scholarly journals Oxysterol-binding Protein and Vesicle-associated Membrane Protein–associated Protein Are Required for Sterol-dependent Activation of the Ceramide Transport Protein

2006 ◽  
Vol 17 (6) ◽  
pp. 2604-2616 ◽  
Author(s):  
Ryan J. Perry ◽  
Neale D. Ridgway
Keyword(s):  
Golgi Apparatus ◽  
Membrane Protein ◽  
Binding Protein ◽  
Chinese Hamster ◽  
Transport Protein ◽  
Cholesterol Homeostasis ◽  
Membrane Microdomains ◽  
Cholesterol Depletion ◽  
Oxysterol Binding Protein ◽  
293 Cells

Sphingomyelin (SM) and cholesterol are coregulated metabolically and associate physically in membrane microdomains involved in cargo sorting and signaling. One mechanism for regulation of this metabolic interface involves oxysterol binding protein (OSBP) via high-affinity binding to oxysterol regulators of cholesterol homeostasis and activation of SM synthesis at the Golgi apparatus. Here, we show that OSBP regulation of SM synthesis involves the endoplasmic reticulum (ER)-to-Golgi ceramide transport protein (CERT). RNA interference (RNAi) experiments in Chinese hamster ovary (CHO)-K1 cells revealed that OSBP and vesicle-associated membrane protein–associated protein (VAP) were required for stimulation of CERT-dependent ceramide transport and SM synthesis by 25-hydroxycholesterol and cholesterol depletion in response to cyclodextrin. Additional RNAi experiments in human embryonic kidney 293 cells supported OSBP involvement in oxysterol-activated SM synthesis and also revealed a role for OSBP in basal SM synthesis. Activation of ER-to-Golgi ceramide transport in CHO-K1 cells required interaction of OSBP with the ER and Golgi apparatus, OSBP-dependent Golgi translocation of CERT, and enhanced CERT–VAP interaction. Regulation of CERT by OSBP, sterols, and VAP reveals a novel mechanism for integrating sterol regulatory signals with ceramide transport and SM synthesis in the Golgi apparatus.

scholarly journals Cholesterol regulates oxysterol binding protein (OSBP) phosphorylation and Golgi localization in Chinese hamster ovary cells: correlation with stimulation of sphingomyelin synthesis by 25-hydroxycholesterol

1998 ◽  
Vol 336 (1) ◽  
pp. 247-256 ◽  
Author(s):  
Margo K. STOREY ◽  
David M. BYERS ◽  
Harold W. COOK ◽  
Neale D. RIDGWAY
Keyword(s):  
Golgi Apparatus ◽  
Chinese Hamster Ovary ◽  
Binding Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Density Lipoprotein ◽  
Cholesterol Content ◽  
Cholesterol Depletion ◽  
Experimental Conditions ◽  
Oxysterol Binding Protein

