Sterol transfer,PI4P consumption,and control of membrane lipid order by endogenous OSBP

The network of proteins that orchestrate the distribution of cholesterol among cellular organelles is not fully characterized. We previously proposed that oxysterol‐binding protein (OSBP) drives cholesterol/PI4P exchange at contact sites between the endoplasmic reticulum (ER) and the trans‐Golgi network (TGN). Using the inhibitor OSW‐1, we report here that the sole activity of endogenous OSBP makes a major contribution to cholesterol distribution, lipid order, and PI4P turnover in living cells. Blocking OSBP causes accumulation of sterols at ER/lipid droplets at the expense of TGN, thereby reducing the gradient of lipid order along the secretory pathway. OSBP consumes about half of the total cellular pool of PI4P, a consumption that depends on the amount of cholesterol to be transported. Inhibiting the spatially restricted PI4‐kinase PI4KIIIβ triggers large periodic traveling waves of PI4P across the TGN. These waves are cadenced by long‐range PI4P production by PI4KIIα and PI4P consumption by OSBP. Collectively, these data indicate a massive spatiotemporal coupling between cholesterol transport and PI4P turnover via OSBP and PI4‐kinases to control the lipid composition of subcellular membranes.     

Multisite phosphorylation of oxysterol-binding protein regulates sterol binding and activation of sphingomyelin synthesis

The endoplasmic reticulum (ER)-Golgi sterol transfer activity of oxysterol-binding protein (OSBP) regulates sphingomyelin (SM) synthesis, as well as post-Golgi cholesterol efflux pathways. The phosphorylation and ER-Golgi localization of OSBP are correlated, suggesting this modification regulates the directionality and/or specificity of transfer activity. In this paper, we report that phosphorylation on two serine-rich motifs, S381-S391 (site 1) and S192, S195, S200 (site 2), specifically controls OSBP activity at the ER. A phosphomimetic of the SM/cholesterol-sensitive phosphorylation site 1 (OSBP-S5E) had increased in vitro cholesterol and 25-hydroxycholesterol-binding capacity, and cholesterol extraction from liposomes, but reduced transfer activity. Phosphatidylinositol 4-phosphate (PI(4)P) and cholesterol competed for a common binding site on OSBP; however, direct binding of PI(4)P was not affected by site 1 phosphorylation. Individual site 1 and site 2 phosphomutants supported oxysterol activation of SM synthesis in OSBP-deficient CHO cells. However, a double site1/2 mutant (OSBP-S381A/S3D) was deficient in this activity and was constitutively colocalized with vesicle-associated membrane protein-associated protein A (VAP-A) in a collapsed ER network. This study identifies phosphorylation regulation of sterol and VAP-A binding by OSBP in the ER, and PI(4)P as an alternate ligand that could be exchanged for sterol in the Golgi apparatus.     

Osh proteins regulate phosphoinositide metabolism at ER-plasma membrane contact sites

Sac1 phosphoinositide (PI) phosphatases are essential regulators of PI-signaling networks. Yeast Sac1, an integral endoplasmic reticulum (ER) membrane protein, controls PI4P levels at the ER, Golgi, and plasma membrane (PM). Whether Sac1 can act in trans and turn over PI4P at the Golgi and PM from the ER remains a paradox. We find that Sac1-mediated PI4P metabolism requires the oxysterol-binding homology (Osh) proteins. The PH domain-containing family member, Osh3, localizes to PM/ER membrane contact sites dependent upon PM PI4P levels. We reconstitute Osh protein-stimulated Sac1 PI phosphatase activity in vitro. We also show that the ER membrane VAP proteins, Scs2/Scs22, control PM PI4P levels and Sac1 activity in vitro. We propose that Osh3 functions at ER/PM contact sites as both a sensor of PM PI4P and an activator of the ER Sac1 phosphatase. Our findings further suggest that the conserved Osh proteins control PI metabolism at additional membrane contact sites.     

