VAMP-associated protein-A regulates partitioning of oxysterol-binding protein-related protein-9 between the endoplasmic reticulum and Golgi apparatus

We recently showed that oxysterol-binding protein (OSBP), one of twelve related PH domain containing proteins with lipid and sterol binding activity, interacts with VAMP-associated protein (VAP)-A on the endoplasmic reticulum (ER). In addition to OSBP, seven OSBP-related proteins (ORPs) bind VAP-A via a conserved E-F/Y-F/Y-DA 'FFAT' motif. We focused on this interaction for ORP9, which is expressed as a full-length (ORP9L) or truncated version missing the PH domain (ORP9S). Mutation analysis showed that the interaction required the ORP9 FFAT motif and the N-terminal conserved domain of VAP. Endogenous ORP9L displayed Golgi localization, which was partially mediated by the PH domain based on limited localization of OPR9-PH-GFP with the Golgi apparatus. When inducibly overexpressed, ORP9S and ORP9L colocalized with VAP-A and caused vacuolation of the ER as well as retention of the ER-Golgi intermediate compartment marker ERGIC-53/p58 in the ER. ORP9L mutated in the VAP-A binding domain (ORP9L-FY-->AA) did not localize to the ER but appeared with giantin and Sec31 on large vesicular structures, suggesting the presence of a hybrid Golgi-COPII compartment. Normal Golgi localization was also observed for ORP9L-FY-->AA. Results show that VAP binding and PH domains target ORP9 to the ER and a Golgi-COPII compartment, respectively, and that ORP9L overexpression in these compartments severely perturbed their organization.     

Targeting of OSBP-related protein 3 (ORP3) to endoplasmic reticulum and plasma membrane is controlled by multiple determinants

The intracellular targeting determinants of oxysterol binding protein (OSBP)-related protein 3 (ORP3) were studied using a series of truncated and point mutated constructs. The pleckstrin homology (PH) domain of ORP3 binds the phosphoinositide-3-kinase (PI3K) products, PI(3,4)P2 and PI(3,4,5)P3. A functional PH domain and flanking sequences are crucial for the plasma membrane (PM) targeting of ORP3. The endoplasmic reticulum (ER) targeting of ORP3 is regulated the by a FFAT motif (EFFDAxE), which mediates interaction with VAMP-associated protein (VAP)-A. The targeting function of the FFAT motif dominates over that of the PH domain. In addition, the exon 10/11 region modulates interaction of ORP3 with the ER and the nuclear membrane. Analysis of a chimeric ORP3:OSBP protein suggests that ligand binding by the C-terminal domain of OSBP induces allosteric changes that activate the N-terminal targeting modules of ORP3. Notably, over-expression of ORP3 together with VAP-A induces stacked ER membrane structures also known as organized smooth ER (OSER). Moreover, lipid starvation promotes formation of dilated peripheral ER (DPER) structures dependent on the ORP3 protein. Based on the present data, we introduce a model for the inter-relationships of the functional domains of ORP3 in the membrane targeting of the protein.     

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.     

Kes1p shares homology with human oxysterol binding protein and participates in a novel regulatory pathway for yeast Golgi-derived transport vesicle biogenesis.

The yeast phosphatidylinositol transfer protein (Sec14p) is required for biogenesis of Golgi-derived transport vesicles and cell viability, and this essential Sec14p requirement is abrogated by inactivation of the CDP-choline pathway for phosphatidylcholine biosynthesis. These findings indicate that Sec14p functions to alleviate a CDP-choline pathway-mediated toxicity to yeast Golgi secretory function. We now report that this toxicity is manifested through the action of yeast Kes1p, a polypeptide that shares homology with the ligand-binding domain of human oxysterol binding protein (OSBP). Identification of Kes1p as a negative effector for Golgi function provides the first direct insight into the biological role of any member of the OSBP family, and describes a novel pathway for the regulation of Golgi-derived transport vesicle biogenesis.     

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.     

