Neale D. Ridgway

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 Δ132–182). 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 °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 °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.

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.

Interactions between metabolism and intracellular distribution of cholesterol and sphingomyelin.

There is ample evidence from experimental models and human metabolic disorders indicating that cholesterol and sphingomyelin (SM) levels are coordinately regulated. Generally it has been observed that altering the cellular content of sphingomyelin or cholesterol results in corresponding changes in mass and/or synthesis of the other lipid. In the case of cholesterol synthesis and trafficking, SM regulates the capacity of membranes to absorb cholesterol and thereby controls sterol flux between the plasma membrane and regulatory pathways in the endoplasmic reticulum. This relationship exemplifies the importance of cholesterol/sphingolipid-rich domains in cholesterol homeostasis, as well as other aspects of cell signaling and transport. Evidence for regulation of sphingomyelin metabolism by cholesterol is less convincing and dependent on the model system under study. Sphingomyelin biosynthetic rates are not dramatically affected by alterations in cholesterol balance suggesting that sphingomyelin or its metabolites serve other indispensable functions in the cell. A notable exception is the robust and specific regulation of both SM and cholesterol synthesis by 25-hydroxycholesterol. This finding is reviewed in the context of the role of oxysterol binding protein and its putative role in cholesterol and SM trafficking between the plasma membrane and Golgi apparatus.

Differential effects of sphingomyelin hydrolysis and cholesterol transport on oxysterol-binding protein phosphorylation and Golgi localization

The deposition of de novo synthesized and lipoprotein-derived cholesterol at the plasma membrane and transport to the endoplasmic reticulum is dependent on sphingomyelin (SM) content. Here we show that hydrolysis of plasma membrane SM in Chinese hamster ovary cells by exogenous bacterial sphingomyelinase resulted in enhanced cholesterol esterification at the endoplasmic reticulum and rapid dephosphorylation of the oxysterol-binding protein (OSBP), a cytosolic/Golgi receptor for oxysterols such as 25-hydroxycholesterol. After sphingomyelinase treatment, restoration of OSBP phosphorylation closely paralleled resynthesis of SM and down-regulation of cholesterol ester synthesis. SM hydrolysis activated an okadaic acid-sensitive phosphatase that was not stimulated in Chinese hamster ovary cells by short chain ceramides. Agents that specifically blocked sphingomyelinase-mediated delivery of cholesterol to acyl-CoA:cholesterol acyltransferase (U18666A) or promoted cholesterol efflux to the medium (cyclodextrin) did not inhibit OSBP dephosphorylation. SM hydrolysis also promoted OSBP translocation from a vesicular compartment to the Golgi apparatus. Cyclodextrin and U18666A also caused OSBP translocation to the Golgi apparatus, suggesting that OSBP movement is coupled to changes in the cholesterol content of the plasma membrane or Golgi apparatus. These results identify OSBP as a potential target of SM turnover and cholesterol mobilization at the plasma membrane and/or Golgi apparatus.

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.

The role of phospholipids in the biological activity and structure of the endoplasmic reticulum.

The endoplasmic reticulum (ER) is an interconnected network of tubular and planar membranes that supports the synthesis and export of proteins, carbohydrates and lipids. Phospholipids, in particular phosphatidylcholine (PC), are synthesized in the ER where they have essential functions including provision of membranes required for protein synthesis and export, cholesterol homeostasis, and triacylglycerol storage and secretion. Coordination of these biological processes is essential, as highlighted by findings that link phospholipid metabolism in the ER with perturbations in lipid storage/secretion and stress responses, ultimately contributing to obesity/diabetes, atherosclerosis and neurological disorders. Phospholipid synthesis is not uniformly distributed in the ER but is localized at membrane interfaces or contact zones with other organelles, and in dynamic, proliferating ER membranes. The topology of phospholipid synthesis is an important consideration when establishing the etiology of diseases that arise from ER dysfunction. This review will highlight our current understanding of the contribution of phospholipid synthesis to proper ER function, and how alterations contribute to aberrant stress responses and disease. This article is part of a Special Issue entitled: Functional and structural diversity of endoplasmic reticulum.

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

Protein kinase D (PKD) is a critical regulator of Golgi structure and function. Biochemical evidence is presented that demonstrates the oxysterol-binding protein OSBP as a novel PKD substrate. Phosphorylation inhibits OSBP Golgi localization, impairs CERT Golgi localization, and promotes Golgi fragmentation.

The role of phosphatidylcholine and choline metabolites to cell proliferation and survival

The reorganization of metabolic pathways in cancer facilitates the flux of carbon and reducing equivalents into anabolic pathways at the expense of oxidative phosphorylation. This provides rapidly dividing cells with the necessary precursors for membrane, protein and nucleic acid synthesis. A fundamental metabolic perturbation in cancer is the enhanced synthesis of fatty acids by channeling glucose and/or glutamine into cytosolic acetyl-CoA and upregulation of key biosynthetic genes. This lipogenic phenotype also extends to the production of complex lipids involved in membrane synthesis and lipid-based signaling. Cancer cells display sensitivity to ablation of fatty acid synthesis possibly as a result of diminished capacity to synthesize complex lipids involved in signaling or growth pathways. Evidence has accrued that phosphatidylcholine, the major phospholipid component of eukaryotic membranes, as well as choline metabolites derived from its synthesis and catabolism, contribute to both proliferative growth and programmed cell death. This review will detail our current understanding of how coordinated changes in substrate availability, gene expression and enzyme activity lead to altered phosphatidylcholine synthesis in cancer, and how these changes contribute directly or indirectly to malignant growth. Conversely, apoptosis targets key steps in phosphatidylcholine synthesis and degradation that are linked to disruption of cell cycle regulation, reinforcing the central role that phosphatidylcholine and its metabolites in determining cell fate.

Oxysterol Binding Protein-dependent Activation of Sphingomyelin Synthesis in the Golgi Apparatus Requires Phosphatidylinositol 4-Kinase IIα

The study identifies a sterol- and oxysterol binding protein (OSBP)-regulated phosphatidylinositol 4-kinase that regulates ceramide transport protein (CERT) activity and sphingomyelin (SM) synthesis. RNA interference silencing experiments identify PI4KIIα; as the mediator of Golgi recruitment of CERT, providing a potential mechanism for coordinating assembly of SM and cholesterol in the Golgi or more distal compartments.

Functional implications of sterol transport by the oxysterol-binding protein gene family.

Cholesterol and its numerous oxygenated derivatives (oxysterols) profoundly affect the biophysical properties of membranes, and positively and negatively regulate sterol homoeostasis through interaction with effector proteins. As the bulk of cellular sterols are segregated from the sensory machinery that controls homoeostatic responses, an important regulatory step involves sterol transport or signalling between membrane compartments. Evidence for rapid, energy-independent transport between organelles has implicated transport proteins, such as the eukaryotic family of OSBP (oxysterol-binding protein)/ORPs (OSBP-related proteins). Since the founding member of this family was identified more than 25 years ago, accumulated evidence has implicated OSBP/ORPs in sterol signalling and/or sterol transport functions. However, recent evidence of sterol transfer activity by OSBP/ORPs suggests that other seemingly disparate functions could be the result of alterations in membrane sterol distribution or ancillary to this primary activity.