Search

Choudhary et al. show lipid droplet (LD) biogenesis from discrete ER subdomains in yeast. Fld1 together with Nem1 localize to discrete ER subdomains independent of each other and of LDs, but both are required to recruit triacylglycerol (TAG)-synthases and LD biogenesis factors for localized TAG production and droplet assembly., Lipid droplets (LDs) are fat storage organelles that originate from the endoplasmic reticulum (ER). Relatively little is known about how sites of LD formation are selected and which proteins/lipids are necessary for the process. Here, we show that LDs induced by the yeast triacylglycerol (TAG)-synthases Lro1 and Dga1 are formed at discrete ER subdomains defined by seipin (Fld1), and a regulator of diacylglycerol (DAG) production, Nem1. Fld1 and Nem1 colocalize to ER–LD contact sites. We find that Fld1 and Nem1 localize to ER subdomains independently of each other and of LDs, but both are required for the subdomains to recruit the TAG-synthases and additional LD biogenesis factors: Yft2, Pex30, Pet10, and Erg6. These subdomains become enriched in DAG. We conclude that Fld1 and Nem1 are both necessary to recruit proteins to ER subdomains where LD biogenesis occurs., Graphical Abstract

Glycosylphosphatidylinositol (GPI) is a glycolipid membrane anchor found on surface proteins in all eukaryotes. It is synthesized in the ER membrane. Each GPI anchor requires three molecules of ethanolamine phosphate (P-Etn), which are derived from phosphatidylethanolamine (PE). We found that efficient GPI anchor synthesis in Saccharomyces cerevisiae requires Csf1; cells lacking Csf1 accumulate GPI precursors lacking P-Etn. Structure predictions suggest Csf1 is a tube-forming lipid transport protein like Vps13. Csf1 is found at contact sites between the ER and other organelles. It interacts with the ER protein Mcd4, an enzyme that adds P-Etn to nascent GPI anchors, suggesting Csf1 channels PE to Mcd4 in the ER at contact sites to support GPI anchor biosynthesis. CSF1 has orthologues in Caenorhabditis elegans (lpd-3) and humans (KIAA1109/TWEEK); mutations in KIAA1109 cause the autosomal recessive neurodevelopmental disorder Alkuraya-Kučinskas syndrome. Knockout of lpd-3 and knockdown of KIAA1109 reduced GPI-anchored proteins on the surface of cells, suggesting Csf1 orthologues in human cells support GPI anchor biosynthesis.

Lipid droplets (LDs) are neutral lipid-containing organelles enclosed in a single monolayer of phospholipids. LD formation begins with the accumulation of neutral lipids within the bilayer of the endoplasmic reticulum (ER) membrane. It is not known how the sites of formation of nascent LDs in the ER membrane are determined. Here we show that multiple C2 domain-containing transmembrane proteins, MCTP1 and MCTP2, are at sites of LD formation in specialized ER subdomains. We show that the transmembrane domain (TMD) of these proteins is similar to a reticulon homology domain. Like reticulons, these proteins tubulate the ER membrane and favor highly curved regions of the ER. Our data indicate that the MCTP TMDs promote LD biogenesis, increasing LD number. MCTPs colocalize with seipin, a protein involved in LD biogenesis, but form more stable microdomains in the ER. The MCTP C2 domains bind charged lipids and regulate LD size, likely by mediating ER-LD contact sites. Together, our data indicate that MCTPs form microdomains within ER tubules that regulate LD biogenesis, size, and ER-LD contacts. Interestingly, MCTP punctae colocalized with other organelles as well, suggesting that these proteins may play a general role in linking tubular ER to organelle contact sites.

Reinisch and Prinz review the emerging understanding of the mechanisms, regulation, and functions of nonvesicular lipid transport in eukaryotic cells., We have long known that lipids traffic between cellular membranes via vesicles but have only recently appreciated the role of nonvesicular lipid transport. Nonvesicular transport can be high volume, supporting biogenesis of rapidly expanding membranes, or more targeted and precise, allowing cells to rapidly alter levels of specific lipids in membranes. Most such transport probably occurs at membrane contact sites, where organelles are closely apposed, and requires lipid transport proteins (LTPs), which solubilize lipids to shield them from the aqueous phase during their transport between membranes. Some LTPs are cup like and shuttle lipid monomers between membranes. Others form conduits allowing lipid flow between membranes. This review describes what we know about nonvesicular lipid transfer mechanisms while also identifying many remaining unknowns: How do LTPs facilitate lipid movement from and into membranes, do LTPs require accessory proteins for efficient transfer in vivo, and how is directionality of transport determined?

Lipid droplets (LDs) are implicated in conditions of lipid and protein dysregulation. The fat storage-inducing transmembrane (FIT; also known as FITM) family induces LD formation. Here, we establish a model system to study the role of the Saccharomyces cerevisiae FIT homologues (ScFIT), SCS3 and YFT2, in the proteostasis and stress response pathways. While LD biogenesis and basal endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) remain unaltered in ScFIT mutants, SCS3 was found to be essential for proper stress-induced UPR activation and for viability in the absence of the sole yeast UPR transducer IRE1. Owing to not having a functional UPR, cells with mutated SCS3 exhibited an accumulation of triacylglycerol within the ER along with aberrant LD morphology, suggesting that there is a UPR-dependent compensatory mechanism that acts to mitigate lack of SCS3. Additionally, SCS3 was necessary to maintain phospholipid homeostasis. Strikingly, global protein ubiquitylation and the turnover of both ER and cytoplasmic misfolded proteins is impaired in ScFITΔ cells, while a screen for interacting partners of Scs3 identifies components of the proteostatic machinery as putative targets. Together, our data support a model where ScFITs play an important role in lipid metabolism and proteostasis beyond their defined roles in LD biogenesis. This article has an associated First Person interview with the first author of the paper.

