The AAA ATPase VPS4/SKD1 Regulates Endosomal Cholesterol Trafficking Independently of ESCRT‐III

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.     

Molecular basis for sterol transport by StART‐like lipid transfer domains

Lipid transport proteins at membrane contact sites, where two organelles are closely apposed, play key roles in trafficking lipids between cellular compartments while distinct membrane compositions for each organelle are maintained. Understanding the mechanisms underlying non‐vesicular lipid trafficking requires characterization of the lipid transporters residing at contact sites. Here, we show that the mammalian proteins in the lipid transfer proteins anchored at a membrane contact site (LAM) family, called GRAMD1a‐c, transfer sterols with similar efficiency as the yeast orthologues, which have known roles in sterol transport. Moreover, we have determined the structure of a lipid transfer domain of the yeast LAM protein Ysp2p, both in its apo‐bound and sterol‐bound forms, at 2.0 Å resolution. It folds into a truncated version of the steroidogenic acute regulatory protein‐related lipid transfer (StART) domain, resembling a lidded cup in overall shape. Ergosterol binds within the cup, with its 3‐hydroxy group interacting with protein indirectly via a water network at the cup bottom. This ligand binding mode likely is conserved for the other LAM proteins and for StART domains transferring sterols.     

Short-range intracellular trafficking of small molecules across endoplasmic reticulum junctions

Intracellular trafficking is not mediated exclusively by vesicles. Additional, non-vesicular mechanisms transport material, in particular small molecules such as lipids and Ca(2+) ions, from one organelle to another. This transport occurs at narrow cytoplasmic gaps called membrane contact sites (MCSs), at which two organelles come into close apposition. Despite the conservation of these structures throughout evolution, little is known about this transport, largely because of a lack of knowledge of almost all molecular components of MCSs. Recently, this situation has started to change because the structural proteins that bridge an MCS are now known in a single case, and proteins implicated in lipid trafficking have been localized to MCSs. In the light of these advances, I hypothesize that the endoplasmic reticulum has a central role in the trafficking of lipids and ions by forming a network of MCSs with most other intracellular organelles.     

Multiple Surface Regions on the Niemann-Pick C2 Protein Facilitate Intracellular Cholesterol Transport

The cholesterol storage disorder Niemann-Pick type C (NPC) disease is caused by defects in either of two late endosomal/lysosomal proteins, NPC1 and NPC2. NPC2 is a 16-kDa soluble protein that binds cholesterol in a 1:1 stoichiometry and can transfer cholesterol between membranes by a mechanism that involves protein-membrane interactions. To examine the structural basis of NPC2 function in cholesterol trafficking, a series of point mutations were generated across the surface of the protein. Several NPC2 mutants exhibited deficient sterol transport properties in a set of fluorescence-based assays. Notably, these mutants were also unable to promote egress of accumulated intracellular cholesterol from npc2^−/− fibroblasts. The mutations mapped to several regions on the protein surface, suggesting that NPC2 can bind to more than one membrane simultaneously. Indeed, we have previously demonstrated that WT NPC2 promotes vesicle-vesicle interactions. These interactions were abrogated, however, by mutations causing defective sterol transfer properties. Molecular modeling shows that NPC2 is highly plastic, with several intense positively charged regions across the surface that could interact favorably with negatively charged membrane phospholipids. The point mutations generated in this study caused changes in NPC2 surface charge distribution with minimal conformational changes. The plasticity, coupled with membrane flexibility, probably allows for multiple cholesterol transfer routes. Thus, we hypothesize that, in part, NPC2 rapidly traffics cholesterol between closely appositioned membranes within the multilamellar interior of late endosomal/lysosomal proteins, ultimately effecting cholesterol egress from this compartment.     

Lipids at membrane contact sites: cell signaling and ion transport

Communication between organelles is essential to coordinate cellular functions and the cell's response to physiological and pathological stimuli. Organellar communication occurs at membrane contact sites (MCSs), where the endoplasmic reticulum (ER) membrane is tethered to cellular organelle membranes by specific tether proteins and where lipid transfer proteins and cell signaling proteins are located. MCSs have many cellular functions and are the sites of lipid and ion transfer between organelles and generation of second messengers. This review discusses several aspects of MCSs in the context of lipid transfer, formation of lipid domains, generation of Ca²⁺ and cAMP second messengers, and regulation of ion transporters by lipids.     

