title: Oxysterol-binding Protein Activation at Endoplasmic Reticulum-Golgi Contact Sites Reorganizes Phosphatidylinositol 4-Phosphate Pools.
abstract: Oxysterol-binding protein (OSBP) exchanges cholesterol and phosphatidylinositol 4-phosphate (PI-4P) at contact sites between the endoplasmic reticulum (ER) and the trans-Golgi/trans-Golgi network. 25-Hydroxycholesterol (25OH) competitively inhibits this exchange reaction in vitro and causes the constitutive localization of OSBP at the ER/Golgi interface and PI-4P-dependent recruitment of ceramide transfer protein (CERT) for sphingomyelin synthesis. We used PI-4P probes and mass analysis to determine how OSBP controls the availability of PI-4P for this metabolic pathway. Treatment of fibroblasts or Chinese hamster ovary (CHO) cells with 25OH caused a 50-70% reduction in Golgi-associated immunoreactive PI-4P that correlated with Golgi localization of OSBP. In contrast, 25OH caused an OSBP-dependent enrichment in Golgi PI-4P that was detected with a pleckstrin homology domain probe. The cellular mass of phosphatidylinositol monophosphates and Golgi PI-4P measured with an unbiased PI-4P probe (P4M) was unaffected by 25OH and OSBP silencing, indicating that OSBP shifts the distribution of PI-4P upon localization to ER-Golgi contact sites. The PI-4P and sterol binding activities of OSBP were both required for 25OH activation of sphingomyelin synthesis, suggesting that 25OH must be exchanged for PI-4P to be concentrated at contact sites. We propose a model wherein 25OH activation of OSBP promotes the binding and retention of PI-4P at ER-Golgi contact sites. This pool of PI-4P specifically recruits pleckstrin homology domain-containing proteins involved in lipid transfer and metabolism, such as CERT.
authors.affiliation: From the Atlantic Research Centre, Departments of Pediatrics and Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada.
authors.forename: Asako
authors.initials: A
authors.lastname: Goto
authors.affiliation: From the Atlantic Research Centre, Departments of Pediatrics and Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada.
authors.forename: Mark
authors.initials: M
authors.lastname: Charman
authors.affiliation: From the Atlantic Research Centre, Departments of Pediatrics and Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada nridgway@dal.ca.
authors.forename: Neale D
authors.identifier: http://orcid.org/0000-0002-0441-6228
authors.initials: ND
authors.lastname: Ridgway
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D003412:Cricetulus; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D017353:Gene Deletion; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D006888:Hydroxycholesterols / Q000378:metabolism; D008954:Models, Biological; D009154:Mutation; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D021381:Protein Transport; D034622:RNA Interference; D011987:Receptors, Steroid / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012867:Skin / Q000166:cytology; D021601:trans-Golgi Network / Q000378:metabolism*; D000098825:Oxysterol Binding Proteins
chemical_list: D006888:Hydroxycholesterols; D018129:Phosphatidylinositol Phosphates; D011987:Receptors, Steroid; D011994:Recombinant Proteins; D000098825:Oxysterol Binding Proteins; C037178:phosphatidylinositol 4-phosphate; C007997:25-hydroxycholesterol
keywords: 25-hydroxycholesterol; Golgi; cholesterol; endoplasmic reticulum (ER); fluorescence recovery after photobleaching (FRAP); lipid transport; oxysterol binding protein; sphingomyelin