Targeting of Golgi-specific pleckstrin homology domains involves both PtdIns 4-kinase-dependent and -independent components

BACKGROUND: Phosphoinositides are required for the recruitment of many proteins to both the plasma membrane and the endosome; however, their role in protein targeting to other organelles is less clear. The pleckstrin homology (PH) domains of oxysterol binding protein (OSBP) and its relatives have been shown to bind to the Golgi apparatus in yeast and mammalian cells. Previous in vitro binding studies identified phosphatidylinositol (PtdIns) (4)P and PtdIns(4,5)P(2) as candidate ligands, but it is not known which is recognized in vivo and whether phosphoinositide specificity can account for Golgi-specific targeting. RESULTS: We have examined the distribution of GFP fusions to the PH domain of OSBP and to related PH domains in yeast strains carrying mutations in individual phosphoinositide kinases. We find that Golgi targeting requires the activity of the PtdIns 4-kinase Pik1p but not phosphorylation of PtdIns at the 3 or 5 positions and that a PH domain specific for PtdIns(4,5)P(2) is targeted exclusively to the plasma membrane. However, a mutant version of the OSBP PH domain that does not bind phosphoinositides in vitro still shows some targeting in vivo. This targeting is independent of Pik1p but dependent on the Golgi GTPase Arf1p. CONCLUSIONS: Phosphorylation of PtdIns at the 4 position but not conversion to PtdIns(4,5)P(2) contributes to recruitment of PH domains to the Golgi apparatus. However, potential phosphoinositide ligands for these PH domains are not restricted to the Golgi, and the OSBP PH domain also recognizes a second determinant that is ARF dependent, indicating that organelle specificity reflects a combinatorial interaction.     

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.     

Novel members of the human oxysterol-binding protein family bind phospholipids and regulate vesicle transport

Oxysterol-binding proteins (OSBPs) are a family of eukaryotic intracellular lipid receptors. Mammalian OSBP1 binds oxygenated derivatives of cholesterol and mediates sterol and phospholipid synthesis through as yet poorly undefined mechanisms. The precise cellular roles for the remaining members of the oxysterol-binding protein family remain to be elucidated. In yeast, a family of OSBPs has been identified based on primary sequence similarity to the ligand binding domain of mammalian OSBP1. Yeast Kes1p, an oxysterol-binding protein family member that consists of only the ligand binding domain, has been demonstrated to regulate the Sec14p pathway for Golgi-derived vesicle transport. Specifically, inactivation of the KES1 gene resulted in the ability of yeast to survive in the absence of Sec14p, a phosphatidylinositol/phosphatidylcholine transfer protein that is normally required for cell viability due to its essential requirement in transporting vesicles from the Golgi. We cloned the two human members of the OSBP family, ORP1 and ORP2, with the highest degree of similarity to yeast Kes1p. We expressed ORP1 and ORP2 in yeast lacking Sec14p and Kes1p function and found that ORP1 complemented Kes1p function with respect to cell growth and Golgi vesicle transport, whereas ORP2 was unable to do so. Phenotypes associated with overexpression of ORP2 in yeast were a dramatic decrease in cell growth and a block in Golgi-derived vesicle transport distinct from that of ORP1. Purification of ORP1 and ORP2 for ligand binding studies demonstrated ORP1 and ORP2 did not bind 25-hydroxycholesterol but instead bound phospholipids with both proteins exhibiting strong binding to phosphatidic acid and weak binding to phosphatidylinositol 3-phosphate. In Chinese hamster ovary cells, ORP1 localized to a cytosolic location, whereas ORP2 was associated with the Golgi apparatus, consistent with our vesicle transport studies that indicated ORP1 and ORP2 function at different steps in the regulation of vesicle transport.     

Efficient trafficking of ceramide from the endoplasmic reticulum to the Golgi apparatus requires a VAMP-associated protein-interacting FFAT motif of CERT

Ceramide is synthesized at the endoplasmic reticulum (ER) and transported to the Golgi apparatus by CERT for its conversion to sphingomyelin in mammalian cells. CERT has a pleck-strin homology (PH) domain for Golgi targeting and a START domain catalyzing the intermembrane transfer of ceramide. The region between the two domains contains a short peptide motif designated FFAT, which is supposed to interact with the ER-resident proteins VAP-A and VAP-B. Both VAPs were actually co-immunoprecipitated with CERT, and the CERT/VAP interaction was abolished by mutations in the FFAT motif. These mutations did not affect the Golgi targeting activity of CERT. Whereas mutations of neither the FFAT motif nor the PH domain inhibited the ceramide transfer activity of CERT in a cell-free system, they impaired the ER-to-Golgi transport of ceramide in intact and in semi-intact cells at near endogenous expression levels. By contrast, when overexpressed, both the FFAT motif and the PH domain mutants of CERT substantially supported the transport of ceramide from the ER to the site where sphingomyelin is produced. These results suggest that the Golgi-targeting PH domain and ER-interacting FFAT motif of CERT spatially restrict the random ceramide transfer activity of the START domain in cells.     

