Oxysterol binding protein activation at endoplasmic reticulum-golgi contact sites reorganizes phosphatidylinositol 4-phosphate pools [Cell Biology]

November 23rd, 2015 by Goto, A., Charman, M., Ridgway, N. D.

Oxysterol binding protein (OSBP) exchanges cholesterol and phosphatidylinositol 4-phosphate (PI-4P) at contact sites between the endoplasmic reticulum (ER) and 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 (PH) domain probe. The cellular mass of phosphatidylinositol mono-phosphates and Golgi PI-4P measured with an unbiased PI-4P probe (P4M) were 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 SM synthesis, suggesting that 25OH must be exchanged for PI-4P in order 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 pool specifically recruits PH domain containing proteins involved in lipid transfer and metabolism, such as CERT.
  • Posted in Journal of Biological Chemistry, Publications
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