Sphingomyelin (SM) and cholesterol content is positively correlated in cellular membranes, and in several pathological and experimental conditions there is evidence for coregulation. The potential role of oxysterols and oxysterol binding protein (OSBP) in mediating the coregulation of cholesterol and SM was examined using Chinese hamster ovary (CHO) and cholesterol auxotrophic, sterol regulatory defective (SRD) 6 cells. SRD 6 cells grown in the presence or absence of cholesterol for 24 h displayed a 30–50% reduction in SM synthesis compared with control CHO 7 cells. SM synthesis in CHO 7 and cholesterol-supplemented SRD 6 cells was stimulated 2-fold by 25-hydroxycholesterol, but cholesterol-starved SRD 6 cells were unresponsive. Basal and 25-hydroxycholesterol-stimulated SM synthesis was also inhibited in lovastatin-treated wild-type CHO-K1 cells. Lack of 25-hydroxycholesterol activation of SM synthesis in cholesterol-starved SRD 6 and lovastatin-treated CHO-K1 cells was correlated with dephosphorylation of OSBP. In SRD 6 cells, this was evident after 12 h of cholesterol depletion, it occurred equally at all phosphorylation sites and was exacerbated by 25-hydroxycholesterol. Unlike CHO 7 cells, where OSBP was observed in small vesicles and the cytoplasm, OSBP in cholesterol-starved SRD 6 cells was constitutively localized in the Golgi apparatus. Supplementation with non-lipoprotein cholesterol promoted redistribution to vesicles and the cytoplasm. Similarly, OSBP in CHO-K1 cells grown in delipidated serum was predominantly in the Golgi apparatus. Low-density lipoprotein (LDL) supplementation of CHO-K1 cells caused the redistribution of OSBP to the cytoplasm and small vesicles, and this effect was blocked by pharmacological agents {3-β-[2-(diethylamino)ethoxy]androst-5-en-17-one and progesterone}, which inhibited LDL cholesterol efflux from lysosomes. The results showed that localization of OSBP between the Golgi apparatus and a cytoplasmic/vesicular compartment was responsive to changes in cholesterol content and trafficking. In cholesterol depleted SRD 6 cells, this was accompanied by dephosphorylation of OSBP and attenuation of 25-hydroxycholesterol activation of SM synthesis.

scholarly journals Oxysterol Binding Protein-dependent Activation of Sphingomyelin Synthesis in the Golgi Apparatus Requires Phosphatidylinositol 4-Kinase IIα

2010 ◽  
Vol 21 (23) ◽  
pp. 4141-4150 ◽  
Author(s):  
Sangeeta Banerji ◽  
Mike Ngo ◽  
Ciaran F. Lane ◽  
Carolyn-Ann Robinson ◽  
Shane Minogue ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Binding Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cholesterol Content ◽  
Pleckstrin Homology Domain ◽  
Cell Fractionation ◽  
Oxysterol Binding Protein ◽  
Transfer Activity ◽  
Phosphatidylinositol 4

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.

scholarly journals Regulation of Oxysterol-binding Protein Golgi Localization through Protein Kinase D–mediated Phosphorylation

2010 ◽  
Vol 21 (13) ◽  
pp. 2327-2337 ◽  
Author(s):  
Sokha Nhek ◽  
Mike Ngo ◽  
Xuemei Yang ◽  
Michelle M. Ng ◽  
Seth J. Field ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Binding Protein ◽  
Critical Role ◽  
Protein Kinase D ◽  
Cholesterol Depletion ◽  
Oxysterol Binding Protein ◽  
Golgi Localization ◽  
Golgi Network

Protein kinase D (PKD) plays a critical role at the trans-Golgi network by regulating the fission of transport carriers destined for the plasma membrane. Two known Golgi-localized PKD substrates, PI4-kinase IIIβ and the ceramide transfer protein CERT, mediate PKD signaling to influence vesicle trafficking to the plasma membrane and sphingomyelin synthesis, respectively. PKD is recruited and activated at the Golgi through interaction with diacylglycerol, a pool of which is generated as a by-product of sphingomyelin synthesis from ceramide. Here we identify a novel substrate of PKD at the Golgi, the oxysterol-binding protein OSBP. Using a substrate-directed phospho-specific antibody that recognizes the optimal PKD consensus motif, we show that PKD phosphorylates OSBP at Ser240 in vitro and in cells. We further show that OSBP phosphorylation occurs at the Golgi. Phosphorylation of OSBP by PKD does not modulate dimerization, sterol binding, or affinity for PI(4)P. Instead, phosphorylation attenuates OSBP Golgi localization in response to 25-hydroxycholesterol and cholesterol depletion, impairs CERT Golgi localization, and promotes Golgi fragmentation.

scholarly journals Role of Oxysterol Binding Protein in Hepatitis C Virus infection

