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.
Banerji
Sangeeta
S
Departments of Pediatrics and Biochemistry and Molecular Biology, Atlantic Research Centre, Dalhousie University, Halifax, NS, Canada.
Ngo
Mike
M
Lane
Ciaran F
CF
Robinson
Carolyn-Ann
CA
Minogue
Shane
S
Ridgway
Neale D
ND
D000818:Animals; D016466:CHO Cells; D002458:Cell Fractionation; D006224:Cricetinae; D003412:Cricetulus / Q000378:metabolism; D020868:Gene Silencing; D006056:Golgi Apparatus / Q000378:metabolism*; D006888:Hydroxycholesterols / Q000494:pharmacology*; D007425:Intracellular Membranes / Q000378:metabolism; D008856:Microscopy, Fluorescence; D015778:Minor Histocompatibility Antigens; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D034741:RNA, Small Interfering; D011987:Receptors, Steroid / Q000378:metabolism*; D013109:Sphingomyelins / Q000096:biosynthesis*; D021601:trans-Golgi Network / Q000737:chemistry; D000098825:Oxysterol Binding Proteins
D006888:Hydroxycholesterols; D015778:Minor Histocompatibility Antigens; D034741:RNA, Small Interfering; D011987:Receptors, Steroid; D013109:Sphingomyelins; D000098825:Oxysterol Binding Proteins; C007997:25-hydroxycholesterol; D017853:Phosphotransferases (Alcohol Group Acceptor); C402182:phosphatidylinositol phosphate 4-kinase