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.     

The OSBP-related proteins: a novel protein family involved in vesicle transport,cellular lipid metabolism,and cell signalling

Proteins/genes showing high sequence homology to the mammalian oxysterol binding protein (OSBP) have been identified in a variety of eukaryotic organisms from yeast to man. The unifying feature of the gene products denoted as OSBP-related proteins (ORPs) is the presence of an OSBP-type ligand binding (LB) domain. The LB domains of OSBP and its closest homologue bind oxysterols, while data on certain other family members suggest interaction with phospholipids. Many ORPs also have a pleckstrin homology (PH) domain in the amino-terminal region. The PH domains of the family members studied in detail are known to interact with membrane phosphoinositides and play an important role in the intracellular targeting of the proteins. It is plausible that the ORPs constitute a regulatory apparatus that senses the status of specific lipid ligands in membranes, using the PH and/or LB domains, and mediates information to yet poorly known downstream machineries. Functional studies carried out on the ORP proteins in different organisms indicate roles of the gene family in diverse cellular processes including control of lipid metabolism, regulation of vesicle transport, and cell signalling events.     

The OSBP-related proteins: a novel protein family involved in vesicle transport,cellular lipid metabolism,and cell signalling

Proteins/genes showing high sequence homology to the mammalian oxysterol binding protein (OSBP) have been identified in a variety of eukaryotic organisms from yeast to man. The unifying feature of the gene products denoted as OSBP-related proteins (ORPs) is the presence of an OSBP-type ligand binding (LB) domain. The LB domains of OSBP and its closest homologue bind oxysterols, while data on certain other family members suggest interaction with phospholipids. Many ORPs also have a pleckstrin homology (PH) domain in the amino-terminal region. The PH domains of the family members studied in detail are known to interact with membrane phosphoinositides and play an important role in the intracellular targeting of the proteins. It is plausible that the ORPs constitute a regulatory apparatus that senses the status of specific lipid ligands in membranes, using the PH and/or LB domains, and mediates information to yet poorly known downstream machineries. Functional studies carried out on the ORP proteins in different organisms indicate roles of the gene family in diverse cellular processes including control of lipid metabolism, regulation of vesicle transport, and cell signalling events.     

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.     

Human VAP-B is involved in hepatitis C virus replication through interaction with NS5A and NS5B

The hepatitis C virus (HCV) nonstructural protein (NS) 5A is a phosphoprotein that associates with various cellular proteins and participates in the replication of the HCV genome. Human vesicle-associated membrane protein-associated protein (VAP) subtype A (VAP-A) is known to be a host factor essential for HCV replication by binding to both NS5A and NS5B. To obtain more information on the NS5A protein in HCV replication, we screened human brain and liver libraries by a yeast two-hybrid system using NS5A as bait and identified VAP-B as an NS5A-binding protein. Immunoprecipitation and mutation analyses revealed that VAP-B binds to both NS5A and NS5B in mammalian cells and forms homo- and heterodimers with VAP-A. VAP-A interacts with VAP-B through the transmembrane domain. NS5A interacts with the coiled-coil domain of VAP-B via 70 residues in the N-terminal and 341 to 344 amino acids in the C-terminal polyproline cluster region. NS5A was colocalized with VAP-B in the endoplasmic reticulum and Golgi apparatus. The specific antibody to VAP-B suppressed HCV RNA replication in a cell-free assay. Overexpression of VAP-B, but not of a mutant lacking its transmembrane domain, enhanced the expression of NS5A and NS5B and the replication of HCV RNA in Huh-7 cells harboring a subgenomic replicon. In the HCV replicon cells, the knockdown of endogenous VAP-B by small interfering RNA decreased expression of NS5B, but not of NS5A. These results suggest that VAP-B, in addition to VAP-A, plays an important role in the replication of the HCV genome.     