Niemann-Pick Type C2 protein contributes to the transport of endosomal cholesterol to mitochondria without interacting with NPC1

Mitochondrial cholesterol is maintained within a narrow range to regulate steroid and oxysterol synthesis and to ensure mitochondrial function. Mitochondria acquire cholesterol through several pathways from different cellular pools. Here we have characterized mitochondrial import of endosomal cholesterol using Chinese hamster ovary cells expressing a CYP11A1 fusion protein that converts cholesterol to pregnenolone at the mitochondrial inner membrane. RNA interference-mediated depletion of the voltage-dependent anion channel 1 in the mitochondrial outer membrane or of Niemann-Pick Type C2 (NPC2) in the endosome lumen decreased arrival of cholesterol at the mitochondrial inner membrane. Expression of NPC2 mutants unable to transfer cholesterol to NPC1 still restored mitochondrial cholesterol import in NPC2-depleted cells. Transport assays in semi-permeabilized cells showed nonvesicular cholesterol trafficking directly from endosomes to mitochondria that did not require cytosolic transport proteins but that was reduced in the absence of NPC2. Our findings indicate that NPC2 delivers cholesterol to the perimeter membrane of late endosomes, where it becomes available for transport to mitochondria without requiring NPC1.     

Sterol, protein and lipid trafficking in Chinese hamster ovary cells with Niemann-Pick type C1 defect

We studied the trafficking of sterols, lipids and proteins in Niemann-Pick type C (NPC) cells. The NPC is an inherited disorder involving the accumulation of sterol and lipids in modified late-endosome/lysosome-like storage organelles. Most sterol accumulation studies in NPC cells have been carried out using low-density lipoprotein (LDL) as the sterol source, and it has been shown that sterol efflux from late endosomes is impaired in NPC cells. In this study, we used a fluorescent sterol analog, dehydroergosterol, which can be quickly and efficiently delivered to the plasma membrane. Thus, we were able to study the trafficking kinetics of the non-LDL-derived sterol pool, and we found that dehydroergosterol accumulates in the storage organelles over the course of several hours in NPC cells. We also found that dialkylindocarbocyanine lipid-mimetic analogs that recycle efficiently from early endosomes in wild-type cells are targeted to late endosomal organelles in NPC cells, and transferrin receptors recycle slowly and inefficiently in NPC cells. These data are consistent with multiple trafficking defects in both early and late endosomes in NPC cells.     

Deficiency of Niemann-Pick Type C-1 Protein Impairs Release of Human Immunodeficiency Virus Type 1 and Results in Gag Accumulation in Late Endosomal/Lysosomal Compartments