Lipid droplets (LDs) have long been regarded as inert cytoplasmic organelles with the primary function of housing excess intracellular lipids. More recently, LDs have been strongly implicated in conditions of lipid and protein dysregulation. The fat storage inducing transmembrane (FIT) family of proteins comprises of evolutionarily conserved endoplasmic reticulum (ER)-resident proteins that have been reported to induce LD formation. Here, we establish a model system to study the role of S. cerevisiae FIT homologues (ScFIT), SCS3 and YFT2, in proteostasis and stress response pathways. While LD biogenesis and basal ER stress-induced unfolded protein response (UPR) remain unaltered in ScFIT mutants, SCS3 was found to be essential for proper stress-induced UPR activation and for viability in the absence of the sole yeast UPR transducer IRE1. Devoid of a functional UPR, scs3 mutants exhibited accumulation of triacylglycerol within the ER along with aberrant LD morphology, suggesting a UPR-dependent compensatory mechanism for LD maturation. Additionally, SCS3 was necessary to maintain phospholipid homeostasis. Strikingly, the absence of the ScFIT proteins results in the downregulation of the closely-related Heat Shock Response (HSR) pathway. In line with this observation, global protein ubiquitination and the turnover of both ER and cytoplasmic misfolded proteins is impaired in ScFIT cells, while a screen for interacting partners of Scs3 identifies components of the proteostatic machinery as putative targets. Taken together, these suggest that ScFIT proteins may modulate proteostasis and stress response pathways with lipid metabolism at the interface between the two cellular processes.

Prinz and Hurley preview work from Li and colleagues, which uses structural analyses to reveal a new method of lipid transfer by VPS13., All lipid transport proteins in eukaryotes are thought to shuttle lipids between cellular membranes. In this issue, Li et al. (2020. J. Cell Biol. https://doi.org/10.1083/jcb.202001161) show that Vps13 has a channel-like domain that may allow lipids to flow between closely apposed membranes at contact sites.

ESCRT-III proteins, which form filaments that deform, bud, and sever membranes, are found in eukaryotes and some archaea. Three studies in this issue of Cell reveal that PspA and Vipp1 are bacterial and cyanobacterial members of the ESCRT-III superfamily, indicating it is even more ubiquitous and ancient than previously thought.

Fat storage-inducing transmembrane (FIT or FITM) proteins have been implicated in the partitioning of triacylglycerol to lipid droplets and the budding of lipid droplets from the ER. At the molecular level, the sole relevant interaction is that FITMs directly bind to triacyglycerol and diacylglycerol, but how they function at the molecular level is not known. Saccharomyces cerevisiae has two FITM homologues: Scs3p and Yft2p. Scs3p was initially identified because deletion leads to inositol auxotrophy, with an unusual sensitivity to addition of choline. This strongly suggests a role for Scs3p in phospholipid biosynthesis. Looking at the FITM family as widely as possible, we found that FITMs are widespread throughout eukaryotes, indicating presence in the last eukaryotic common ancestor. Protein alignments also showed that FITM sequences contain the active site of lipid phosphatase/phosphotransferase (LPT) enzymes. This large family transfers phosphate-containing headgroups either between lipids or in exchange for water. We confirmed the prediction that FITMs are related to LPTs by showing that single amino-acid substitutions in the presumptive catalytic site prevented their ability to rescue growth of the mutants on low inositol/high choline media when over-expressed. The substitutions also prevented rescue of other phenotypes associated with loss of FITM in yeast, including mistargeting of Opi1p, defective ER morphology, and aberrant lipid droplet budding. These results suggest that Scs3p, Yft2p and FITMs in general are LPT enzymes involved in an as yet unknown critical step in phospholipid metabolism.

The VPS13 proteins (VPS13A–D) are thought to mediate lipid transport between organelles and are linked to distinct neurological disorders in humans. Baldwin et al. found that, in addition to known involvement in mitochondrial morphology, VPS13D is essential for peroxisome biogenesis., The VPS13 gene family consists of VPS13A–D in mammals. Although all four genes have been linked to human diseases, their cellular functions are poorly understood, particularly those of VPS13D. We generated and characterized knockouts of each VPS13 gene in HeLa cells. Among the individual knockouts, only VPS13D-KO cells exhibit abnormal mitochondrial morphology. Additionally, VPS13D loss leads to either partial or complete peroxisome loss in several transformed cell lines and in fibroblasts derived from a VPS13D mutation–carrying patient with recessive spinocerebellar ataxia. Our data show that VPS13D regulates peroxisome biogenesis.

Lipid droplets (LDs) are implicated in conditions of lipid and protein dysregulation. The fat storage inducing transmembrane (FIT) family induces LD formation. Here, we establish a model system to study the role of S. cerevisiae FIT homologues (ScFIT), SCS3 and YFT2, in proteostasis and stress response pathways. While LD biogenesis and basal endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) remain unaltered in ScFIT mutants, SCS3 was found essential for proper stress-induced UPR activation and for viability in the absence of the sole yeast UPR transducer IRE1. Devoid of a functional UPR, muted SCS3 exhibited accumulation of triacylglycerol within the ER along with aberrant LD morphology, suggesting a UPR-dependent compensatory mechanism. Additionally, SCS3 was necessary to maintain phospholipid homeostasis. Strikingly, global protein ubiquitination and the turnover of both ER and cytoplasmic misfolded proteins is impaired in ScFITΔ cells, while a screen for interacting partners of Scs3 identifies components of the proteostatic machinery as putative targets. Together, our data support a model where ScFITs play an important role in lipid metabolism and proteostasis beyond their defined roles in LD biogenesis.

Organelles compartmentalize eukaryotic cells, enhancing their ability to respond to environmental and developmental changes. One way in which organelles communicate and integrate their activities is by forming close contacts, often called 'membrane contact sites' (MCSs). Interest in MCSs has grown dramatically in the past decade as it is has become clear that they are ubiquitous and have a much broader range of critical roles in cells than was initially thought. Indeed, functions for MCSs in intracellular signalling (particularly calcium signalling, reactive oxygen species signalling and lipid signalling), autophagy, lipid metabolism, membrane dynamics, cellular stress responses and organelle trafficking and biogenesis have now been reported.

Lipid droplets (LDs) are ubiquitous organelles that fulfill essential roles in response to metabolic cues. The identification of several neutral lipid synthesizing and regulatory protein complexes have propelled significant advance on the mechanisms of LD biogenesis in the endoplasmic reticulum (ER). However, our understanding of signaling networks, especially transcriptional mechanisms, regulating membrane biogenesis is very limited. Here, we show that the nutrient-sensing Target of Rapamycin Complex 1 (TORC1) regulates LD formation at a transcriptional level, by targeting DGA1 expression, in a Sit4-, Mks1-, and Sfp1-dependent manner. We show that cytosolic pH (pHc), co-regulated by the plasma membrane H+-ATPase Pma1 and the vacuolar ATPase (V-ATPase), acts as a second messenger, upstream of protein kinase A (PKA), to adjust the localization and activity of the major transcription factor repressor Opi1, which in turn controls the metabolic switch between phospholipid metabolism and lipid storage. Together, this work delineates hitherto unknown molecular mechanisms that couple nutrient availability and pHc to LD formation through a transcriptional circuit regulated by major signaling transduction pathways This research was funded by FCT—Fundação para a Ciência e a Tecnologia. V.T. (CEECIND/00724/2017 andCEECIND/00724/2017/CP1386/CT0006) and T.M. (SFRH/BD/136996/2018) were supported by FCT. This work was also funded by national funds through FCT, under the project UIDB/04293/2020. W.A.P is supported by the Intramural Research Program of The National Institute of Diabetes and Digestive and Kidney Diseases.

