Structural Insight into Eukaryotic Sterol Transport through Niemann-Pick Type C Proteins.

Niemann-Pick type C (NPC) proteins are essential for sterol homeostasis, believed to drive sterol integration into the lysosomal membrane before redistribution to other cellular membranes. Here, using a combination of crystallography, cryo-electron microscopy, and biochemical and in vivo studies on the Saccharomyces cerevisiae NPC system (NCR1 and NPC2), we present a framework for sterol membrane integration. Sterols are transferred between hydrophobic pockets of vacuolar NPC2 and membrane-protein NCR1. NCR1 has its N-terminal domain (NTD) positioned to deliver a sterol to a tunnel connecting NTD to the luminal membrane leaflet 50 Å away. A sterol is caught inside this tunnel during transport, and a proton-relay network of charged residues in the transmembrane region is linked to this tunnel supporting a proton-driven transport mechanism. We propose a model for sterol integration that clarifies the role of NPC proteins in this essential eukaryotic pathway and that rationalizes mutations in patients with Niemann-Pick disease type C.

Exploring the ATG9A interactome uncovers interaction with VPS13A.

ATG9A, a transmembrane protein of the core autophagy pathway, cycles between the Golgi, endosomes and a vesicular compartment. ATG9A was recently shown to act as a lipid scramblase, and this function is thought to require its interaction with another core autophagy protein, ATG2A, which acts as a lipid transfer protein. Together, ATG9A and ATG2A are proposed to function to expand the growing autophagosome. However, ATG9A is implicated in other pathways including membrane repair and lipid droplet homeostasis. To elucidate other ATG9A interactors within the autophagy pathway, or interactors beyond autophagy, we performed an interactome analysis through mass spectrometry. This analysis revealed a host of proteins involved in lipid synthesis and trafficking, including ACSL3, VPS13A and VPS13C. Furthermore, we show that ATG9A directly interacts with VPS13A and forms a complex that is distinct from the ATG9A-ATG2A complex.

Arabidopsis trigalactosyldiacylglycerol1 mutants reveal a critical role for phosphtidylcholine remodeling in lipid homeostasis.

Lipid remodeling plays a critical role in plant response to abiotic stress and metabolic perturbations. Key steps in this process involve modifications of phosphatidylcholine (PC) acyl chains mediated by lysophosphatidylcholine: acyl-CoA acyltransferases (LPCATs) and phosphatidylcholine: diacylglycerol cholinephosphotransferase (ROD1). To assess their importance in lipid homeostasis, we took advantage of the trigalactosyldiacylglycerol1 (tgd1) mutant that exhibits marked increases in fatty acid synthesis and fatty acid flux through PC due to a block in inter-organelle lipid trafficking. Here, we showed that the increased fatty acid synthesis in tgd1 is due to posttranslational activation of the plastidic acetyl-coenzyme A carboxylase. Genetic analysis showed that knockout of LPCAT1 and 2 resulted in a lethal phenotype in tgd1. In addition, plants homozygous for lpcat2 and heterozygous for lpcat1 in the tgd1 background showed reduced levels of PC and triacylglycerols (TAG) and alterations in their fatty acid profiles. We further showed that disruption of ROD1 in tgd1 resulted in changes in fatty acid composition of PC and TAG, decreased leaf TAG content and reduced seedling growth. Together, our results reveal a critical role of LPCATs and ROD1 in maintaining cellular lipid homeostasis under conditions, in which fatty acid production largely exceeds the cellular demand for membrane lipid synthesis.

Inhibition of cellular factor TM6SF2 suppresses secretion pathways of Hepatitis B, Hepatitis C and Hepatitis D viruses.

Chronic viral hepatitis is caused by hepatitis B virus, hepatitis C virus or hepatitis D virus (HBV, HCV, and HDV). Despite different replication strategies, all these viruses rely on secretion through the host endoplasmic reticulum-Golgi pathway, providing potential host targets for antiviral therapy. Knockdown of transmembrane 6 superfamily member 2 (TM6SF2) in virus cell culture models reduced secretion of infectious HCV virions, HDV virions and HBV subviral particles. Moreover, in a cohort of people with hepatitis B a TM6SF2 polymorphism (rs58542926 CT/TT, which causes protein misfolding and reduced TM6SF2 in the liver) correlated with lower concentrations of subviral particles in blood, complementing our previous work showing decreased HCV viral load in people with this polymorphism. In conclusion, the host protein TM6SF2 plays a key role in secretion of HBV, HCV and HDV, providing the potential for novel pan-viral agents to treat people with chronic viral hepatitis.