Oxysterol binding protein-dependent activation of sphingomyelin synthesis in the golgi apparatus requires phosphatidylinositol 4-kinase IIα

Cholesterol and sphingomyelin (SM) associate in raft domains and are metabolically coregulated. One aspect of coordinate regulation occurs in the Golgi apparatus where oxysterol binding protein (OSBP) mediates sterol-dependent activation of ceramide transport protein (CERT) activity and SM synthesis. Because CERT transfer activity is dependent on its phosphatidylinositol 4 phosphate [PtdIns(4)P]-specific pleckstrin homology domain, we investigated whether OSBP activation of CERT involved a Golgi-associated PtdIns 4-kinase (PI4K). Cell fractionation experiments revealed that Golgi/endosome-enriched membranes from 25-hydroxycholesterol-treated Chinese hamster ovary cells had increased activity of a sterol-sensitive PI4K that was blocked by small interfering RNA silencing of OSBP. Consistent with this sterol-requirement, OSBP silencing also reduced the cholesterol content of endosome/trans-Golgi network (TGN) fractions containing PI4KIIα. PI4KIIα, but not PI4KIIIβ, was required for oxysterol-activation of SM synthesis and recruitment of CERT to the Golgi apparatus. However, neither PI4KIIα nor PI4KIIIβ expression was required for 25-hydroxycholesterol-dependent translocation of OSBP to the Golgi apparatus. The presence of OSBP, CERT, and PI4KIIα in the TGN of oxysterol-stimulated cells suggests that OSBP couples sterol binding or transfer activity with regulation of PI4KIIα activity, leading to CERT recruitment to the TGN and increased SM synthesis.     

Metabolic Activation of the HOG MAP Kinase Pathway by Snf1/AMPK Regulates Lipid Signaling at the Golgi

Phosphatidylinositol‐4‐phosphate (PI(4)P) is an important regulator of Golgi function. Metabolic regulation of Golgi PI(4)P requires the lipid phosphatase Sac1 that translocates between endoplasmic reticulum (ER) and Golgi membranes. Localization of Sac1 responds to changes in glucose levels, yet the upstream signaling pathways that regulate Sac1 traffic are unknown. Here, we report that mitogen‐activated protein kinase (MAPK) Hog1 transmits glucose signals to the Golgi and regulates localization of Sac1. We find that Hog1 is rapidly activated by both glucose starvation and glucose stimulation, which is independent of the well‐characterized response to osmotic stress but requires the upstream element Ssk1 and is controlled by Snf1, the yeast homolog of AMP‐activated kinase (AMPK). Elimination of either Hog1 or Snf1 slows glucose‐induced translocation of Sac1 lipid phosphatase from the Golgi to the ER and thus delays PI(4)P accumulation at the Golgi. We conclude that a novel cross‐talk between the HOG pathway and Snf1/AMPK is required for the metabolic control of lipid signaling at the Golgi.     

Golgi localization and phosphorylation of oxysterol binding protein in Niemann-Pick C and U18666A-treated cells.

Oxysterol binding protein (OSBP) translocation between Golgi and vesicular/cytoplasmic compartments is affected by conditions that alter cholesterol and sphingomyelin homeostasis, indicating a role in lipid and sterol regulation in this organelle. In this study, we show that OSBP dissociation from the Golgi apparatus was inhibited when LDL cholesterol efflux from lysosomes was blocked in Niemann-Pick C (NPC) or U18666A [3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one]-treated fibroblasts. Dissociation of OSBP from the Golgi apparatus in response to LDL was independent of de novo cholesterol biosynthesis. OSBP did not localize with filipin-stained lysosomal cholesterol, and the NPC defect did not alter OSBP expression or phosphorylation. However, OSBP in the Golgi apparatus was progressively dephosphorylated (as assessed by a molecular mass shift on SDS-PAGE) in U18666A-treated fibroblasts or Chinese hamster ovary cells as a result of combined inhibition of LDL cholesterol transport and de novo cholesterol synthesis. In vivo phosphopeptide mapping and mutagenesis of OSBP was used to identify the cholesterol-sensitive phosphorylation sites at serines 381, 384, and 387 that were responsible for the altered mobility on SDS-PAGE. NPC-1 protein-mediated release of LDL-derived cholesterol and de novo biosynthesis regulates OSBP localization and phosphorylation. This indicates that OSBP responds to or senses altered cellular sterol content and transport.     