Human immunodeficiency virus type 1 (HIV-1) relies on cholesterol-laden lipid raft membrane microdomains for entry into and egress out of susceptible cells. In the present study, we examine the need for intracellular cholesterol trafficking pathways with respect to HIV-1 biogenesis using Niemann-Pick type C-1 (NPC1)-deficient (NPCD) cells, wherein these pathways are severely compromised, causing massive accumulation of cholesterol in late endosomal/lysosomal (LE/L) compartments. We have found that induction of an NPC disease-like phenotype through treatment of various cell types with the commonly used hydrophobic amine drug U18666A resulted in profound suppression of HIV-1 release. Further, NPCD Epstein-Barr virus-transformed B lymphocytes and fibroblasts from patients with NPC disease infected with a CD4-independent strain of HIV-1 or transfected with an HIV-1 proviral clone, respectively, replicated HIV-1 poorly compared to normal cells. Infection of the NPCD fibroblasts with a vesicular stomatitis virus G-pseudotyped strain of HIV-1 produced similar results, suggesting a postentry block to HIV-1 replication in these cells. Examination of these cells using confocal microscopy showed an accumulation and stabilization of Gag in LE/L compartments. Additionally, normal HIV-1 production could be restored in NPCD cells upon expression of a functional NPC1 protein, and overexpression of NPC1 increased HIV-1 release. Taken together, our findings demonstrate that intact intracellular cholesterol trafficking pathways mediated by NPC1 are needed for efficient HIV-1 production.Human immunodeficiency virus type 1 (HIV-1) is a complex retrovirus highly dependent upon a myriad of cellular mechanisms for successful virus replication. Cholesterol plays a pivotal role throughout the HIV-1 life cycle (23, 40, 41, 64). HIV-1 entry, assembly, and budding processes occur at cholesterol-enriched membrane microdomains known as lipid rafts, and depletion of cellular cholesterol markedly and specifically reduces HIV-1 particle production. Virion-associated cholesterol is required for fusion and subsequent infection of susceptible cells (41), and cholesterol-sequestering drugs, such as β-cyclodextrin, render the virus incompetent for cell entry (4, 25, 57). Therefore, intracellular cholesterol trafficking pathways that allow nascent HIV-1 particles to acquire lipids appear critical for virus replication.Recent evidence supports a critical role for cholesterol trafficking and homeostasis in viral replication, showing that the HIV-1 accessory protein Nef increases synthesis and transport of cholesterol to both lipid rafts and progeny virions and induces multiple genes involved in cholesterol synthesis (80, 88). More recent studies have revealed that binding of Nef to the ATP-binding cassette transporter A1 (ABCA1) leads to impairment of ABCA1-dependent cholesterol efflux and an accumulation of lipids within the cell (51).Mammalian cells acquire cholesterol primarily from endocytosed low-density lipoproteins (LDL). The Niemann-Pick type C-1 (NPC1) protein is well known for its role in intracellular trafficking of LDL-derived free unesterified cholesterol. Dysfunctional NPC1 activity leads to development of NPC disease, a rare, autosomal recessive, neurodegenerative disorder characterized by the massive accumulation of cholesterol and glycosphingolipids in late endosomal/lysosomal (LE/L) compartments (61). In normal cells, endocytosed LDLs are delivered to the LE/Ls, where they are hydrolyzed and free cholesterol is released. Homeostasis is achieved when cholesterol is then rapidly transported out of the LE/Ls to the plasma membrane and endoplasmic reticulum (ER) (17, 19, 42, 73, 85), or first to the trans-Golgi (TG) network (TGN) and then to the ER (76). In NPC1-deficient (NPCD) cells, the cholesterol does not exit the endocytic pathway, resulting in its accumulation within LE/L structures.In 95% of NPC patients, the disease is caused by mutations in the NPC1 gene, while the remaining 5% harbor mutations in the NPC2 gene (50, 72, 79). One of the most frequently found and extensively characterized NPC1 mutations is the I1061T mutation (37, 38, 86). This mutation results in misfolding of the NPC1 protein, leading to its degradation and causing an 85% decrease in cellular NPC1 expression (20). Cells with such low levels of functional NPC1 maintain only 38% of normal sphingomyelinase activity and have impaired cholesterol esterification and trafficking.NPC1 is a large, multispanning protein that resides in the limiting membrane of the LE and binds cholesterol via its N-terminal domain (31). While the complete physiological function of NPC1 is still unclear, NPC1 does share homology with the resistance-nodulation-division family of prokaryotic permeases and may function as a transmembrane efflux pump to transport cargos in LEs (9, 75). Other studies suggest that NPC1 might also function in vesicle-mediated pathways for cargo transportation from LEs to other intracellular sites (21, 33). Recent studies by Infante et al. have propelled forward our understanding of how NPC1 works together with NPC2, also known to bind cholesterol, to support cholesterol efflux from the LE (32). Their findings provide a basis for either of two possible models, with respect to cholesterol trafficking: (i) NPC1 binds cholesterol found within the LE and mediates either direct export or transfer to NPC2 for delivery to a cholesterol efflux transporter, such as ABCA1; or (ii) NPC2 is the first to bind cholesterol and then mediate its delivery to NPC1 for direct export or transfer to ABCA1. These recent findings underscore the highly critical role of these proteins in maintaining intracellular cholesterol homeostasis.In addition to its role in sterol trafficking, some studies suggest that the NPC pathway may be directly involved in trafficking multiple proteins from LE/L compartments. LEs act as sorting stations to deliver endocytosed molecules to L''s for degradation, while at the same time retrieving other classes of proteins and lipids for transport back to nondegradative compartments (3, 14, 15, 28, 63, 69, 78). LE compartments also serve as sorting stations for HIV-1 viral proteins and represent a major site for HIV-1 assembly and budding (7, 12, 16, 22, 24, 57, 59).The endosomal trafficking defects observed in NPCD cells extend to proteins such as IGF2/MPR, NPC1, and annexin II, all of which utilize the endosomal recycling pathway (42, 74). Electron microscopy studies have shown that within the LEs of NPCD cells these proteins are trapped in the cholesterol-enriched membrane-bound vesicular structures (47). Cholesterol and glycosphingolipid accumulation within NPCD cells appears to disrupt Rab9 GTPase function in LE-to-TGN transport, trapping Rab9-associated proteins, such as vimentin, Tip47, and the mannose-6-phosphate receptor in LEs (18, 83). Overexpression of Rab7 and Rab9 GTPases can reverse the cholesterol accumulation phenotype caused by NPCD (8, 84). These observations suggest that NPC1, directly or indirectly, plays a role in protein export from LEs. It is unknown whether NPC1 is involved in the export of HIV-1 proteins from LEs; however, the Rab9 GTPase-mediated pathway is known to be required for HIV-1 replication (53). This strongly suggests that HIV assembly will be hindered when the NPC pathway is disrupted.Given the function of NPC1 in mediating intracellular cholesterol trafficking within the LE and given the need of HIV-1 for cholesterol, NPC1 involvement in HIV-1 biogenesis is highly likely. In the present study, using cells treated with U18666A or NPCD cells, we show that impaired NPC1 function results in profound suppression of HIV-1 replication. Further, our findings demonstrate that the NPC1 protein is essential for proper trafficking of the HIV-1 Gag protein during the late stages of assembly and budding. It appears that in NPCD cells, in which cholesterol and cellular proteins accumulate in LE/L compartments, the viral Gag protein fails to traffic properly and accumulates within these compartments, resulting in decreased particle production. Our findings not only reinforce the dependence of HIV-1 on cholesterol homeostasis but also support a role for NPC1 in HIV-1 viral protein trafficking and particle release from infected cells.     