The role of lipids in exosome biology and intercellular communication: Function, analytics and applications.

Exosomes are extracellular vesicles that in recent years have received special attention for their regulatory functions in numerous biological processes. Recent evidence suggests a correlation between the composition of exosomes in body fluids and the progression of some disorders, such as cancer, diabetes and neurodegenerative diseases. In consequence, numerous studies have been performed to evaluate the composition of these vesicles, aiming to develop new biomarkers for diagnosis and to find novel therapeutic targets. On their part, lipids represent one of the most important components of exosomes, with important structural and regulatory functions during exosome biogenesis, release, targeting and cellular uptake. Therefore, exosome lipidomics has emerged as an innovative discipline for the discovery of novel lipid species with biomedical applications. This review summarizes the current knowledge about exosome lipids and their roles in exosome biology and intercellular communication. Furthermore, it presents the state-of-the-art analytical procedures used in exosome lipidomics while emphasizing how this emerging discipline is providing new insights for future applications of exosome lipids in biomedicine.

The tocopherol transfer protein mediates vitamin E trafficking between cerebellar astrocytes and neurons.

Alpha-tocopherol (vitamin E) is an essential nutrient that functions as a major lipid-soluble antioxidant in humans. The alpha-tocopherol transfer protein (TTP) binds α-tocopherol with high affinity and selectivity and regulates whole-body distribution of the vitamin. Heritable mutations in the TTPA gene result in familial vitamin E deficiency, elevated indices of oxidative stress, and progressive neurodegeneration that manifest primarily in spinocerebellar ataxia. Although the essential role of vitamin E in neurological health has been recognized for over 50 years, the mechanisms by which this essential nutrient is transported in the central nervous system are poorly understood. Here we found that, in the murine cerebellum, TTP is selectively expressed in glial fibrillary acidic protein-positive astrocytes, where it facilitates efflux of vitamin E to neighboring neurons. We also show that induction of oxidative stress enhances the transcription of the TtpA gene in cultured cerebellar astrocytes. Furthermore, secretion of vitamin E from astrocytes is mediated by an ABC-type transporter, and uptake of the vitamin into neurons involves the low-density lipoprotein receptor-related protein 1. Taken together, our data indicate that TTP-expressing astrocytes control the delivery of vitamin E from astrocytes to neurons, and that this process is homeostatically responsive to oxidative stress. These are the first observations that address the detailed molecular mechanisms of vitamin E transport in the central nervous system, and these results have important implications for understanding the molecular underpinnings of oxidative stress-related neurodegenerative diseases.

Lipid exchange in crystal-confined fatty acid binding proteins: X-ray evidence and molecular dynamics explanation.

Fatty acid binding proteins (FABPs) are responsible for the long-chain fatty acids (FAs) transport inside the cell. However, despite the years, since their structure is known and the many studies published, there is no definitive answer about the stages of the lipid entry-exit mechanism. Their structure forms a ß -barrel of 10 anti-parallel strands with a cap in a helix-turn-helix motif, and there is some consensus on the role of the so-called portal region, involving the second α -helix from the cap ( α 2), ß C- ß D, and ß E- ß F turns in FAs exchange. To test the idea of a lid that opens, we performed a soaking experiment on an h-FABP crystal in which the cap is part of the packing contacts, and its movement is strongly restricted. Even in these conditions, we observed the replacement of palmitic acid by 2-Bromohexadecanoic acid (Br-palmitic acid). Our MD simulations reveal a two-step lipid entry process: (i) The travel of the lipid head through the cavity in the order of tens of nanoseconds, and (ii) The accommodation of its hydrophobic tail in hundreds to thousands of nanoseconds. We observed this even in the cases in which the FAs enter the cavity by their tail. During this process, the FAs do not follow a single trajectory, but multiple ones through which they get into the protein cavity. Thanks to the complementary views between experiment and simulation, we can give an approach to a mechanistic view of the exchange process.

Structures and Mechanisms of a Novel Bacterial Transport System for Fatty Acids.