Liu et al. show that ER–Golgi tethering increases during ER stress in yeast. The protein Nvj2p is required for this tethering, which promotes nonvesicular ceramide transport from the ER to the Golgi to alleviate ceramide toxicity., Ceramides are key intermediates in sphingolipid biosynthesis and potent signaling molecules. However, excess ceramide is toxic, causing growth arrest and apoptosis. In this study, we identify a novel mechanism by which cells prevent the toxic accumulation of ceramides; they facilitate nonvesicular ceramide transfer from the endoplasmic reticulum (ER) to the Golgi complex, where ceramides are converted to complex sphingolipids. We find that the yeast protein Nvj2p promotes the nonvesicular transfer of ceramides from the ER to the Golgi complex. The protein is a tether that generates close contacts between these compartments and may directly transport ceramide. Nvj2p normally resides at contacts between the ER and other organelles, but during ER stress, it relocalizes to and increases ER–Golgi contacts. ER–Golgi contacts fail to form during ER stress in cells lacking Nvj2p. Our findings demonstrate that cells regulate ER–Golgi contacts in response to stress and reveal that nonvesicular ceramide transfer out of the ER prevents the buildup of toxic amounts of ceramides.

Joshi et al. show that Pex30p and Pex31p contain reticulon-like ER tubulating domains. Like reticulons, they localize to the edges of ER sheets and tubules but are only present in subdomains. These subdomains are devoid of reticulons and are the sites of pre-peroxisome vesicle biogenesis., Saccharomyces cerevisiae contains three conserved reticulon and reticulon-like proteins that help maintain ER structure by stabilizing high membrane curvature in ER tubules and the edges of ER sheets. A mutant lacking all three proteins has dramatically altered ER morphology. We found that ER shape is restored in this mutant when Pex30p or its homologue Pex31p is overexpressed. Pex30p can tubulate membranes both in cells and when reconstituted into proteoliposomes, indicating that Pex30p is a novel ER-shaping protein. In contrast to the reticulons, Pex30p is low abundance, and we found that it localizes to subdomains in the ER. We show that these ER subdomains are the sites where most preperoxisomal vesicles (PPVs) are generated. In addition, overproduction or deletion of Pex30p or Pex31p alters the size, shape, and number of PPVs. Our findings suggest that Pex30p and Pex31p help shape and generate regions of the ER where PPV biogenesis occurs.

Nascent lipid droplet (LD) formation occurs in the endoplasmic reticulum (ER) membrane but it is not known how sites of biogenesis are determined. We previously identified ER domains in S. cerevisiae containing the reticulon homology domain (RHD) protein Pex30 that are regions where preperoxisomal vesicles (PPVs) form. Here, we show that Pex30 domains are also sites where most nascent LDs form. Mature LDs usually remain associated with Pex30 subdomains, and the same Pex30 subdomain can simultaneously associate with a LD and a PPV or peroxisome. We find that in higher eukaryotes multiple C2 domain containing transmembrane protein (MCTP2) is similar to Pex30: it contains an RHD and resides in ER domains where most nascent LD biogenesis occurs and that often associate with peroxisomes. Together, these findings indicate that most LDs and PPVs form and remain associated with conserved ER subdomains, and suggest a link between LD and peroxisome biogenesis., Lipid droplets (LDs) and peroxisomes are both generated by budding off the endoplasmic reticulum (ER). Here, the authors show that the yeast protein Pex30 marks ER subdomains where both LD and peroxisome biogenesis occurs, and identify MCTP2 as the putative mammalian Pex30 ortholog.

Recent studies have reported intrinsic metabolic reprogramming in Pkd1 knock-out cells, implicating dysregulated cellular metabolism in the pathogenesis of polycystic kidney disease. However, the exact nature of the metabolic changes and their underlying cause remains controversial. We show herein that Pkd1 k o /ko renal epithelial cells have impaired fatty acid utilization, abnormal mitochondrial morphology and function, and that mitochondria in kidneys of ADPKD patients have morphological alterations. We further show that a C-terminal cleavage product of polycystin-1 (CTT) translocates to the mitochondria matrix and that expression of CTT in Pkd1 ko/ko cells rescues some of the mitochondrial phenotypes. Using Drosophila to model in vivo effects, we find that transgenic expression of mouse CTT results in decreased viability and exercise endurance but increased CO2 production, consistent with altered mitochondrial function. Our results suggest that PC1 may play a direct role in regulating mitochondrial function and cellular metabolism and provide a framework to understand how impaired mitochondrial function could be linked to the regulation of tubular diameter in both physiological and pathological conditions.

Cytological and biochemical analyses show that Ylr072w, here renamed Lipid transfer at contact site 1 (Ltc1), is a sterol transport protein localized to both ER–mitochondria and ER–vacuole contact sites in partnership with the organelle-specific components Tom70/81 and Vac8, respectively., Organelle contact sites perform fundamental functions in cells, including lipid and ion homeostasis, membrane dynamics, and signaling. Using a forward proteomics approach in yeast, we identified new ER–mitochondria and ER–vacuole contacts specified by an uncharacterized protein, Ylr072w. Ylr072w is a conserved protein with GRAM and VASt domains that selectively transports sterols and is thus termed Ltc1, for Lipid transfer at contact site 1. Ltc1 localized to ER–mitochondria and ER–vacuole contacts via the mitochondrial import receptors Tom70/71 and the vacuolar protein Vac8, respectively. At mitochondria, Ltc1 was required for cell viability in the absence of Mdm34, a subunit of the ER–mitochondria encounter structure. At vacuoles, Ltc1 was required for sterol-enriched membrane domain formation in response to stress. Increasing the proportion of Ltc1 at vacuoles was sufficient to induce sterol-enriched vacuolar domains without stress. Thus, our data support a model in which Ltc1 is a sterol-dependent regulator of organelle and cellular homeostasis via its dual localization to ER–mitochondria and ER–vacuole contact sites.

