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.
Highlights
0.2% of mammalian genomes encode proteins that are implicated in lipid transfer
Mammalian oxysterol-binding protein (OSBP) and yeast homologues are proposed to use existing phosphatidylinositol 4-phosphate (PI-4P) gradients for the countercurrent transfer of other lipids and sterols between membranes [9, 11, 35]. 25OH inhibits the OSBP-mediated exchange of cholesterol and PI-4P between liposomes in vitro, but it activates an alternative pathway for the transport and synthesis of sphingolipids and phosphoinositides in the Golgi apparatus [12, 21,22,23]
PI-4P detection in the Golgi apparatus with an antibody was inhibited by OSBP expression and PI-4P binding activity and was evident when endogenous OSBP was activated at the endoplasmic reticulum (ER)-Golgi interface by 25OH
Summary
0.2% of mammalian genomes encode proteins that are implicated in lipid transfer. B, fluorescence intensity of PI-4P immunostaining in the cell (total), cytoplasm, and Golgi apparatus was quantified and expressed relative to the no addition controls. Quantification of fluorescence intensity indicated that Golgi-associated mCherry-P4M was not significantly affected in CHO-shNT or CHO-shOSBP cells after 25OH treatment for 4 h (Fig. 4B).
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