Coordinated lipid transfer between the endoplasmic reticulum and the Golgi complex requires the VAP proteins and is essential for Golgi-mediated transport

Lipid transport between intracellular organelles is mediated by vesicular and nonvesicular transport mechanisms and is critical for maintaining the identities of different cellular membranes. Nonvesicular lipid transport between the endoplasmic reticulum (ER) and the Golgi complex has been proposed to affect the lipid composition of the Golgi membranes. Here, we show that the integral ER-membrane proteins VAP-A and VAP-B affect the structural and functional integrity of the Golgi complex. Depletion of VAPs by RNA interference reduces the levels of phosphatidylinositol-4-phosphate (PI4P), diacylglycerol, and sphingomyelin in the Golgi membranes, and it leads to substantial inhibition of Golgi-mediated transport events. These effects are coordinately mediated by the lipid-transfer/binding proteins Nir2, oxysterol-binding protein (OSBP), and ceramide-transfer protein (CERT), which interact with VAPs via their FFAT motif. The effect of VAPs on PI4P levels is mediated by the phosphatidylinositol/phosphatidylcholine transfer protein Nir2, which is required for Golgi targeting of OSBP and CERT and the subsequent production of diacylglycerol and sphingomyelin. We propose that Nir2, OSBP, and CERT function coordinately at the ER-Golgi membrane contact sites, thereby affecting the lipid composition of the Golgi membranes and consequently their structural and functional identities.     

Phosphatidylinositol‐4 kinase III beta and oxysterol‐binding protein accumulate unesterified cholesterol on poliovirus‐induced membrane structure

Studies on anti‐picornavirus compounds have revealed an essential role of a novel cellular pathway via host phosphatidylinositol‐4 kinase III beta (PI4KB) and oxysterol‐binding protein (OSBP) family I in poliovirus (PV) replication. However, the molecular role for this pathway in PV replication has yet to be determined. Here, viral and host proteins modulating production of phosphatidylinositol 4‐phosphate (PI4P) and accumulation of unesterified cholesterol (UC) in cells were analyzed and the role of the PI4KB/OSBP pathway in PV replication characterized. Virus protein 2BC was identified as a novel interactant of PI4KB. PI4KB and VCP/p97 bind to a partially overlapped region of 2BC with different sensitivity to a 2C inhibitor. Production of PI4P and accumulation of UC were enhanced by virus protein 2BC, but suppressed by virus proteins 3A and 3AB. In PV‐infected cells, a PI4KB inhibitor suppressed production of PI4P, and both a PI4KB inhibitor and an OSBP ligand suppressed accumulation of UC on virus‐induced membrane structure. Inhibition of PI4KB activity caused dissociation of OSBP from virus‐induced membrane structure in PV‐infected cells. Synthesis of viral nascent RNA in PV‐infected cells was not affected in the presence of PI4KB inhibitor and OSBP ligand; however, transient pre‐treatment of PV‐infected cells with these inhibitors suppressed viral RNA synthesis. These results suggest that virus proteins modulate PI4KB activity and provide PI4P for recruitment of OSBP to accumulate UC on virus‐induced membrane structure for formation of a virus replication complex.     

Oxysterol Binding Protein–related Protein 9 (ORP9) Is a Cholesterol Transfer Protein That Regulates Golgi Structure and Function

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) constitute a large gene family that differentially localize to organellar membranes, reflecting a functional role in sterol signaling and/or transport. OSBP partitions between the endoplasmic reticulum (ER) and Golgi apparatus where it imparts sterol-dependent regulation of ceramide transport and sphingomyelin synthesis. ORP9L also is localized to the ER–Golgi, but its role in secretion and lipid transport is unknown. Here we demonstrate that ORP9L partitioning between the trans-Golgi/trans-Golgi network (TGN), and the ER is mediated by a phosphatidylinositol 4-phosphate (PI-4P)-specific PH domain and VAMP-associated protein (VAP), respectively. In vitro, both OSBP and ORP9L mediated PI-4P–dependent cholesterol transport between liposomes, suggesting their primary in vivo function is sterol transfer between the Golgi and ER. Depletion of ORP9L by RNAi caused Golgi fragmentation, inhibition of vesicular somatitus virus glycoprotein transport from the ER and accumulation of cholesterol in endosomes/lysosomes. Complete cessation of protein transport and cell growth inhibition was achieved by inducible overexpression of ORP9S, a dominant negative variant lacking the PH domain. We conclude that ORP9 maintains the integrity of the early secretory pathway by mediating transport of sterols between the ER and trans-Golgi/TGN.     