Visualization of nascent lipid droplets reveals that they form lens-like structures inside the ER membrane bilayer and that FIT proteins are necessary for lipid droplet protrusion toward the cytoplasm., Lipid droplets (LDs) are found in all cells and play critical roles in lipid metabolism. De novo LD biogenesis occurs in the endoplasmic reticulum (ER) but is not well understood. We imaged early stages of LD biogenesis using electron microscopy and found that nascent LDs form lens-like structures that are in the ER membrane, raising the question of how these nascent LDs bud from the ER as they grow. We found that a conserved family of proteins, fat storage-inducing transmembrane (FIT) proteins, is required for proper budding of LDs from the ER. Elimination or reduction of FIT proteins in yeast and higher eukaryotes causes LDs to remain in the ER membrane. Deletion of the single FIT protein in Caenorhabditis elegans is lethal, suggesting that LD budding is an essential process in this organism. Our findings indicated that FIT proteins are necessary to promote budding of nascent LDs from the ER.

Cholesterol biosynthesis in the endoplasmic reticulum (ER) is tightly controlled by multiple mechanisms to regulate cellular cholesterol levels. Squalene monooxygenase (SM) is the second rate-limiting enzyme in cholesterol biosynthesis and is regulated both transcriptionally and post-translationally. SM undergoes cholesterol-dependent proteasomal degradation when cholesterol is in excess. The first 100 amino acids of SM (designated SM N100) are necessary for this degradative process and represent the shortest cholesterol-regulated degron identified to date. However, the fundamental intrinsic characteristics of this degron remain unknown. In this study, we performed a series of deletions, point mutations, and domain swaps to identify a 12-residue region (residues Gln-62-Leu-73), required for SM cholesterol-mediated turnover. Molecular dynamics and circular dichroism revealed an amphipathic helix within this 12-residue region. Moreover, 70% of the variation in cholesterol regulation was dependent on the hydrophobicity of this region. Of note, the earliest known Doa10 yeast degron, Deg1, also contains an amphipathic helix and exhibits 42% amino acid similarity with SM N100. Mutating SM residues Phe-35/Ser-37/Leu-65/Ile-69 into alanine, based on the key residues in Deg1, blunted SM cholesterol-mediated turnover. Taken together, our results support a model whereby the amphipathic helix in SM N100 attaches reversibly to the ER membrane depending on cholesterol levels; with excess, the helix is ejected and unravels, exposing a hydrophobic patch, which then serves as a degradation signal. Our findings shed new light on the regulation of a key cholesterol synthesis enzyme, highlighting the conservation of critical degron features from yeast to humans.

Significance Mitochondria constantly connect through membrane fusion. The merging of the outer mitochondrial membrane requires mitofusin (MFN) proteins. MFN is a membrane-anchored GTPase, but whether it is sufficient to achieve fusion, and if so how, is largely unknown. We have taken advantage of a similar GTPase named atlastin (ATL), which mediates fusion of the endoplasmic reticulum (ER), as its mechanism is better understood. Domain swapping experiments show that MFN is capable of fusing membranes, even on the ER. The C-terminal tail of MFN contains an amphipathic helix that promotes fusion. MFN is properly inserted into the mitochondrial membrane with the help of the helix and neighboring hydrophobic residues. These findings provide insight into how mitochondria fuse., Mitochondria constantly divide and fuse. Homotypic fusion of the outer mitochondrial membranes requires the mitofusin (MFN) proteins, a family of dynamin-like GTPases. MFNs are anchored in the membrane by transmembrane (TM) segments, exposing both the N-terminal GTPase domain and the C-terminal tail (CT) to the cytosol. This arrangement is very similar to that of the atlastin (ATL) GTPases, which mediate fusion of endoplasmic reticulum (ER) membranes. We engineered various MFN-ATL chimeras to gain mechanistic insight into MFN-mediated fusion. When MFN1 is localized to the ER by TM swapping with ATL1, it functions in the maintenance of ER morphology and fusion. In addition, an amphipathic helix in the CT of MFN1 is exchangeable with that of ATL1 and critical for mitochondrial localization of MFN1. Furthermore, hydrophobic residues N-terminal to the TM segments of MFN1 play a role in membrane targeting but not fusion. Our findings provide important insight into MFN-mediated membrane fusion.

Nascent lipid droplet (LD) formation occurs in the ER membrane1-4. It is not known whether LD biogenesis occurs stochastically in the ER or at subdomains with unique protein and lipid composition. We previously identified ER subdomains in S. cerevisiae that contain Pex30, a reticulon-like ER-resident membrane protein5. There are ~25 Pex30-containing puncta in the ER per cell. These sites are regions where preperoxisomal vesicles (PPVs) are generated5. Here we show that Pex30 subdomains are also the location where most nascent LDs form. Mature LDs remain associated with Pex30 subdomains and the same Pex30 subdomain can simultaneously associate with a LD and a PPV. Pex30 subdomains become highly enriched in diacylglycerol (DAG) during LD biogenesis, indicating they have a unique lipid composition. We find that in higher eukaryotes multiple C2 domain containing transmembrane protein (MCTP2) is the functional homologue of Pex30; MCTP2 resides in ER subdomains where most nascent LD biogenesis occurs and that are often associated with peroxisomes. Together, these findings indicate that most LDs and PPVs form and remain associated with conserved ER subdomains and suggest a link between LD and peroxisome biogenesis.

Murley et al. show that sterol transport proteins regulate target-of-rapamycin signaling at membrane contact sites in budding yeast. Ltc3/4 localize to a previously unknown region of the plasma membrane and, with other proteins, inhibit TORC2-Ypk1 signaling. At ER–vacuole contact sites, Ltc1 regulates the formation of sterol-enriched membrane domains that correlate with reduced TORC1 signaling., Membrane contact sites (MCSs) function to facilitate the formation of membrane domains composed of specialized lipids, proteins, and nucleic acids. In cells, membrane domains regulate membrane dynamics and biochemical and signaling pathways. We and others identified a highly conserved family of sterol transport proteins (Ltc/Lam) localized at diverse MCSs. In this study, we describe data indicating that the yeast family members Ltc1 and Ltc3/4 function at the vacuole and plasma membrane, respectively, to create membrane domains that partition upstream regulators of the TORC1 and TORC2 signaling pathways to coordinate cellular stress responses with sterol homeostasis.

