Golgi localization and phosphorylation of oxysterol binding protein in Niemann-Pick C and U18666A-treated cells.
Oxysterol binding protein (OSBP) translocation between Golgi and vesicular/cytoplasmic compartments is affected by conditions that alter cholesterol and sphingomyelin homeostasis, indicating a role in lipid and sterol regulation in this organelle. In this study, we show that OSBP dissociation from the Golgi apparatus was inhibited when LDL cholesterol efflux from lysosomes was blocked in Niemann-Pick C (NPC) or U18666A [3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one]-treated fibroblasts. Dissociation of OSBP from the Golgi apparatus in response to LDL was independent of de novo cholesterol biosynthesis. OSBP did not localize with filipin-stained lysosomal cholesterol, and the NPC defect did not alter OSBP expression or phosphorylation. However, OSBP in the Golgi apparatus was progressively dephosphorylated (as assessed by a molecular mass shift on SDS-PAGE) in U18666A-treated fibroblasts or Chinese hamster ovary cells as a result of combined inhibition of LDL cholesterol transport and de novo cholesterol synthesis. In vivo phosphopeptide mapping and mutagenesis of OSBP was used to identify the cholesterol-sensitive phosphorylation sites at serines 381, 384, and 387 that were responsible for the altered mobility on SDS-PAGE. NPC-1 protein-mediated release of LDL-derived cholesterol and de novo biosynthesis regulates OSBP localization and phosphorylation. This indicates that OSBP responds to or senses altered cellular sterol content and transport.
Mohammadi
A
A
Department of Pediatrics and Department of Biochemistry and Molecular Biology, Atlantic Research Centre, Rm 306, CRC Bldg., Dalhousie University, 5849 University Ave., Halifax, Nova Scotia, Canada B3H 4H7.
Perry
R J
RJ
Storey
M K
MK
Cook
H W
HW
Byers
D M
DM
Ridgway
N D
ND
D000736:Androstenes / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D002784:Cholesterol / Q000096:biosynthesis*; D008078:Cholesterol, LDL / Q000378:metabolism*; D018680:Cholinergic Antagonists / Q000494:pharmacology; D006224:Cricetinae; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D016297:Mutagenesis, Site-Directed / Q000502:physiology; D009542:Niemann-Pick Diseases / Q000378:metabolism*; D010766:Phosphorylation; D021381:Protein Transport / Q000502:physiology; D011987:Receptors, Steroid / Q000378:metabolism*; D000098825:Oxysterol Binding Proteins
D000736:Androstenes; D008078:Cholesterol, LDL; D018680:Cholinergic Antagonists; D011987:Receptors, Steroid; D000098825:Oxysterol Binding Proteins; C006261:3-beta-(2-(diethylamino)ethoxy)androst-5-en-17-one; D002784:Cholesterol