OSBP- and FAN-mediated sterol requirement for spermatogenesis in Drosophila

Members of the oxysterol binding protein (OSBP) family are involved in diverse biological processes, including non-vesicular sterol transport and vesicle trafficking. The mechanisms by which OSBPs integrate functionally with developmental and physiological processes remain elusive. Here, we report the in vivo analysis of OSBP function in the model organism Drosophila. Osbp mutants are male-sterile and exhibit defects in individualization, the process by which each spermatid is packaged into its own membrane. Overexpression of OSBP leads to post-eclosion behaviour defects that can be suppressed by co-expression of endoplasmic reticulum-specific VAP family proteins. Most notably, FAN, a testis-specific VAP protein, acts together with OSBP genetically and physically to regulate the individualization process. OSBP-positive and sterol-enriched speckles are found at the leading edge of the individualization complex in wild type but not in Osbp or fan mutants, suggesting that sterol trafficking might play key roles during the membrane-remodelling phase of individualization. In addition, Osbp mutants that are fed additional sterols partially recover fertility, implying that male sterility is attributable to sterol shortage. Thus, we have identified an OSBP- and FAN-mediated sterol requirement in Drosophila spermatogenesis.     

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.     

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.     

Requirement for Golgi-localized PI(4)P in fusion of COPII vesicles with Golgi compartments

The role of specific membrane lipids in transport between endoplasmic reticulum (ER) and Golgi compartments is poorly understood. Using cell-free assays that measure stages in ER-to-Golgi transport, we screened a variety of enzyme inhibitors, lipid-modifying enzymes, and lipid ligands to investigate requirements in yeast. The pleckstrin homology (PH) domain of human Fapp1, which binds phosphatidylinositol-4-phosphate (PI(4)P) specifically, was a strong and specific inhibitor of anterograde transport. Analysis of wild type and mutant PH domain proteins in addition to recombinant versions of the Sac1p phosphoinositide-phosphatase indicated that PI(4)P was required on Golgi membranes for fusion with coat protein complex II (COPII) vesicles. PI(4)P inhibition did not prevent vesicle tethering but significantly reduced formation of soluble n-ethylmaleimide sensitive factor adaptor protein receptor (SNARE) complexes between vesicle and Golgi SNARE proteins. Moreover, semi-intact cell membranes containing elevated levels of the ER-Golgi SNARE proteins and Sly1p were less sensitive to PI(4)P inhibitors. Finally, in vivo analyses of a pik1 mutant strain showed that inhibition of PI(4)P synthesis blocked anterograde transport from the ER to early Golgi compartments. Together, the data presented here indicate that PI(4)P is required for the SNARE-dependent fusion stage of COPII vesicles with the Golgi complex.     

Oxysterol‐binding protein recruitment and activity at the endoplasmic reticulum‐Golgi interface are independent of Sac1

Oxysterol‐binding protein (OSBP) localizes to endoplasmic reticulum (ER)‐Golgi contact sites where it transports cholesterol and phosphatidylinositol 4‐phosphate (PI‐4P), and activates lipid transport and biosynthetic activities. The PI‐4P phosphatase Sac1 cycles between the ER and Golgi apparatus where it potentially regulates OSBP activity. Here we examined whether the ER‐Golgi distribution of endogenous or ectopically expressed Sac1 influences OSBP activity. OSBP and Sac1 co‐localized at apparent ER‐Golgi contact sites in response to 25‐hydroxycholesterol (25OH), cholesterol depletion and p38 MAPK inhibitors. A Sac1 mutant that is unable to exit the ER did not localize with OSBP, suggesting that sterol perturbations cause Sac1 transport to the Golgi apparatus. Ectopic expression of Sac1 in the ER or Golgi apparatus, or Sac1 silencing, did not affect OSBP localization to ER‐Golgi contact sites, OSBP‐dependent activation of sphingomyelin synthesis, or cholesterol esterification in the ER. p38 MAPK inhibition and retention of Sac1 in the Golgi apparatus also caused OSBP phosphorylation and OSBP‐dependent activation of sphingomyelin synthesis at ER‐Golgi contacts. These results demonstrate that Sac1 expression in either the ER or Golgi apparatus has a minimal impact on the PI‐4P that regulates OSBP activity or recruitment to contact sites.