Organelle contacts: Sub‐organelle zones to facilitate rapid and accurate inter‐organelle trafficking of lipids

When one person wants to communicate securely with another, he/she should contact the other person directly. This rule applies not only to human society, but also to the intracellular micro‐society. In the past two decades, it has become increasingly clear that the sub‐organelle regions called membrane contact sites (MCSs) are pivotal for inter‐organelle transport of lipids in cells, as highlighted in the thematic review series “Interorganelle trafficking of lipids” held in Traffic in 2014–2015. In this commentary, we will describe how the currently prevailing model for lipid trafficking at MCSs was generated, and comment on three important issues that have not been explored: (a1) the principles guiding the generation of an asymmetrical inter‐organelle flow of lipids in cells, (b2) the advantages in lipid trafficking at organelle contacts, and (c3) the dynamic network of inter‐organelle lipid trafficking.     

Modulation of cellular cholesterol transport and homeostasis by Rab11

To analyze the contribution of vesicular trafficking pathways in cellular cholesterol transport we examined the effects of selected endosomal Rab proteins on cholesterol distribution by filipin staining. Transient overexpression of Rab11 resulted in prominent accumulation of free cholesterol in Rab11-positive organelles that sequestered transferrin receptors and internalized transferrin. Sphingolipids were selectively redistributed as pyrene-sphingomyelin and sulfatide cosequestered with Rab11-positive endosomes, whereas globotriaosyl ceramide and GM2 ganglioside did not. Rab11 overexpression did not perturb the transport of 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine-perchlorate–labeled low-density lipoprotein (LDL) to late endosomes or the Niemann-Pick type C1 (NPC1)-induced late endosomal cholesterol clearance in NPC patient cells. However, Rab11 overexpression inhibited cellular cholesterol esterification in an LDL-independent manner. This effect could be overcome by introducing cholesterol to the plasma membrane by using cyclodextrin as a carrier. These results suggest that in Rab11-overexpressing cells, deposition of cholesterol in recycling endosomes results in its impaired esterification, presumably due to defective recycling of cholesterol to the plasma membrane. The findings point to the importance of the recycling endosomes in regulating cholesterol and sphingolipid trafficking and cellular cholesterol homeostasis.     

Cnm1: A bridge between mitochondria and nuclear ER

Few membrane contact sites have been defined at the molecular level. By using a high-throughput, microscopy-based screen, Eisenberg-Bord, Zung et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202104100) identify Cnm1 as a novel tethering protein that mediates contact between mitochondria and the nuclear ER in response to phospholipid levels.