Growth control of Golgi phosphoinositides by reciprocal localization of sac1 lipid phosphatase and pik1 4-kinase

Compartment-specific control of phosphoinositide lipids is essential for cell function. The Sac1 lipid phosphatase regulates endoplasmic reticulum (ER) and Golgi phosphatidylinositol-4-phosphate [PI(4)P] in response to nutrient levels and cell growth stages. During exponential growth, Sac1p interacts with Dpm1p at the ER but shuttles to the Golgi during starvation. Here, we report that a C-terminal region in Sac1p is required for retention in the perinuclear ER, whereas the N-terminal domain is responsible for Golgi localization. We also show that starvation-induced shuttling of Sac1p to the Golgi depends on the coat protein complex II and the Rer1 adaptor protein. Starvation-induced shuttling of Sac1p to the Golgi specifically eliminates a pool of PI(4)P generated by the lipid kinase Pik1p. In addition, absence of nutrients leads to a rapid dissociation of Pik1p, together with its non-catalytical subunit Frq1p, from Golgi membranes. Reciprocal rounds of association/dissociation of the Sac1p lipid phosphatase and the Pik1p/Frq1p lipid kinase complex are responsible for growth-dependent control of Golgi phosphoinositides. Sac1p and Pik1p/Frq1p are therefore elements of a unique machinery that synchronizes ER and Golgi function in response to different growth conditions.     

Sac1, a lipid phosphatase at the interface of vesicular and nonvesicular transport

The lipid phosphatase Sac1 dephosphorylates phosphatidylinositol 4‐phosphate (PI4P), thereby holding levels of this crucial membrane signaling molecule in check. Sac1 regulates multiple cellular processes, including cytoskeletal organization, membrane trafficking and cell signaling. Here, we review the structure and regulation of Sac1, its roles in cell signaling and development and its links to health and disease. Remarkably, many of the diverse roles attributed to Sac1 can be explained by the recent discovery of its requirement at membrane contact sites, where its consumption of PI4P is proposed to drive interorganelle transfer of other cellular lipids, thereby promoting normal lipid homeostasis within cells.      

Regulation of Oxysterol-binding Protein Golgi Localization through Protein Kinase D–mediated Phosphorylation

Protein kinase D (PKD) plays a critical role at the trans-Golgi network by regulating the fission of transport carriers destined for the plasma membrane. Two known Golgi-localized PKD substrates, PI4-kinase IIIβ and the ceramide transfer protein CERT, mediate PKD signaling to influence vesicle trafficking to the plasma membrane and sphingomyelin synthesis, respectively. PKD is recruited and activated at the Golgi through interaction with diacylglycerol, a pool of which is generated as a by-product of sphingomyelin synthesis from ceramide. Here we identify a novel substrate of PKD at the Golgi, the oxysterol-binding protein OSBP. Using a substrate-directed phospho-specific antibody that recognizes the optimal PKD consensus motif, we show that PKD phosphorylates OSBP at Ser240 in vitro and in cells. We further show that OSBP phosphorylation occurs at the Golgi. Phosphorylation of OSBP by PKD does not modulate dimerization, sterol binding, or affinity for PI(4)P. Instead, phosphorylation attenuates OSBP Golgi localization in response to 25-hydroxycholesterol and cholesterol depletion, impairs CERT Golgi localization, and promotes Golgi fragmentation.     

Mutant SOD1 inhibits ER‐Golgi transport in amyotrophic lateral sclerosis

Cu/Zn‐superoxide dismutase is misfolded in familial and sporadic amyotrophic lateral sclerosis, but it is not clear how this triggers endoplasmic reticulum (ER) stress or other pathogenic processes. Here, we demonstrate that mutant SOD1 (mSOD1) is predominantly found in the cytoplasm in neuronal cells. Furthermore, we show that mSOD1 inhibits secretory protein transport from the ER to Golgi apparatus. ER‐Golgi transport is linked to ER stress, Golgi fragmentation and axonal transport and we also show that inhibition of ER‐Golgi trafficking preceded ER stress, Golgi fragmentation, protein aggregation and apoptosis in cells expressing mSOD1. Restoration of ER‐Golgi transport by over‐expression of coatomer coat protein II subunit Sar1 protected against inclusion formation and apoptosis, thus linking dysfunction in ER‐Golgi transport to cellular pathology. These findings thus link several cellular events in amyotrophic lateral sclerosis into a single mechanism occurring early in mSOD1 expressing cells.