Bacterial acquisition of metabolites is largely facilitated by transporters with unique substrate scopes. The tripartite ATP-independent periplasmic (TRAP) transporters comprise a large family of bacterial proteins that facilitate the uptake of a variety of small molecules. It has been reported that some TRAP systems encode a fourth protein, the T component. The T-component, or TatT, is predicted to be a periplasmic-facing lipoprotein that enables the uptake of metabolites from the outer membrane. However, no substrates were revealed for any TatT and their functional role(s) remained enigmatic. We recently identified a homolog in Methylococcus capsulatus that binds to sterols, and herein, we report two additional homologs that demonstrate a preference for long-chain fatty acids. Our bioinformatics, quantitative analyses of protein-ligand interactions, and high-resolution crystal structures suggest that TatTs might facilitate the trafficking of hydrophobic or lipophilic substrates and represent a new class of bacterial lipid and fatty acid transporters.

CYP311A1 in the anterior midgut is involved in lipid distribution and microvillus integrity in Drosophila melanogaster.

Lipids are either taken up from food sources or produced internally in specialized tissues such as the liver. Among others, both routes of lipid metabolism involve cytochrome P450 monooxygenases (CYPs). We sought to analyze the function of Cyp311a1 that has been shown to be expressed in the midgut of the fruit fly Drosophila melanogaster. Using a GFP-tagged version of CYP311A1 that is expressed under the control of its endogenous promoter, we show that Cyp311a1 localizes to the endoplasmic reticulum in epithelial cells of the anterior midgut. In larvae with reduced Cyp311a1 expression in the anterior midgut, compared to control larvae, the apical plasma membrane of the respective epithelial cells contains less and shorter microvilli. In addition, we observed reduction of neutral lipids in the fat body, the insect liver, and decreased phosphatidylethanolamine (PE) and triacylglycerols (TAG) amounts in the whole body of these larvae. Probably as a consequence, they cease to grow and eventually die. The microvillus defects in larvae with reduced Cyp311a1 expression are restored by supplying PE, a major phospholipid of plasma membranes, to the food. Moreover, the growth arrest phenotype of these larvae is partially rescued. Together, these results suggest that the anterior midgut is an import hub in lipid distribution and that the midgut-specific CYP311A1 contributes to this function by participating in shaping microvilli in a PE-dependent manner.

Intravital lipid droplet labeling and imaging reveals the phenotypes and functions of individual macrophages in vivo.

Macrophages play pivotal roles in the maintenance of tissue homeostasis. However, the reactivation of macrophages toward proinflammatory states correlates with a plethora of inflammatory diseases, including atherosclerosis, obesity, neurodegeneration, and bone marrow (BM) failure syndromes. The lack of methods to reveal macrophage phenotype and function in vivo impedes the translational research of these diseases. Here, we found that proinflammatory macrophages accumulate intracellular lipid droplets (LDs) relative to resting or noninflammatory macrophages both in vitro and in vivo, indicating that LD accumulation serves as a structural biomarker for macrophage phenotyping. To realize the staining and imaging of macrophage LDs in vivo, we developed a fluorescent fatty acid analog-loaded poly(lactic-co-glycolic acid) nanoparticle to label macrophages in mice with high efficiency and specificity. Using these novel nanoparticles, we achieved in situ functional identification of single macrophages in BM, liver, lung, and adipose tissues under conditions of acute or chronic inflammation. Moreover, with this intravital imaging platform, we further realized in vivo phenotyping of individual macrophages in the calvarial BM of mice under systemic inflammation. In conclusion, we established an efficient in vivo LD labeling and imaging system for single macrophage phenotyping, which will aid in the development of diagnostics and therapeutic monitoring. Moreover, this method also provides new avenues for the study of lipid trafficking and dynamics in vivo.

Proteins associated with the Arabidopsis thaliana plastid rhomboid-like protein RBL10.