Organelles communicate through the exchange of biological materials by vesicular trafficking or at sites of close membrane apposition known as membrane contact sites (MCSs). While the molecular machinery mediating vesicular trafficking has been well characterized, our knowledge of the molecules involved in forming and regulating MCSs is limited. MCSs physically tether two or more organelles via protein–protein or protein–lipid interactions, contain defined proteomes, and perform specific biological functions (1). While MCSs have been appreciated microscopically since the 1950s, only recently have advances in technology permitted the discovery of the molecular composition of some MCSs (2). A major breakthrough occurred when a synthetic biology screen identified the ER–mitochondria encounter structure (ERMES), which forms an MCS between the ER and mitochondria (3). ERMES has since been shown to be involved in phospholipid transport between mitochondria and the ER (4). While ERMES is one of the best characterized MCSs, there are still many questions as to the precise molecules being transported at ER–mitochondria contacts and how directionality of transport is achieved. Subsequent studies using split fluorescent proteins revealed that nearly all organelles appear to form MCSs of some kind (5). Thus, despite progress in defining the components and functions of a few MCSs, there are still many MCSs whose molecular identities are completely unknown.Recently, a study in mammalian cells identified an MCS between the nucleus and mitochondria that plays a role in adapting cells to stress via the mitochondrial retrograde signaling response (6). The proteins that form this MCS are not conserved in yeast, however, suggesting that alternative mechanisms for nucleus–mitochondria contacts exist in other organisms. In this issue, Eisenberg-Bord, Zung et al., set out to identify proteins involved in forming an MCS between mitochondria and the nuclear ER that is distinct from ERMES-mediated ER–mitochondria contacts (7). First, high-resolution cryo-electron tomographs revealed that mitochondria form contacts with the nucleus that have an average separation of ∼20 nm, which is within the expected range for a bona fide MCS (1). To identify the molecular composition of this contact site, the authors generated a synthetic reporter that is specific to nucleus-mitochondria contacts by fusing one part of a split fluorescent protein to an outer mitochondrial membrane protein and the other to a peripheral nuclear protein. A high-throughput, microscopy-based genetic screen was then used to compare the localization of the synthetic reporter to fluorescently tagged versions of all yeast proteins. Candidates were refined by determining which proteins caused an expansion of the nucleus–mitochondria contact site upon overexpression, a phenotype that has been observed with other MCS proteins (8). Based on these results, the best candidate for a molecular tether between mitochondria and the nucleus was Ybr063c.Ybr063c is a 46-kD nonessential protein of uncharacterized function that contains predicted transmembrane domains. The authors first demonstrated that Ybr063c is an integral membrane protein residing on the nuclear membrane. In support of Ybr063c forming a nucleus–mitochondria contact site that is distinct from ERMES, Ybr063c did not colocalize with ERMES subunits nor did overexpression of Ybr063c alter the size of ERMES patches. Remarkably, overexpression of Ybr063c resulted in the mitochondrial network becoming tightly associated with the nuclear membrane. Based on these results, the authors concluded that Ybr063c functions as a molecular tether between mitochondria and the nucleus and the protein was renamed Cnm1 for contact nucleus mitochondria 1.Through further genetic screens, Eisenberg-Bord, Zung et al., identified several genes that are required to cluster mitochondria around the nucleus when Cnm1 is overexpressed. Interestingly, several of these genes are known to function in phosphatidylcholine (PC) metabolism. Deletion of these components resulted in a decrease in Cnm1 expression, which alters the extent of nucleus-mitochondria contacts. Overexpression of Cnm1 in genetic conditions that reduce PC levels resulted in exaggerated growth defects. These results raise the possibility that Cnm1-mediated nuclear–mitochondria contacts may be involved in the transport of PC from the ER to mitochondria. Thus, while the functional importance is unknown, Cnm1-mediated nuclear–mitochondria contacts respond to PC levels.The genetic screens also identified a single resident