2009 ◽  
Vol 83 (18) ◽  
pp. 9237-9246 ◽  
Author(s):  
Yutaka Amako ◽  
Ali Sarkeshik ◽  
Hak Hotta ◽  
John Yates ◽  
Aleem Siddiqui
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Golgi Apparatus ◽  
Binding Protein ◽  
Hcv Rna ◽  
Ph Domain ◽  
Particle Release ◽  
Oxysterol Binding Protein ◽  
Rnp Complex

ABSTRACT Hepatitis C virus (HCV) RNA genome replicates within the ribonucleoprotein (RNP) complex in the modified membranous structures extended from endoplasmic reticulum. A proteomic analysis of HCV RNP complexes revealed the association of oxysterol binding protein (OSBP) as one of the components of these complexes. OSBP interacted with the N-terminal domain I of the HCV NS5A protein and colocalized to the Golgi compartment with NS5A. An OSBP-specific short hairpin RNA that partially downregulated OSBP expression resulted in a decrease of the HCV particle release in culture supernatant with little effect on viral RNA replication. The pleckstrin homology (PH) domain located in the N-terminal region of OSBP targeted this protein to the Golgi apparatus. OSBP deletion mutation in the PH (ΔPH) domain failed to localize to the Golgi apparatus and inhibited the HCV particle release. These studies suggest a possible functional role of OSBP in the HCV maturation process.

scholarly journals Translocation of oxysterol binding protein to Golgi apparatus triggered by ligand binding.

1992 ◽  
Vol 116 (2) ◽  
pp. 307-319 ◽  
Author(s):  
N D Ridgway ◽  
P A Dawson ◽  
Y K Ho ◽  
M S Brown ◽  
J L Goldstein
Keyword(s):  
Golgi Apparatus ◽  
Leucine Zipper ◽  
Binding Protein ◽  
Brefeldin A ◽  
Sterol Metabolism ◽  
Animal Cells ◽  
Transfected Cells ◽  
Oxysterol Binding Protein ◽  
Golgi Membranes ◽  
Lentil Lectin

A cDNA encoding a cytoplasmic oxysterol binding protein was expressed at high levels by transfection in animal cells. This protein binds oxysterols such as 25-hydroxycholesterol that regulate sterol metabolism by transcriptional and posttranscriptional effects. In the transfected cells, some of the oxysterol binding protein (OSBP) was distributed diffusely in the cytoplasm, and some was bound to small vesicles near the nucleus, as revealed by indirect immunofluorescence. Upon addition of 25-hydroxycholesterol, most of the OSBP became concentrated in large perinuclear structures that stained with lentil lectin, a protein that stains the Golgi apparatus. The structures that contained OSBP were disrupted by brefeldin A, confirming their identification as Golgi. A mutant OSBP lacking the COOH-terminal oxysterol binding domain localized to the Golgi spontaneously, suggesting that this domain normally occludes the domain that binds to the Golgi and that sterols relieve this occlusion. The previously noted potential leucine zipper sequence in OSBP was not required for Golgi localization, nor was it essential for homodimer formation. We conclude that OSBP is triggered to bind extrinsically to Golgi membranes when it binds oxysterols and speculate that this translocation may play a role in the transport, metabolism, or regulatory actions of oxysterols.

scholarly journals Altered regulation of cholesterol and cholesteryl ester synthesis in Chinese-hamster ovary cells overexpressing the oxysterol-binding protein is dependent on the pleckstrin homology domain

1997 ◽  
Vol 326 (1) ◽  
pp. 205-213 ◽  
Author(s):  
Thomas A. LAGACE ◽  
David M. BYERS ◽  
Harold W. COOK ◽  
Neale D. RIDGWAY
Keyword(s):  
Golgi Apparatus ◽  
Cholesteryl Ester ◽  
Chinese Hamster Ovary ◽  
Potential Role ◽  
Chinese Hamster ◽  
Cholesterol Esterification ◽  
Ester Synthesis ◽  
Ph Domain ◽  
Wild Type ◽  
Oxysterol Binding Protein