Rhomboid-like proteins are intramembrane proteases with a variety of regulatory roles in cells. Though many rhomboid-like proteins are predicted in plants, their detailed molecular mechanisms or cellular functions are not yet known. Of the 13 predicted rhomboids in Arabidopsis thaliana, one, RBL10, affects lipid metabolism in the chloroplast, because in the respective rbl10 mutant the transfer of phosphatidic acid through the inner envelope membrane is disrupted. Here we show that RBL10 is part of a high-molecular-weight complex of 250 kDa or greater in size. Nine likely components of this complex are identified by two independent methods and include Acyl Carrier Protein 4 (ACP4) and Carboxyltransferase Interactor1 (CTI1), which have known roles in chloroplast lipid metabolism. The acp4 mutant has decreased C16:3 fatty acid content of monogalactosyldiacylglycerol, similar to the rbl10 mutant, prompting us to offer a mechanistic model of how an interaction between ACP4 and RBL10 might affect chloroplast lipid assembly. We also demonstrate the presence of a seventh transmembrane domain in RBL10, refining the currently accepted topology of this protein. Taken together, the identity of possible RBL10 complex components as well as insights into RBL10 topology and distribution in the membrane provide a stepping-stone towards a deeper understanding of RBL10 function in Arabidopsis lipid metabolism.

A peroxisomal sterol carrier protein 2 (Scp2) contributes to lipid trafficking in differentiation and virulence of the insect pathogenic fungus Beauveria bassiana.

Sterol carrier protein 2 (SCP2) represents a family of proteins binding a variety of lipids and plays essential roles in cellular physiology. However, its physiological roles are largely unknown in filamentous fungi. In this study, we functionally characterized an orthologous Scp2 gene in the filamentous insect pathogenic fungus Beauveria bassiana (BbScp2). BbScp2 was verified to be a peroxisomal protein and displayed different affinities to various lipids, with strong affinity to palmitic acid (PA) and ergosterol (ES). No significant binding activity was detected between protein and oleic acid (OA) or linoleic acid (LA). Ablation of BbScp2 did not cause significant effects on fungal growth on various carbon sources, but resulted in a modest reduction in conidial (49%) and blastospore yield (45%). In addition, exogenous lipids could recover the defectives in conidiation of ΔBbScp2 mutant strain. BbScp2 was required for the cytomembrane functionality in germlings, and its loss resulted in a more significant decrease in virulence indicated by cuticle infection assay than intrahemocoel injection assay. Our findings indicate that Scp2 links the lipid trafficking to the asexual differentiation and virulence of B. bassiana.

A real-time, click chemistry imaging approach reveals stimulus-specific subcellular locations of phospholipase D activity.

The fidelity of signal transduction requires spatiotemporal control of the production of signaling agents. Phosphatidic acid (PA) is a pleiotropic lipid second messenger whose modes of action differ based on upstream stimulus, biosynthetic source, and site of production. How cells regulate the local production of PA to effect diverse signaling outcomes remains elusive. Unlike other second messengers, sites of PA biosynthesis cannot be accurately visualized with subcellular precision. Here, we describe a rapid, chemoenzymatic approach for imaging physiological PA production by phospholipase D (PLD) enzymes. Our method capitalizes on the remarkable discovery that bulky, hydrophilic trans-cyclooctene-containing primary alcohols can supplant water as the nucleophile in the PLD active site in a transphosphatidylation reaction of PLD's lipid substrate, phosphatidylcholine. The resultant trans-cyclooctene-containing lipids are tagged with a fluorogenic tetrazine reagent via a no-rinse, inverse electron-demand Diels-Alder (IEDDA) reaction, enabling their immediate visualization by confocal microscopy in real time. Strikingly, the fluorescent reporter lipids initially produced at the plasma membrane (PM) induced by phorbol ester stimulation of PLD were rapidly internalized via apparent nonvesicular pathways rather than endocytosis, suggesting applications of this activity-based imaging toolset for probing mechanisms of intracellular phospholipid transport. By instead focusing on the initial 10 s of the IEDDA reaction, we precisely pinpointed the subcellular locations of endogenous PLD activity as elicited by physiological agonists of G protein-coupled receptor and receptor tyrosine kinase signaling. These tools hold promise to shed light on both lipid trafficking pathways and physiological and pathological effects of localized PLD signaling.

Involvement of a Cluster of Basic Amino Acids in Phosphorylation-Dependent Functional Repression of the Ceramide Transport Protein CERT.