Lipid droplets (LDs) store fats and play critical roles in lipid and energy homeostasis. They form between the leaflets of the endoplasmic reticulum (ER) membrane and consist of a neutral lipid core wrapped in a phospholipid monolayer with proteins. Two types of ER-LD architecture are thought to exist and be essential for LD functioning. Maturing LDs either emerge from the ER into the cytoplasm, remaining attached to the ER by a narrow membrane neck, or stay embedded in the ER and are surrounded by ER membrane. Here, we identify a lipid-based mechanism that controls which of these two architectures is favored. Theoretical modeling indicated that the intrinsic molecular curvatures of ER phospholipids can determine whether LDs remain embedded in or emerge from the ER; lipids with negative intrinsic curvature such as diacylglycerol (DAG) and phosphatidylethanolamine favor LD embedding, while those with positive intrinsic curvature, like lysolipids, support LD emergence. This prediction was verified by altering the lipid composition of the ER in S. cerevisiae using mutants and the addition of exogenous lipids. We found that fat-storage-inducing transmembrane protein 2 (FIT2) homologs become enriched at sites of LD generation when biogenesis is induced. DAG accumulates at sites of LD biogenesis, and FIT2 proteins may promote LD emergence from the ER by reducing DAG levels at these sites. Altogether, our findings suggest that cells regulate LD integration in the ER by modulating ER lipid composition, particularly at sites of LD biogenesis and that FIT2 proteins may play a central role in this process.

Regions of close apposition between two organelles, often referred to as membrane contact sites (MCSs), mostly form between the endoplasmic reticulum and a second organelle, although contacts between mitochondria and other organelles have also begun to be characterized. Although these contact sites have been noted since cells first began to be visualized with electron microscopy, the functions of most of these domains long remained unclear. The last few years have witnessed a dramatic increase in our understanding of MCSs, revealing the critical roles they play in intracellular signaling, metabolism, the trafficking of metabolites, and organelle inheritance, division, and transport.

Stable raftlike lipid domains form and segregate membrane proteins in the yeast vacuole in response to various stresses., It has been proposed that membrane rafts, which are sterol- and sphingolipid-enriched liquid-ordered (Lo) domains, segregate proteins in membranes and play critical roles in numerous processes in cells. However, rafts remain controversial because they are difficult to observe in cells without invasive methods and seem to be very small (nanoscale) and short lived, leading many to question whether they exist or are physiologically relevant. In this paper, we show that micrometer-scale, stable lipid domains formed in the yeast vacuole membrane in response to nutrient deprivation, changes in the pH of the growth medium, and other stresses. All vacuolar membrane proteins tested segregated to one of two domains. These domains formed quasi-symmetrical patterns strikingly similar to those found in liposomes containing coexisting Lo and liquid-disordered regions. Indeed, we found that one of these domains is probably sterol enriched and Lo. Domain formation was shown to be regulated by the pH-responsive Rim101 signaling pathway and may also require vesicular trafficking to vacuoles.

Understanding organelle biogenesis is a central focus of cell biology. Whereas some are generated from existing organelles, others can be generated de novo. Most de novo organelle biogenesis occurs in the endoplasmic reticulum (ER). Here, we review the role of the ER in the generation of peroxisomes, lipid droplets, and omegasomes, which are platforms for autophagosome production, and discuss how ER subdomains with specific protein and lipid composition form and promote organelle biogenesis.

International audience; Lipid trafficking between mitochondria and other organelles is required for mitochondrial membrane biogenesis and signaling. This lipid exchange occurs by poorly understood nonvesicular mechanisms. In yeast and mammalian cells, this lipid exchange is thought to take place at contact sites between mitochondria and the ER or vacuolar membranes. Some proteins involved in the tethering between membranes or in the transfer of lipids in mitochondria have been identified. However, in plants, little is known about the synthesis of mitochondrial membranes. Mitochondrial membrane biogenesis is particularly important and noteworthy in plants as the lipid composition of mitochondrial membranes is dramatically changed during phosphate starvation and other stresses. This review focuses on the principal pathways involved in the synthesis of the most abundant mitochondrial glycerolipids in plants and the lipid trafficking that is required for plant mitochondria membrane biogenesis.

In higher eukaryotes, the endoplasmic reticulum (ER) contains a network of membrane tubules, which transitions into sheets during mitosis. Network formation involves curvature-stabilizing proteins, including the reticulons (Rtns), as well as the membrane-fusing GTPase atlastin (ATL) and the lunapark protein (Lnp). Here, we have analyzed how these proteins cooperate. ATL is needed to not only form, but also maintain, the ER network. Maintenance requires a balance between ATL and Rtn, as too little ATL activity or too high Rtn4a concentrations cause ER fragmentation. Lnp only affects the abundance of three-way junctions and tubules. We suggest a model in which ATL-mediated fusion counteracts the instability of free tubule ends. ATL tethers and fuses tubules stabilized by the Rtns, and transiently sits in newly formed three-way junctions. Lnp subsequently moves into the junctional sheets and forms oligomers. Lnp is inactivated by mitotic phosphorylation, which contributes to the tubule-to-sheet conversion of the ER. DOI: http://dx.doi.org/10.7554/eLife.18605.001, eLife digest The endoplasmic reticulum is a compartment within the cells of plants, animals and other eukaryotes. This compartment plays a number of roles within cells, for example, serving as the site where many proteins and fat molecules are built. Most often the endoplasmic reticulum exists as a network of thin tubules. However, this shape changes during the lifetime of a single cell, and the endoplasmic reticulum converts into flattened structures known as sheets when the cell divides. Three classes of proteins are known to affect the shape of the endoplasmic reticulum. Proteins called reticulons (called Rtns for short) stabilize the highly curved membranes that make up the thin tubules, while proteins called atlastins (ATLs) fuse these tubules together to form the interconnected network. However, the exact role of the third protein – called lunapark (Lnp) – is unknown. Moreover, it is not clear how these three proteins work together to coordinate their individual activity to shape the endoplasmic reticulum. Now, Wang, Tukachinsky, Romano et al. have used mammalian cells grown in the laboratory and extracts from the eggs of the frog Xenopus laevis to study these three proteins in more details. Unexpectedly, the experiments showed that ATL’s activity was not only required to form a tubular network but also to maintain it. When ATL was inactivated, the network disassembled into small spheres called vesicles. Increasing the amount of Rtn within the endoplasmic reticulum also caused it to disassemble, but increasing the amount of ATL could reverse this fragmentation. Thus, maintaining the tubular network requires a balance between the activities of the ATL and Rtn proteins, with ATL appearing to tether and fuse tubules that are stabilized by the Rtns. Wang et al. also found that the tubular network of the endoplasmic reticulum can form without Lnp, but fewer tubules and junctions are formed. These findings suggest that Lnp might act to stabilize the junctions between tubules. Further experiments showed that Lnp is modified by the addition of phosphate groups before the cell begins to divide. Wang et al. propose that this modification switches Lnp off and helps the endoplasmic reticulum to convert into sheets. Further work is now needed to investigate exactly how Rtn, ATL, and Lnp shape the endoplasmic reticulum. These future experiments will likely have to use simpler systems, in which the purified proteins are incorporated into artificial membranes. DOI: http://dx.doi.org/10.7554/eLife.18605.002