     

Phosphatidylinositol 4‐kinase III beta is the target of oxoglaucine and pachypodol (Ro 09‐0179) for their anti‐poliovirus activities,and is located at upstream of the target step of brefeldin A

In recent years, phosphatidylinositol 4‐kinase III beta (PI4KB) has emerged as a conserved target of anti‐picornavirus compounds. In the present study, PI4KB was identified as the direct target of the plant‐derived anti‐picornavirus compounds, oxoglaucine and pachypodol (also known as Ro 09‐0179). PI4KB was also identified as the target via which pachypodol interferes with brefeldin A (BFA)‐induced Golgi disassembly in non‐infected cells. Oxysterol‐binding protein (OSBP) inhibitor also has interfering activity against BFA. It seems that this interference is not essential for the anti‐poliovirus (PV) activities of BFA and PI4KB/OSBP inhibitors. BFA inhibited early to late phase PV replication (0 to 6 hr postinfection) as well as PI4KB inhibitor, but with some delay compared to guanidine hydrochloride treatment. In contrast with PI4KB/OSBP inhibitors, BFA inhibited viral nascent RNA synthesis, suggesting that BFA targets some step of viral RNA synthesis located downstream of the PI4KB/OSBP pathway in PV replication. Our results suggest that PI4KB is a major target of anti‐picornavirus compounds identified in vitro for their anti‐picornavirus activities and for some uncharacterized biological phenomena caused by these compounds, and that BFA and PI4KB/OSBP inhibitors synergistically repress PV replication by targeting distinct steps in viral RNA replication.     

New insights into the OSBP‒VAP cycle

VAP-A is a major endoplasmic reticulum (ER) receptor that allows this organelle to engage numerous membrane contact sites with other organelles. One highly studied example is the formation of contact sites through VAP-A interaction with Oxysterol-binding protein (OSBP). This lipid transfer protein transports cholesterol from the ER to the trans-Golgi network owing to the counter-exchange of the phosphoinositide PI(4)P. In this review, we highlight recent studies that advance our understanding of the OSBP cycle and extend the model of lipid exchange to other cellular contexts and other physiological and pathological conditions.     

Molecular mechanisms and regulation of ceramide transport

De novo biosynthesis of sphingolipids begins in the endoplasmic reticulum (ER) and continues in the Golgi apparatus and plasma membrane. A crucial step in sphingolipid biosynthesis is the transport of ceramide by vesicular and non-vesicular mechanisms from its site of synthesis in the ER to the Golgi apparatus. The recent discovery of the ceramide transport protein CERT has revealed a novel pathway for the delivery of ceramide to the Golgi apparatus for sphingomyelin (SM) synthesis. In addition to a ceramide-binding START domain, CERT has FFAT (referring to two phenylalanines [FF] in an acidic tract) and pleckstrin homology (PH) domains that recognize the ER integral membrane protein VAMP-associated protein (VAP) and Golgi-associated PtdIns 4-phosphate, respectively. Mechanisms for vectorial transport involving dual-organellar targeting and sites of deposition of ceramide in the Golgi apparatus are proposed. Similar Golgi-ER targeting motifs are also present in the oxysterol-binding protein (OSBP), which regulates ceramide transport and SM synthesis in an oxysterol-dependent manner. Consequently, this emerges as a potential mechanism for integration of sphingolipid and cholesterol metabolism. The identification of organellar targeting motifs in other related lipid-binding/transport proteins indicate that concepts learned from the study of ceramide transport can be applied to other lipid transport processes.     

Golgi‐mediated Glycosylation Determines Residency of the T2 RNase Rny1p in Saccharomyces cerevisiae

The role of glycosylation in the function of the T2 family of RNases is not well understood. In this work, we examined how glycosylation affects the progression of the T2 RNase Rny1p through the secretory pathway in Saccharomyces cerevisiae. We found that Rny1p requires entering into the ER first to become active and uses the adaptor protein Erv29p for packaging into COPII vesicles and transport to the Golgi apparatus. While inside the ER, Rny1p undergoes initial N‐linked core glycosylation at four sites, N37, N70, N103 and N123. Rny1p transport to the Golgi results in the further attachment of high‐glycans. Whereas modifications with glycans are dispensable for the nucleolytic activity of Rny1p, Golgi‐mediated modifications are critical for its extracellular secretion. Failure of Golgi‐specific glycosylation appears to direct Rny1p to the vacuole as an alternative destination and/or site of terminal degradation. These data reveal a previously unknown function of Golgi glycosylation in a T2 RNase as a sorting and secretion signal .      