Ceramide transport protein (CERT) mediates ceramide transfer from the endoplasmic reticulum to the Golgi for sphingomyelin (SM) biosynthesis. CERT is inactivated by multiple phosphorylation at the serine-repeat motif (SRM), and mutations that impair the SRM phosphorylation are associated with a group of inherited intellectual disorders in humans. It has been suggested that the N-terminal phosphatidylinositol 4-monophosphate [PtdIns(4)P] binding domain and the C-terminal ceramide-transfer domain of CERT physically interfere with each other in the SRM phosphorylated state, thereby repressing the function of CERT; however, it remains unclear which regions in CERT are involved in the SRM phosphorylation-dependent repression of CERT. Here, we identified a previously uncharacterized cluster of lysine/arginine residues that were predicted to be located on the outer surface of a probable coiled-coil fold in CERT. Substitutions of the basic amino acids in the cluster with alanine released the SRM-dependent repression of CERT activities, i.e., the synthesis of SM, PtdIns(4)P-binding, vesicle-associated membrane protein-associated protein (VAP) binding, ceramide-transfer activity, and localization to the Golgi, although the effect on SM synthesis activity was only partially compromised by the alanine substitutions, which moderately destabilized the trimeric status of CERT. These results suggest that the basic amino acid cluster in the coiled-coil region is involved in the regulation of CERT function.

The phosphoinositide code is read by a plethora of protein domains.

INTRODUCTION: The proteins that decipher nucleic acid- and protein-based information are well known, however, those that read membrane-encoded information remain understudied. Here, we report 70 different human, microbial and viral protein folds that recognize phosphoinositides (PIs), comprising the readers of a vast membrane code. AREAS COVERED: Membrane recognition is best understood for FYVE, PH and PX domains, which exemplify hundreds of PI code readers. Comparable lipid interaction mechanisms may be mediated by kinases, adjacent C1 and C2 domains, trafficking arrestins, GAT and VHS modules, membrane-perturbing annexins, BAR, CHMP, ENTH, HEAT, syntaxin and Tubby helical bundles, multipurpose FERM, EH, MATH, PHD, PDZ, PROPPIN, PTB and SH2 domains, as well as systems that regulate receptors, GTPases and actin filaments, transfer lipids, and assemble bacterial and viral particles. EXPERT OPINION: The elucidation of how membranes are recognized has extended the genetic code to the PI code. Novel discoveries include PIP-stop and MET-stop residues to which phosphates and metabolites are attached to block phosphatidylinositol phosphate (PIP) recognition, memteins as functional membrane protein apparatuses and lipidons as lipid 'codons' recognized by membrane readers. At least 5% of the human proteome senses such membrane signals and allows eukaryotic organelles and pathogens to operate and replicate.

Neurodegeneration in Niemann-Pick Type C Disease: An Updated Review on Pharmacological and Non-Pharmacological Approaches to Counteract Brain and Cognitive Impairment.

Niemann-Pick type C (NPC) disease is an autosomal recessive storage disorder, characterized by abnormal sequestration of unesterified cholesterol in the late endo-lysosomal system of cells. Progressive neurological deterioration and the onset of symptoms, such as ataxia, seizures, cognitive decline, and severe dementia, are pathognomonic features of the disease. In addition, different pathological similarities, including degeneration of hippocampal and cortical neurons, hyperphosphorylated tau, and neurofibrillary tangle formation, have been identified between NPC disease and other neurodegenerative pathologies. However, the underlying pathophysiological mechanisms are not yet well understood, and even a real cure to counteract neurodegeneration has not been identified. Therefore, the combination of current pharmacological therapies, represented by miglustat and cyclodextrin, and non-pharmacological approaches, such as physical exercise and appropriate diet, could represent a strategy to improve the quality of life of NPC patients. Based on this evidence, in our review we focused on the neurodegenerative aspects of NPC disease, summarizing the current knowledge on the molecular and biochemical mechanisms responsible for cognitive impairment, and suggesting physical exercise and nutritional treatments as additional non-pharmacologic approaches to reduce the progression and neurodegenerative course of NPC disease.

Vacuole membrane protein 1 marks endoplasmic reticulum subdomains enriched in phospholipid synthesizing enzymes and is required for phosphoinositide distribution.