International audience; The mitochondrion is an organelle originating from an endosymbiotic event and playing a role in several fundamental processes such as energy production, metabolite syntheses, and programmed cell death. This organelle is delineated by two membranes whose synthesis requires an extensive exchange of phospholipids with other cellular organelles such as endoplasmic reticulum (ER) and vacuolar membranes in yeast. These transfers of phospholipids are thought to occur by a non-vesicular pathway at contact sites between two closely apposed membranes. In plants, little is known about the biogenesis of mitochondrial membranes. Contact sites between ER and mitochondria are suspected to play a similar role in phospholipid trafficking as in yeast, but this has never been demonstrated. In contrast, it has been shown that plastids are able to transfer lipids to mitochondria during phosphate starvation. However, the proteins involved in such transfer are still unknown. Here, we identified in Arabidopsis thaliana a large lipid-enriched complex called the mitochondrial transmembrane lipoprotein (MTL) complex. The MTL complex contains proteins located in the two mitochondrial membranes and conserved in all eukaryotic cells, such as the TOM complex and AtMic60, a component of the MICOS complex. We demonstrate that AtMic60 contributes to the export of phosphatidylethanolamine from mitochondria and the import of galactoglycerolipids from plastids during phosphate starvation. Furthermore, AtMic60 promotes lipid desorption from membranes, likely as an initial step for lipid transfer, and binds to Tom40, suggesting that AtMic60 could regulate the tethering between the inner and outer membranes of mitochondria.

Budding yeast Sey1p functions analogously to mammalian atlastins in mediating ER fusion through a mechanism that is redundant with a second, ER SNARE-mediated fusion mechanism., The endoplasmic reticulum (ER) forms a network of tubules and sheets that requires homotypic membrane fusion to be maintained. In metazoans, this process is mediated by dynamin-like guanosine triphosphatases (GTPases) called atlastins (ATLs), which are also required to maintain ER morphology. Previous work suggested that the dynamin-like GTPase Sey1p was needed to maintain ER morphology in Saccharomyces cerevisiae. In this paper, we demonstrate that Sey1p, like ATLs, mediates homotypic ER fusion. The absence of Sey1p resulted in the ER undergoing delayed fusion in vivo and proteoliposomes containing purified Sey1p fused in a GTP-dependent manner in vitro. Sey1p could be partially replaced by ATL1 in vivo. Like ATL1, Sey1p underwent GTP-dependent dimerization. We found that the residual ER–ER fusion that occurred in cells lacking Sey1p required the ER SNARE Ufe1p. Collectively, our results show that Sey1p and its homologues function analogously to ATLs in mediating ER fusion. They also indicate that S. cerevisiae has an alternative fusion mechanism that requires ER SNAREs.

Membrane contact sites (MCSs), where the membranes of two organelles are closely apposed, are regions where small molecules such as lipids or calcium are exchanged between organelles. We have identified a conserved membrane-binding domain found exclusively in proteins at MCSs in Saccharomyces cerevisiae. The synaptotagmin-like-mitochondrial-lipid binding protein (SMP) domain is conserved across species. We show that all seven proteins that contain this domain in yeast localize to one of three MCSs. Human proteins with SMP domains also localize to MCSs when expressed in yeast. The SMP domain binds membranes and is necessary for protein targeting to MCSs. Proteins containing this domain could be involved in lipid metabolism. This is the first protein domain found exclusively in proteins at MCSs.

ORP5 works together with Niemann Pick C-1 to facilitate exit of cholesterol from endosomes and lysosomes., Oxysterol-binding protein (OSBP) and its related proteins (ORPs) constitute a large and evolutionarily conserved family of lipid-binding proteins that target organelle membranes to mediate sterol signaling and/or transport. Here we characterize ORP5, a tail-anchored ORP protein that localizes to the endoplasmic reticulum. Knocking down ORP5 causes cholesterol accumulation in late endosomes and lysosomes, which is reminiscent of the cholesterol trafficking defect in Niemann Pick C (NPC) fibroblasts. Cholesterol appears to accumulate in the limiting membranes of endosomal compartments in ORP5-depleted cells, whereas depletion of NPC1 or both ORP5 and NPC1 results in luminal accumulation of cholesterol. Moreover, trans-Golgi resident proteins mislocalize to endosomal compartments upon ORP5 depletion, which depends on a functional NPC1. Our results establish the first link between NPC1 and a cytoplasmic sterol carrier, and suggest that ORP5 may cooperate with NPC1 to mediate the exit of cholesterol from endosomes/lysosomes.

Oxysterol-binding protein (OSBP)-related proteins (ORPs) are lipid-binding proteins that are conserved from yeast to humans. They are implicated in many cellular processes including signaling, vesicular trafficking, lipid metabolism, and nonvesicular sterol transport. All ORPs contain an OSBP-related domain (ORD) that has a hydrophobic pocket that binds a single sterol. ORDs also contain additional membrane-binding surfaces, some of which bind phosphoinositides and may regulate sterol binding. Studies in yeast suggest that ORPs function as sterol transporters, perhaps in regions where organelle membranes are closely apposed. Yeast ORPs also participate in vesicular trafficking, although their role is unclear. In mammalian cells, some ORPs function as sterol sensors that regulate the assembly of protein complexes in response to changes in cholesterol levels. This review will summarize recent advances in our understanding of how ORPs bind lipids and membranes and how they function in diverse cellular processes.

