PUFAs. Because phosphatidylglycerol (PG) was reported as a precursor for BMP synthesis in RAW macrophages, we examined the effects of PG supplementation on both FA composition and amount of BMP in this cell line. Supplementation with dioleoyl-PG (18:1/18:1-PG) induced BMP accumulation, together with an increase of oleate proportion. Supplementation with high concentrations of didocosahexaenoyl-PG (22:6/22:6-PG) led to a marked enrichment of DHA in BMP, resulting in the formation of diDHA molecular species. However, the amount of BMP was selectively decreased. Similar effects were observed after supplementation with high concentrations of nonesterified DHA. Addition of vitamin E prevented the decrease of BMP and further increased its DHA content. Supplementation with 22:6/22:6-PG promoted BMP accumulation with an enhanced proportion of 22:6/22:6-BMP. DHA-rich BMP was significantly degraded after cell exposure to oxidant conditions, in contrast to oleic acid-rich BMP, which was not affected. Using a cell-free system, we showed that 22:6/22:6-BMP is highly oxidizable and partially protects cholesterol oxidation, compared with 18:1/18:1-BMP. Our data suggest that high DHA content in BMP led to specific degradation of this PL, possibly through the diDHA molecular species, which is very prone to peroxidation and, as such, a potential antioxidant in its immediate vicinity.  相似文献   

14.
    
Ubiquitin (Ub) attachment to cell surface proteins causes their lysosomal degradation by incorporating them into lumenal membranes of multivesicular bodies (MVBs). Two yeast endosomal protein complexes have been proposed as Ub-sorting \"receptors,\" the Vps27-Hse1 complex and the ESCRT-I complex. We used NMR spectroscopy and mutagenesis studies to map the Ub-binding surface for Vps27 and Vps23. Mutations in Ub that ablate only Vps27 binding or Vps23 binding blocked the ability of Ub to serve as an MVB sorting signal, supporting the idea that both the Vps27-Hse1 and ESCRT-I complexes interact with ubiquitinated cargo. Vps27 also bound Vps23 directly via two PSDP motifs present within the Vps27 COOH terminus. Loss of Vps27-Vps23 association led to less efficient sorting into the endosomal lumen. However, sorting of vacuolar proteases or the overall biogenesis of the MVB were not grossly affected. In contrast, disrupting interaction between Vps27 and Hse1 caused severe defects in carboxy peptidase Y sorting and MVB formation. These results indicate that both Ub-sorting complexes are coupled for efficient recognition of ubiquitinated cargo.  相似文献   

15.
    
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.  相似文献   

16.
Endosomal compartments sort and deliver exogenous lipoprotein-derived cholesterol to the endoplasmic reticulum for regulating cellular cholesterol homeostasis. A large number of studies have focused on the removal of endosomal cholesterol, since its accumulation leads to devastating human diseases. Recent studies suggest that cytoplasmic sterol-binding proteins may be involved in endosomal cholesterol transport. In particular, endosome/lysosome-localized or -associated cholesterol-binding proteins may serve as key mediators of cholesterol removal in a non-vesicular manner. Further characterization of these cholesterol-binding proteins will shed light on the molecular mechanisms that regulate endosomal cholesterol sorting.  相似文献   

17.
    
The ionic nature of endosomes varies considerably in character along the endocytic pathway. Counter-ion flux across the limiting membrane of endosomes has long been considered essential for full acidification and normal endosome/lysosomal function. The proximal functions of luminal ions, however, have been difficult to assess. The recent development of transgenic mice carrying mutations in the intracellular chloride channels (ClCs) has provided a tool to uncouple Cl(-) influx from endosomal acidification. Intriguingly, many of the defects of the endo-lysomal system attributed to aberrant pH persist in the Cl(-)-deficient mice implying a direct regulatory role for Cl(-) influx in endosome function. These observations may begin to explain the abundance of endosomal ion transporters, including ClCs, sodium-proton exchangers, two-pore channels and mucolipins, that have been localized to endo-lysosomes, and the extensive changes in luminal ion composition therein. In this review, we summarize what is known regarding the mediators of endosomal ion flux, and discuss the implications of changing ionic content on endo-lysosomal function.  相似文献   

18.
    
Niemann-Pick, type C1 (NPC1) is a fatal, neurodegenerative disease, which belongs to the family of lysosomal diseases. In NPC1, endo/lysosomal accumulation of unesterified cholesterol and sphingolipids arise from improper intracellular trafficking resulting in multi-organ dysfunction. With the proximity between the brain and cerebrospinal fluid (CSF), performing differential proteomics provides a means to shed light to changes occurring in the brain. In this study, CSF samples obtained from NPC1 individuals and unaffected controls were used for protein biomarker identification. A subset of these individuals with NPC1 are being treated with miglustat, a glycosphingolipid synthesis inhibitor. Of the 300 identified proteins, 71 proteins were altered in individuals with NPC1 compared to controls including cathepsin D, and members of the complement family. Included are a report of 10 potential markers for monitoring therapeutic treatment. We observed that pro-neuropeptide Y (NPY) was significantly increased in NPC1 individuals relative to healthy controls; however, individuals treated with miglustat displayed levels comparable to healthy controls. In further investigation, NPY levels in a NPC1 mouse model corroborated our findings. We posit that NPY could be a potential therapeutic target for NPC1 due to its multiple roles in the central nervous system such as attenuating neuroinflammation and reducing excitotoxicity.  相似文献   

19.
    
Two key questions in the autophagy field are the mechanisms that underlie the signals for autophagy initiation and the source of membrane for expansion of the nascent membrane, the phagophore. In this review, we discuss recent findings highlighting the role of the classical endosomal pathway, from plasma membrane to lysosome, in the formation and expansion of the phagophore and subsequent degradation of the autophagosome contents. We also highlight the striking conservation of regulatory factors between the two pathways, including those regulating membrane budding and fusion, and the role of the lysosome in sensing the nutrient status of the cell, regulating mTORC1 activity, and ultimately the initiation of autophagy. Editor's suggested further reading in BioEssays The evolution of dynamin to regulate clathrin‐mediated endocytosis Abstract  相似文献   

20.
    
The obligate intracellular liver stage of the Plasmodium parasite represents a bottleneck in the parasite life cycle and remains a promising target for therapeutic intervention. During this stage, parasites undergo dramatic morphological changes and achieve one of the fastest replication rates among eukaryotic species. Nevertheless, relatively little is known about the parasite interactions with the host hepatocyte. Using immunofluorescence, live cell imaging and electron microscopy, we show that Plasmodium berghei parasites are surrounded by vesicles from the host late endocytic pathway. We found that these vesicles are acidic and contain the membrane markers Rab7a, CD63 and LAMP1. When host cell vesicle acidification was disrupted using ammonium chloride or Concanamycin A during the late liver stage of infection, parasite survival was not affected, but schizont size was significantly decreased. Furthermore, when the host cell endocytic pathway was loaded with BSA-gold, gold particles were found within the parasite cytoplasm, showing the transport of material from the host endocytic pathway toward the parasite interior. These observations reveal a novel Plasmodium-host interaction and suggest that vesicles from the host endolysosomal pathway could represent an important source of nutrients exploited by the fast-growing late liver stage parasites.  相似文献   

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