The multispanning membrane protein vacuole membrane protein 1 (VMP1) marks and regulates endoplasmic reticulum (ER)-domains associated with diverse ER-organelle membrane contact sites. A proportion of these domains associate with endosomes during their maturation and remodeling. We found that these VMP1 domains are enriched in choline/ethanolamine phosphotransferase and phosphatidylinositol synthase (PIS1), 2 ER enzymes required for the synthesis of various phospholipids. Interestingly, the lack of VMP1 impairs the formation of PIS1-enriched ER domains, suggesting a role in the distribution of phosphoinositides. In fact, depletion of VMP1 alters the distribution of PtdIns4P and proteins involved in the trafficking of PtdIns4P. Consistently, in these conditions, defects were observed in endosome trafficking and maturation as well as in Golgi morphology. We propose that VMP1 regulates the formation of ER domains enriched in lipid synthesizing enzymes. These domains might be necessary for efficient distribution of PtdIns4P and perhaps other lipid species. These findings, along with previous reports that involved VMP1 in regulating PtdIns3P during autophagy, expand the role of VMP1 in lipid trafficking and explain the pleiotropic effects observed in VMP1-deficient mammalian cells and other model systems.

Lipid trafficking across the Gram-negative cell envelope.

The outer membrane (OM) of Gram-negative bacteria exhibits unique lipid asymmetry, with lipopolysaccharides (LPS) residing in the outer leaflet and phospholipids (PLs) in the inner leaflet. This asymmetric bilayer protects the bacterium against intrusion of many toxic substances, including antibiotics and detergents, yet allows acquisition of nutrients necessary for growth. To build the OM and ensure its proper function, the cell produces OM constituents in the cytoplasm or inner membrane and transports these components across the aqueous periplasmic space separating the two membranes. Of note, the processes by which the most basic membrane building blocks, i.e. PLs, are shuttled across the cell envelope remain elusive. This review highlights our current understanding (or lack thereof) of bacterial PL trafficking, with a focus on recent developments in the field. We adopt a mechanistic approach and draw parallels and comparisons with well-characterized systems, particularly OM lipoprotein and LPS transport, to illustrate key challenges in intermembrane lipid trafficking. Pathways that transport PLs across the bacterial cell envelope are fundamental to OM biogenesis and homeostasis and are potential molecular targets that could be exploited for antibiotic development.

Cellular endo-lysosomal dysfunction in the pathogenesis of non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD), an increasingly devastating human disorder, is characterized by intrahepatic fat accumulation. Although important progress has been made in understanding NAFLD, the fundamental mechanisms involved in the pathogenesis of NAFLD have not been fully explained. The endo-lysosomal trafficking network is central to lipid metabolism, protein degradation and signal transduction, which are involved in a variety of diseases. In recent years, many genes and pathways in the endo-lysosomal trafficking network and involved in lysosomal biogenesis have been associated with the development and progression of NAFLD. Mutations of these genes and impaired signalling lead to dysfunction in multiple steps of the endo-lysosomal network (endocytic trafficking, membrane fusion and lysosomal degradation), resulting in the accumulation of pathogenic proteins. In this review, we will focus on how alterations in these genes and pathways affect endo-lysosomal trafficking as well as the pathophysiology of NAFLD.

StAR-related lipid transfer domain 11 (STARD11)-mediated ceramide transport mediates extracellular vesicle biogenesis.

Extracellular vesicles are important carriers of cellular materials and have critical roles in cell-to-cell communication in both health and disease. Ceramides are implicated in extracellular vesicle biogenesis, yet the cellular machinery that mediates the formation of ceramide-enriched extracellular vesicles remains unknown. We demonstrate here that the ceramide transport protein StAR-related lipid transfer domain 11 (STARD11) mediates the release of palmitate-stimulated extracellular vesicles having features consistent with exosomes. Using palmitate as a model of lipotoxic diseases and as a substrate for ceramide biosynthesis in human and murine liver cell lines and primary mouse hepatocytes, we found that STARD11-deficient cells release fewer extracellular vesicles. Moreover, STARD11 reciprocally regulated exosome ceramide enrichment and cellular ceramide depletion. We further observed that in STARD11 knockout cells intracellular ceramide accumulates and that this apparent inability to transfer cellular ceramide into extracellular vesicles reduces cellular viability. Using endogenous markers, we uncovered structural and functional colocalization of the endoplasmic reticulum (ER), STARD11, and multivesicular bodies. This colocalization increased following palmitate treatment, suggesting a functional association that may mediate ceramide trafficking from the ER to the multivesicular body. However, the size and number of multivesicular bodies were comparable in WT and STARD11-knockout cells. In conclusion, we propose a model of how STARD11 mediates ceramide trafficking in palmitate-treated cells and stimulates exosome biogenesis.