SummaryThe endoplasmic reticulum (ER) consists of the nuclear envelope and a peripheral network of tubules and membrane sheets. The tubules are shaped by the curvature-stabilizing proteins reticulons and DP1/Yop1p, but how the sheets are formed is unclear. Here, we identify several sheet-enriched membrane proteins in the mammalian ER, including proteins that translocate and modify newly synthesized polypeptides, as well as coiled-coil membrane proteins that are highly upregulated in cells with proliferated ER sheets, all of which are localized by membrane-bound polysomes. These results indicate that sheets and tubules correspond to rough and smooth ER, respectively. One of the coiled-coil proteins, Climp63, serves as a “luminal ER spacer” and forms sheets when overexpressed. More universally, however, sheet formation appears to involve the reticulons and DP1/Yop1p, which localize to sheet edges and whose abundance determines the ratio of sheets to tubules. These proteins may generate sheets by stabilizing the high curvature of edges.

Iron is an essential cofactor for enzymes involved in numerous cellular processes, yet little is known about the impact of iron deficiency on cellular metabolism or iron proteins. Previous studies have focused on changes in transcript and proteins levels in iron-deficient cells, yet these changes may not reflect changes in transport activity or flux through a metabolic pathway. We analyzed the metabolomes and transcriptomes of yeast grown in iron-rich and iron-poor media to determine which biosynthetic processes are altered when iron availability falls. Iron deficiency led to changes in glucose metabolism, amino acid biosynthesis, and lipid biosynthesis that were due to deficiencies in specific iron-dependent enzymes. Iron-sulfur proteins exhibited loss of iron cofactors, yet amino acid synthesis was maintained. Ergosterol and sphingolipid biosynthetic pathways had blocks at points where heme and diiron enzymes function, whereas Ole1, the essential fatty acid desaturase, was resistant to iron depletion. Iron-deficient cells exhibited depletion of most iron enzyme activities, but loss of activity during iron deficiency did not consistently disrupt metabolism. Amino acid homeostasis was robust, but iron deficiency impaired lipid synthesis, altering the properties and functions of cellular membranes.

Cellular membranes can assume a number of highly dynamic shapes. Many cellular processes also require transient membrane deformations. Membrane shape is determined by the complex interactions of proteins and lipids. A number of families of proteins that directly bend membranes have been identified. Most associate transiently with membranes and deform them. These proteins work by one or more of three types of mechanisms. First, some bend membranes by inserting amphipathic domains into one of the leaflets of the bilayer; increasing the area of only one leaflet causes the membrane to bend. Second, some proteins form a rigid scaffold that deforms the underlying membrane or stabilizes an already bent membrane. Third, some proteins may deform membranes by clustering lipids or by affecting lipid ordering in membranes. Still other proteins may use novel but poorly understood mechanisms. In this review, we summarize what is known about how different families of proteins bend membranes.

SummaryThe endoplasmic reticulum (ER) consists of tubules that are shaped by the reticulons and DP1/Yop1p, but how the tubules form an interconnected network is unknown. Here, we show that mammalian atlastins, which are dynamin-like, integral membrane GTPases, interact with the tubule-shaping proteins. The atlastins localize to the tubular ER and are required for proper network formation in vivo and in vitro. Depletion of the atlastins or overexpression of dominant-negative forms inhibits tubule interconnections. The Sey1p GTPase in S. cerevisiae is likely a functional ortholog of the atlastins; it shares the same signature motifs and membrane topology and interacts genetically and physically with the tubule-shaping proteins. Cells simultaneously lacking Sey1p and a tubule-shaping protein have ER morphology defects. These results indicate that formation of the tubular ER network depends on conserved dynamin-like GTPases. Since atlastin-1 mutations cause a common form of hereditary spastic paraplegia, we suggest ER-shaping defects as a neuropathogenic mechanism.

The oxysterol binding protein homologue Kes1p has been implicated in nonvesicular sterol transport in Saccharomyces cerevisiae. Kes1p also represses formation of protein transport vesicles from the trans-Golgi network (TGN) through an unknown mechanism. Here, we show that potential phospholipid translocases in the Drs2/Dnf family (type IV P-type ATPases [P4-ATPases]) are downstream targets of Kes1p repression. Disruption of KES1 suppresses the cold-sensitive (cs) growth defect of drs2Δ, which correlates with an enhanced ability of Dnf P4-ATPases to functionally substitute for Drs2p. Loss of Kes1p also suppresses a drs2-ts allele in a strain deficient for Dnf P4-ATPases, suggesting that Kes1p antagonizes Drs2p activity in vivo. Indeed, Drs2-dependent phosphatidylserine translocase (flippase) activity is hyperactive in TGN membranes from kes1Δ cells and is potently attenuated by addition of recombinant Kes1p. Surprisingly, Drs2p also antagonizes Kes1p activity in vivo. Drs2p deficiency causes a markedly increased rate of cholesterol transport from the plasma membrane to the endoplasmic reticulum (ER) and redistribution of endogenous ergosterol to intracellular membranes, phenotypes that are Kes1p dependent. These data suggest a homeostatic feedback mechanism in which appropriately regulated flippase activity in the Golgi complex helps establish a plasma membrane phospholipid organization that resists sterol extraction by a sterol binding protein.

The ORP lipid-binding domain can contact two membranes simultaneously to facilitate sterol extraction or delivery at one membrane in response to the lipid composition of the other., Sterols are transferred between cellular membranes by vesicular and poorly understood nonvesicular pathways. Oxysterol-binding protein–related proteins (ORPs) have been implicated in sterol sensing and nonvesicular transport. In this study, we show that yeast ORPs use a novel mechanism that allows regulated sterol transfer between closely apposed membranes, such as organelle contact sites. We find that the core lipid-binding domain found in all ORPs can simultaneously bind two membranes. Using Osh4p/Kes1p as a representative ORP, we show that ORPs have at least two membrane-binding surfaces; one near the mouth of the sterol-binding pocket and a distal site that can bind a second membrane. The distal site is required for the protein to function in cells and, remarkably, regulates the rate at which Osh4p extracts and delivers sterols in a phosphoinositide-dependent manner. Together, these findings suggest a new model of how ORPs could sense and regulate the lipid composition of adjacent membranes.