mitochondrial protein, Tom70, as affecting the ability of overexpressed Cnm1 to cluster mitochondria around the nucleus. Subsequent experiments demonstrated that localization of Cnm1 to the nuclear membrane and Tom70 to the mitochondrial membrane is required to tether mitochondria to the nucleus upon overexpression of Cnm1. Thus, Cnm1 and Tom70 mediate an MCS between mitochondria and the nucleus.The identification of Cnm1-mediated nucleus–mitochondria contacts opens many questions about the function and composition of the contact site and how it operates in the broader context of mitochondrial–nuclear communication. While identifying the functions of MCSs has proven challenging, the genetic screens conducted in this study provide an excellent starting point by elucidating a link between Cnm1 and PC metabolism. The authors propose that Cnm1-mediated contacts could function in the direct transport of PC from the ER to mitochondria (Fig. 1). In this model, ERMES, which likely functions in earlier steps of PC synthesis by transporting phosphatidylethanolamine (PE) or phosphatidylserine (PS), would have a distinct but related function in organizing and maintaining a pipeline for the transport of lipids between the ER and mitochondria (Fig. 1). This model is speculative, however, and future experiments will be necessary to define the role of Cnm1 in PC metabolism.Open in a separate windowFigure 1.The ER and vacuole form multiple MCSs with mitochondria in budding yeast. The ER is depicted in green, and the mitochondrial network is depicted in gray. ERMES mediates an MCS between tubular ER and mitochondria. In addition to functions that are distinct from lipid trafficking, ERMES-mediated MCSs likely function to transport PS or PE between the organelles. Cnm1 mediates an MCS specifically between the nuclear ER and mitochondria and potentially functions in PC transport. The Vps13-Mcp1 vCLAMP mediates an MCS between mitochondria and the vacuole that likely functions in lipid transport and may have redundant functions with ERMES. The Vps39-Tom40 vCLAMP is a separate MCS between mitochondria and the vacuole that responds to different stress conditions, though its function is unknown.There is a growing body of evidence that two organelles can form multiple MCSs that are spatially and functionally distinct. In addition to ERMES and Cnm1-mediated mitochondria–ER contacts, in yeast, two distinct MCSs have been described between mitochondria and the vacuole that are referred to as vacuolar and mitochondrial patches, or vCLAMPs. One, mediated by Vam6 and Tom40, has been implicated in responding to cellular stress while the other, mediated by Mcp1 and Vps13, may have overlapping functions with the ERMES complex (8, 9; Fig. 1). Interestingly, many of the proteins present at MCSs have been shown to be multifunctional (2). For example, the vCLAMP component Vam6 is also a subunit of the homotypic fusion and protein-sorting (HOPS) complex while its binding partner Tom40 is the central subunit of the translocase of outer membrane (TOM) complex (8). Thus, while these complexes have distinct biological functions in vacuolar protein sorting and mitochondrial protein import respectively, individual subunits have moonlighting functions in the formation, and perhaps function, of MCSs. Eisenberg-Bord, Zung et al., now reveal that Tom70, another component of the TOM complex, also plays a role in the formation of nucleus–mitochondria contacts. This raises the exciting possibility that cells use these multifunctional proteins to coordinate functions such as mitochondrial protein import with lipid trafficking. A crucial next step will be to determine how the multiple functions of these proteins are coordinated to maintain organelle homeostasis.Nuclear–mitochondrial communication is a critical aspect of eukaryotic cellular life that allows cells to adapt to different environmental conditions and energy needs. A breakdown in communication between mitochondria and the nucleus has been implicated in several diseases, including cancers (10). The formation of a nucleus–mitochondria MCS likely facilitates the exchange of lipids or small molecules that stimulate signaling pathways to help cells respond to environmental changes or mitochondrial damage (6, 7). Identifying the molecules that regulate these contacts and clarifying the physiological contexts under which these contacts function is crucial to our understanding of human disease. Thus, the identification of a nucleus-mitochondria MCS represents a significant breakthrough in our understanding of nucleus–mitochondria communication.     