Oxysterol-binding protein and vesicle-associated membrane protein-associated protein are required for sterol-dependent activation of the ceramide transport protein

Sphingomyelin (SM) and cholesterol are coregulated metabolically and associate physically in membrane microdomains involved in cargo sorting and signaling. One mechanism for regulation of this metabolic interface involves oxysterol binding protein (OSBP) via high-affinity binding to oxysterol regulators of cholesterol homeostasis and activation of SM synthesis at the Golgi apparatus. Here, we show that OSBP regulation of SM synthesis involves the endoplasmic reticulum (ER)-to-Golgi ceramide transport protein (CERT). RNA interference (RNAi) experiments in Chinese hamster ovary (CHO)-K1 cells revealed that OSBP and vesicle-associated membrane protein-associated protein (VAP) were required for stimulation of CERT-dependent ceramide transport and SM synthesis by 25-hydroxycholesterol and cholesterol depletion in response to cyclodextrin. Additional RNAi experiments in human embryonic kidney 293 cells supported OSBP involvement in oxysterol-activated SM synthesis and also revealed a role for OSBP in basal SM synthesis. Activation of ER-to-Golgi ceramide transport in CHO-K1 cells required interaction of OSBP with the ER and Golgi apparatus, OSBP-dependent Golgi translocation of CERT, and enhanced CERT-VAP interaction. Regulation of CERT by OSBP, sterols, and VAP reveals a novel mechanism for integrating sterol regulatory signals with ceramide transport and SM synthesis in the Golgi apparatus.     

Vesicle-associated membrane protein-associated protein-A (VAP-A) interacts with the oxysterol-binding protein to modify export from the endoplasmic reticulum

Oxysterol-binding protein (OSBP) is 1 of 12 related proteins implicated in the regulation of vesicle transport and sterol homeostasis. A yeast two-hybrid screen using full-length OSBP as bait was undertaken to identify partner proteins that would provide clues to the function of OSBP. This resulted in the cloning of vesicle-associated membrane protein-associated protein-A (VAP-A), a syntaxin-like protein implicated in endoplasmic reticulum (ER)/Golgi vesicle transport, and phospholipid regulation in mammalian cells and yeast, respectively. By using a combination of yeast two-hybrid, glutathione S-transferase pull-down and immunoprecipitation experiments, the VAP-A-binding region in OSBP was localized to amino acids 351-442. This region did not include the pleckstrin homology (PH) domain but overlapped with the N terminus of the oxysterol binding and OSBP homology domains. C- and N-terminal truncations or deletions of VAP prevented interaction with OSBP but did not affect VAP multimerization. Although the OSBP PH domain was not necessary for VAP-A binding in vitro, interaction with VAP-A was enhanced in cells by mutation of the conserved PH domain tryptophan (OSBP W174A) or deletion of the C-terminal half of the PH domain (OSBP Delta 132-182). OSBP W174A retained oxysterol binding activity, association with phospholipid vesicles via the PH domain, and localized with VAP in unusual ER-associated structures. At 40 degrees C, misfolded ts045-vesicular stomatitis virus G protein fused to green fluorescent protein was co-localized with VAP-A/OSBP W174A structures on the ER but was exported to the Golgi when folded normally at 32 degrees C. A fluorescent ceramide analogue also accumulated in these ER inclusions, and export to the Golgi was partially inhibited as indicated by decreased Golgi staining and a 30% reduction in sphingomyelin synthesis. These studies show that OSBP binding to the ER and Golgi apparatus is regulated by its PH domain and VAP interactions, and the complex is involved at a stage of protein and ceramide transport from the ER.     

CARTS biogenesis requires VAP–lipid transfer protein complexes functioning at the endoplasmic reticulum–Golgi interface

Vesicle-associated membrane protein–associated protein (VAP) is an endoplasmic reticulum (ER)-resident integral membrane protein that controls a nonvesicular mode of ceramide and cholesterol transfer from the ER to the Golgi complex by interacting with ceramide transfer protein and oxysterol-binding protein (OSBP), respectively. We report that VAP and its interacting proteins are required for the processing and secretion of pancreatic adenocarcinoma up-regulated factor, whose transport from the trans-Golgi network (TGN) to the cell surface is mediated by transport carriers called “carriers of the trans-Golgi network to the cell surface” (CARTS). In VAP-depleted cells, diacylglycerol level at the TGN was decreased and CARTS formation was impaired. We found that VAP forms a complex with not only OSBP but also Sac1 phosphoinositide phosphatase at specialized ER subdomains that are closely apposed to the trans-Golgi/TGN, most likely reflecting membrane contact sites. Immobilization of ER–Golgi contacts dramatically reduced CARTS production, indicating that association–dissociation dynamics of the two membranes are important. On the basis of these findings, we propose that the ER–Golgi contacts play a pivotal role in lipid metabolism to control the biogenesis of transport carriers from the TGN.