The endoplasmic reticulum (ER) is highly plastic, and increased expression of distinct single ER-resident membrane proteins, such as HMG-CoA reductase (HMGR), can induce a dramatic restructuring of ER membranes into highly organized arrays. Studies on the ER-remodeling behavior of the two yeast HMGR isozymes, Hmg1p and Hmg2p, suggest that they could be mechanistically distinct. We examined the features of Hmg2p required to generate its characteristic structures, and we found that the molecular requirements are similar to those of Hmg1p. However, the structures generated by Hmg1p and Hmg2p have distinct cell biological features determined by the transmembrane regions of the proteins. In parallel, we conducted a genetic screen to identify HER genes (required for Hmg2p-induced ER Remodeling), further confirming that the mechanisms of membrane reorganization by these two proteins are distinct because most of the HER genes were required for Hmg2p but not Hmg1p-induced ER remodeling. One of the HER genes identified was PSD1, which encodes the phospholipid biosynthetic enzyme phosphatidylserine decarboxylase. This direct connection to phospholipid biosynthesis prompted a more detailed examination of the effects of Hmg2p on phospholipid mutants and composition. Our analysis revealed that overexpression of Hmg2p caused significant and specific growth defects in nulls of the methylation pathway for phosphatidylcholine biosynthesis that includes the Psd1p enzyme. Furthermore, increased expression of Hmg2p altered the composition of cellular phospholipids in a manner that implied a role for PSD1. These phospholipid effects, unlike Hmg2p-induced ER remodeling, required the enzymatic activity of Hmg2p. Together, our results indicate that, although related, Hmg2p- and Hmg1p-induced ER remodeling are mechanistically distinct.

We recently identified a class of membrane proteins, the reticulons and DP1/Yop1p, which shape the tubular endoplasmic reticulum (ER) in yeast and mammalian cells. These proteins are highly enriched in the tubular portions of the ER and virtually excluded from other regions. To understand how they promote tubule formation, we characterized their behavior in cellular membranes and addressed how their localization in the ER is determined. Using fluorescence recovery after photobleaching, we found that yeast Rtn1p and Yop1p are less mobile in the membrane than normal ER proteins. Sucrose gradient centrifugation and cross-linking analyses show that they form oligomers. Mutants of yeast Rtn1p, which no longer localize exclusively to the tubular ER or are even totally inactive in inducing ER tubules, are more mobile and oligomerize less extensively. The mammalian reticulons and DP1 are also relatively immobile and can form oligomers. The conserved reticulon homology domain that includes the two membrane-embedded segments is sufficient for the localization of the reticulons to the tubular ER, as well as for their diffusional immobility and oligomerization. Finally, ATP depletion in both yeast and mammalian cells further decreases the mobilities of the reticulons and DP1. We propose that oligomerization of the reticulons and DP1/Yop1p is important for both their localization to the tubular domains of the ER and for their ability to form tubules.

Sterols such as cholesterol are important components of cellular membranes. They are not uniformly distributed among organelles and maintaining the proper distribution of sterols is critical for many cellular functions. Both vesicular and non-vesicular pathways move sterols between membranes and into and out of cells. There is growing evidence that a number of non-vesicular transport pathways operate in cells and, in the past few years, a number of proteins have been proposed to facilitate this transfer. Some are soluble sterol transfer proteins that may move sterol between membranes. Others are integral membranes proteins that mediate sterol efflux, uptake from cells, and perhaps intracellular sterol transfer as well. In most cases, the mechanisms and regulation of these proteins remains poorly understood. This review summarizes our current knowledge of these proteins and how they could contribute to intracellular sterol trafficking and distribution.

Sterols such as cholesterol are a significant component of eukaryotic cellular membranes, and their unique physical properties influence a wide variety of membrane processes. It is known that the concentration of sterol within the membrane varies widely between organelles, and that the cell actively maintains this distribution through various transport processes. Vesicular pathways such as secretion or endocytosis may account for this traffic, but increasing evidence highlights the importance of nonvesicular routes as well. The structure of an oxysterol-binding protein homologue (OSH) in yeast (Osh4p/Kes1p) has recently been solved, identifying it as a sterol binding protein, and there is evidence consistent with the role of a cytoplasmic, nonvesicular sterol transporter. Yeast have seven such proteins, which appear to have distinct but overlapping functions with regard to maintaining intracellular sterol distribution and homeostasis. Control of sterol distribution can have far-reaching effects on membrane-related functions, and Osh proteins have been implicated in a variety of processes such as secretory vesicle budding from the Golgi and establishment of cell polarity. This review summarizes the current body of knowledge regarding this family and its potential functions, placing it in the context of known and hypothesized pathways of sterol transport in yeast.

Most membrane-bound organelles have elaborate, dynamic shapes and often include regions with distinct morphologies. These complex structures are relatively conserved throughout evolution, which indicates that they are important for optimal organelle function. Various mechanisms of determining organelle shape have been proposed - proteins that stabilize highly curved membranes, the tethering of organelles to other cellular components and the regulation of membrane fission and fusion might all contribute.

Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are two of the most abundant phospholipids in cells. Although both lipids can be synthesized in the endoplasmic reticulum (ER), in S. cerevisiae PE can also be produced in mitochondria and endosomes; this PE can be transported back to the ER where it is converted to PC. In this study we found that dithiothreitol (DTT), which induces ER stress, decreases PE export from mitochondria to the ER. This results in decreased levels of total cellular PC and mitochondrial PC. These decreases were not caused by changes in levels of PC synthesizing or degrading enzymes. PE export from mitochondria to the ER during ER stress was further reduced in cells lacking Mdm10p, a component of an ER-mitochondrial tethering complex that may facilitated lipid exchange between these compartments. We also found that reducing mitochondrial PC levels induces mitophagy. In conclusion, we show that ER stress affected PE export from mitochondria to ER and the Mdm10p is important for this process.

Sterols are moved between cellular membranes by nonvesicular pathways whose functions are poorly understood. In yeast, one such pathway transfers sterols from the plasma membrane (PM) to the endoplasmic reticulum (ER). We show that this transport requires oxysterol-binding protein (OSBP)–related proteins (ORPs), which are a large family of conserved lipid-binding proteins. We demonstrate that a representative member of this family, Osh4p/Kes1p, specifically facilitates the nonvesicular transfer of cholesterol and ergosterol between membranes in vitro. In addition, Osh4p transfers sterols more rapidly between membranes containing phosphoinositides (PIPs), suggesting that PIPs regulate sterol transport by ORPs. We confirmed this by showing that PM to ER sterol transport slows dramatically in mutants with conditional defects in PIP biosynthesis. Our findings argue that ORPs move sterols among cellular compartments and that sterol transport and intracellular distribution are regulated by PIPs.