Physiological and coordinate downregulation of the NPC1 and NPC2 genes are associated with the sequestration of LDL‐derived cholesterol within endocytic compartments

The Niemann‐Pick C1 and C2 (NPC1 and NPC2) proteins have a central role in regulating the transport of lipoprotein‐derived cholesterol from endocytic compartments to the endoplasmic reticulum for esterification by acyl‐CoA:cholesterol acyltransferase (ACAT) and feedback inhibition of the sterol regulatory element‐binding protein (SREBP) pathway. Since the NPC1 gene/protein has recently been shown to be downregulated by feedback inhibition of the SREBP pathway, the present study was performed to determine whether physiological downregulation of the NPC1 gene/protein alters the transport and metabolism of low‐density lipoprotein (LDL)‐derived cholesterol in human fibroblasts. To perform this study, three different culture conditions were used that included fibroblasts grown in lipoprotein‐deficient serum (LPDS), LPDS supplemented with LDL, and LPDS supplemented with LDL, followed by equilibration in the absence of LDL to allow the transport of LDL‐derived cholesterol from endocytic compartments and equilibration of cellular sterol pools. The results from this study indicated that in addition to the NPC1 gene/protein, the NPC2 gene/protein was also downregulated by LDL‐derived cholesterol‐dependent feedback inhibition and that downregulation of both the NPC1 and NPC2 genes/proteins was associated with the sequestration of LDL‐derived cholesterol within endocytic compartments, including late endosomes/lysosomes after equilibration. Therefore, it is proposed that physiological and coordinate downregulation of the NPC1 and NPC2 genes/proteins promotes the sequestration of LDL‐derived cholesterol within endocytic compartments and serves a role in maintaining intracellular cholesterol homeostasis. J. Cell. Biochem. 108: 1102–1116, 2009. © 2009 Wiley‐Liss, Inc.     

Interorganellar communication and membrane contact sites in protozoan parasites

A key characteristic of eukaryotic cells is the presence of organelles with discrete boundaries and functions. Such subcellular compartmentalization into organelles necessitates platforms for communication and material exchange between each other which often involves vesicular trafficking and associated processes. Another way is via the close apposition between organellar membranes, called membrane contact sites (MCSs). Apart from lipid transfer, MCSs have been implicated to mediate in various cellular processes including ion transport, apoptosis, and organelle dynamics. In mammalian and yeast cells, contact sites have been reported between the membranes of the following: the endoplasmic reticulum (ER) and the plasma membrane (PM), ER and the Golgi apparatus, ER and endosomes (i.e., vacuoles, lysosomes), ER and lipid droplets (LD), the mitochondria and vacuoles, the nucleus and vacuoles, and the mitochondria and lipid droplets, whereas knowledge of MCSs in non-model organisms such as protozoan parasites is extremely limited. Growing evidence suggests that MCSs play more general and conserved roles in cell physiology. In this mini review, we summarize and discuss representative MCSs in divergent parasitic protozoa, and highlight the universality, diversity, and the contribution of MCSs to parasitism.     

Peroxisome proliferator-activated receptor alpha controls cellular cholesterol trafficking in macrophages

The mobilization of cholesterol from intracellular pools to the plasma membrane is a determinant that governs its availability for efflux to extracellular acceptors. NPC1 and NPC2 are proteins localized in the late endosome and control cholesterol transport from the lysosome to the plasma membrane. Here, we report that NPC1 and NPC2 gene expression is induced by oxidized LDL (OxLDL) in human macrophages. Because OxLDLs contain natural activators of peroxisome proliferator-activated receptor alpha (PPARalpha), a fatty acid-activated nuclear receptor, the regulation of NPC1 and NPC2 by PPARalpha and the consequences on cholesterol trafficking were further studied. NPC1 and NPC2 expression is induced by synthetic PPARalpha ligands in human macrophages. Furthermore, PPARalpha activation leads to an enrichment of cholesterol in the plasma membrane. By contrast, incubation with progesterone, which blocks postlysosomal cholesterol trafficking, as well as NPC1 and NPC2 mRNA depletion using small interfering RNA, abolished ABCA1-dependent cholesterol efflux induced by PPARalpha activators. These observations identify a novel regulatory role for PPARalpha in the control of cholesterol availability for efflux that, associated with its ability to inhibit cholesterol esterification and to stimulate ABCA1 and scavenger receptor class B type I expression, may contribute to the stimulation of reverse cholesterol transport.     