Oxysterol-binding protein (OSBP) is a high-affinity receptor for a variety of oxysterols, such as 25-hydroxycholesterol, that down-regulate cholesterol synthesis and stimulate cholesterol esterification. To examine a potential role for OSBP in regulating cholesterol metabolism, we stably overexpressed this protein in Chinese-hamster ovary (CHO)-K1 cells. Compared with mock-transfected controls, several cell lines overexpressing wild-type OSBP (CHO-OSBP) displayed a 50% decrease in cholesteryl ester synthesis when cultured in medium with delipidated serum, 25-hydroxycholesterol or low-density lipoprotein (LDL) . CHO-OSBP cells showed a 40–60% decrease in acyl-CoA:cholesterol acyltransferase activity and mRNA, a 50% elevation in mRNA for three sterol-regulated genes [LDL receptor, 3-hydroxy-3-methylgluraryl (HMG)-CoA reductase and HMG-CoA synthase], and an 80% increase in [14C]acetate incorporation into cholesterol. CHO-K1 cells overexpressing two OSBP mutants with a complete or N-terminal deletion of the pleckstrin homology (PH) domain had cholesterol esterification and synthesis rates that were similar to those shown by mock-transfected controls. Unlike wild-type OSBP, both PH domain mutants displayed diffuse cytoplasmic immunofluorescence staining and did not translocate to the Golgi apparatus in the presence of 25-hydroxycholesterol. CHO-K1 cells overexpressing OSBP have pronounced alterations in cholesterol esterification and synthesis, indicating a potential role for this receptor in cholesterol homoeostasis. The phenotype observed in cells overexpressing OSBP is dependent on the PH domain, which appears to be necessary for ligand-dependent localization of OSBP to the Golgi apparatus.

scholarly journals Oxysterol-binding protein (OSBP) is required for the perinuclear localization of intra-Golgi v-SNAREs

2013 ◽  
Vol 24 (22) ◽  
pp. 3534-3544 ◽  
Author(s):  
Taki Nishimura ◽  
Yasunori Uchida ◽  
Rieko Yachi ◽  
Tetyana Kudlyk ◽  
Vladimir Lupashin ◽  
...  
Keyword(s):  
Cholesterol Level ◽  
Binding Protein ◽  
Cholesterol Depletion ◽  
Oxysterol Binding Protein ◽  
Golgi Localization ◽  
Protein Receptors ◽  
Intracellular Organelles ◽  
Endoplasmic Reticulum Targeting ◽  
Related Proteins ◽  
Interfering Rna

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) have been implicated in the distribution of sterols among intracellular organelles. OSBP regulates the Golgi cholesterol level, but how it relates to Golgi function is elusive. Here we report that OSBP is essential for the localization of intra-Golgi soluble vesicle N-ethylmaleimide-sensitive fusion attachment protein receptors (v-SNAREs). Depletion of OSBP by small interfering RNA causes mislocalization of intra-Golgi v-SNAREs GS28 and GS15 throughout the cytoplasm without affecting the perinuclear localization of Golgi target-SNARE syntaxin5 and reduces the abundance of a Golgi enzyme, mannosidase II (Man II). GS28 mislocalization and Man II reduction are also induced by cellular cholesterol depletion. Three domains of OSBP—an endoplasmic reticulum–targeting domain, a Golgi-targeting domain, and a sterol-binding domain—are all required for Golgi localization of GS28. Finally, GS28 mislocalization and Man II reduction in OSBP-depleted cells are largely restored by depletion of ArfGAP1, a regulator of the budding of coat protein complex (COP)-I vesicles. From these results, we postulate that Golgi cholesterol level, which is controlled by OSBP, is essential for Golgi localization of intra-Golgi v-SNAREs by ensuring proper COP-I vesicle transport.

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