Thematic Review Series: Recent Advances in the Treatment of Lysosomal Storage Diseases: Niemann-Pick C disease and mobilization of lysosomal cholesterol by cyclodextrin

Niemann-Pick type C (NPC) disease is a lysosomal storage disease in which endocytosed cholesterol becomes sequestered in late endosomes/lysosomes (LEs/Ls) because of mutations in either the NPC1 or NPC2 gene. Mutations in either of these genes can lead to impaired functions of the NPC1 or NPC2 proteins and progressive neurodegeneration as well as liver and lung disease. NPC1 is a polytopic protein of the LE/L limiting membrane, whereas NPC2 is a soluble protein in the LE/L lumen. These two proteins act in tandem and promote the export of cholesterol from LEs/Ls. Consequently, a defect in either NPC1 or NPC2 causes cholesterol accumulation in LEs/Ls. In this review, we summarize the molecular mechanisms leading to NPC disease, particularly in the CNS. Recent exciting data on the mechanism by which the cholesterol-sequestering agent cyclodextrin can bypass the functions of NPC1 and NPC2 in the LEs/Ls, and mobilize cholesterol from LEs/Ls, will be highlighted. Moreover, the possible use of cyclodextrin as a valuable therapeutic agent for treatment of NPC patients will be considered.     

High‐Throughput Quantitation of Intracellular Trafficking and Organelle Disruption by Flow Cytometry

Current methods for the quantitation of membrane protein trafficking rely heavily on microscopy, which has limited quantitative capacity for analyses of cell populations and is cumbersome to perform. Here we describe a simple flow cytometry‐based method that circumvents these limitations. The method utilizes fluorescent pulse‐width measurements as a highly sensitive indicator to monitor the changes in intracellular distributions of a fluorescently labelled molecule in a cell. Pulse‐width analysis enabled us to discriminate cells with target proteins in different intracellular locations including Golgi, lyso‐endosomal network and the plasma membrane, as well as detecting morphological changes in organelles such as Golgi perturbation. The movement of endogenous and exogenous retrograde cargo was tracked from the plasma membrane‐to‐endosomes‐to‐Golgi, by decreasing pulse‐width values. A block in transport upon RNAi‐mediated ablation of transport machinery was readily quantified, demonstrating the versatility of this technique to identify pathway inhibitors. We also showed that pulse‐width can be exploited to sort and recover cells based on different intracellular staining patterns, e.g. early endosomes and Golgi, opening up novel downstream applications. Overall, the method provides new capabilities for viewing membrane transport in thousands of cells per minute, unbiased analysis of the trafficking of cargo, and the potential for rapid screening of inhibitors of trafficking pathways.      

细胞内胆固醇运输

作为生物膜的重要成分,细胞内不同膜上胆固醇含量的高低直接影响生物膜的生物物理特性和细胞信号的传递,与细胞正常的生理功能密切相关。外源内吞的胆固醇和内源合成的胆固醇通过囊泡介导和非囊泡介导的胆固醇运输途径在不同细胞膜之间转运,从而维持了不同细胞器上胆固醇的浓度梯度。一系列胆固醇结合和转运蛋白在细胞内胆固醇的运输中发挥了重要作用。本文旨在总结细胞内胆固醇运输途径与参与胆固醇运输的重要分子及相关作用机制。     

Modeling Fusion/Fission‐Dependent Intracellular Transport of Fluid Phase Markers

A fundamental feature of eukaryotic cells is the presence of distinct membrane‐bound compartments having unique protein and lipid composition. These compartments are interconnected by active trafficking mechanisms that must direct macromolecules to defined locations, and at the same time maintain the protein and lipid composition of each organelle. It is well accepted that Rab proteins play a central role in intracellular transport regulating the recognition, fusion and fission of organelles. However, how the transport is achieved is not completely understood. We propose a model whereby a soluble component in the luminal compartment is transported along different Rab‐containing organelles that interact according to the following simple principles: (i) only organelles with the same or compatible Rab membrane domains can fuse; (ii) after fusion, an asymmetric fission occurs producing a tubule and a round‐shaped vesicle; and (iii) Rab membrane domains distribute asymmetrically between the two resulting organelles. When this model was tested in a simulation, efficient unidirectional transport was observed, while the compartment identity was preserved. All three principles were absolutely necessary for transport. The model is compatible with Rab association/dissociation dynamics and with Rab conversion. In simulations mimicking a simplified endocytic pathway, soluble and membrane‐associated markers were efficiently transported preserving the identity of the interacting compartments.     

The Cytosolic Adaptor AP‐1A Is Essential for the Trafficking and Function of Niemann‐Pick Type C Proteins

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.