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 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 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 Multisite phosphorylation of oxysterol-binding protein regulates sterol binding and activation of sphingomyelin synthesis

2012 ◽  
Vol 23 (18) ◽  
pp. 3624-3635 ◽  
Author(s):  
Asako Goto ◽  
Xinwei Liu ◽  
Carolyn-Ann Robinson ◽  
Neale D. Ridgway
Keyword(s):  
Binding Capacity ◽  
Binding Protein ◽  
Protein A ◽  
Phosphorylation Site ◽  
Oxysterol Binding Protein ◽  
Transfer Activity ◽  
Individual Site ◽  
Direct Binding ◽  
Phosphatidylinositol 4

The endoplasmic reticulum (ER)-Golgi sterol transfer activity of oxysterol-binding protein (OSBP) regulates sphingomyelin (SM) synthesis, as well as post-Golgi cholesterol efflux pathways. The phosphorylation and ER-Golgi localization of OSBP are correlated, suggesting this modification regulates the directionality and/or specificity of transfer activity. In this paper, we report that phosphorylation on two serine-rich motifs, S381-S391 (site 1) and S192, S195, S200 (site 2), specifically controls OSBP activity at the ER. A phosphomimetic of the SM/cholesterol-sensitive phosphorylation site 1 (OSBP-S5E) had increased in vitro cholesterol and 25-hydroxycholesterol–binding capacity, and cholesterol extraction from liposomes, but reduced transfer activity. Phosphatidylinositol 4-phosphate (PI(4)P) and cholesterol competed for a common binding site on OSBP; however, direct binding of PI(4)P was not affected by site 1 phosphorylation. Individual site 1 and site 2 phosphomutants supported oxysterol activation of SM synthesis in OSBP-deficient CHO cells. However, a double site1/2 mutant (OSBP-S381A/S3D) was deficient in this activity and was constitutively colocalized with vesicle-associated membrane protein–associated protein A (VAP-A) in a collapsed ER network. This study identifies phosphorylation regulation of sterol and VAP-A binding by OSBP in the ER, and PI(4)P as an alternate ligand that could be exchanged for sterol in the Golgi apparatus.

scholarly journals The oxysterol binding protein Kes1p regulates Golgi apparatus phosphatidylinositol-4-phosphate function

2007 ◽  
Vol 104 (39) ◽  
pp. 15352-15357 ◽  
Author(s):  
G. D. Fairn ◽  
A. J. Curwin ◽  
C. J. Stefan ◽  
C. R. McMaster
Keyword(s):  
Golgi Apparatus ◽  
Binding Protein ◽  
Oxysterol Binding Protein ◽  
Phosphatidylinositol 4

scholarly journals Un marché d’échange de lipides

2020 ◽  
Vol 36 (2) ◽  
pp. 130-136
Author(s):  
Joëlle Bigay ◽  
Bruno Mesmin ◽  
Bruno Antonny
Keyword(s):  
Binding Protein ◽  
Oxysterol Binding Protein ◽  
Phosphatidylinositol 4

Le cholestérol est synthétisé dans le réticulum endoplasmique (RE) puis transporté vers les compartiments cellulaires dont la fonction en nécessite un taux élevé. Nous décrivons ici le mécanisme de transport du cholestérol du RE vers le réseau trans golgien (TGN) par la protéine OSBP (oxysterol binding protein). Celle-ci présente deux activités complémentaires : elle arrime les deux compartiments, RE et TGN, en formant un site de contact où les deux membranes sont à une vingtaine de nanomètres de distance ; puis elle échange le cholestérol du RE avec un lipide présent dans le TGN, le phosphatidylinositol 4-phosphate (PI4P). Dans le RE, le PI4P est hydrolysé, rendant le cycle d’échange irréversible. OSBP est donc au cœur d’un marché d’échange de lipides dans lequel un cholestérol transporté « coûte » un PI4P. Des molécules à activités antivirales ou anticancéreuses ont pour cible OSBP, suggérant une importance dans différents contextes physiopathologiques du cycle d’OSBP, dont les bases générales sont partagées par d’autres protéines transporteurs de lipides.

scholarly journals Chinese hamster ovary cell lysosomes retain pinocytized horseradish peroxidase and in situ-radioiodinated proteins.

1984 ◽  
Vol 4 (2) ◽  
pp. 296-301 ◽  
Author(s):  
B Storrie ◽  
M Sachdeva ◽  
V S Viers
Keyword(s):  
Horseradish Peroxidase ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Fluid Phase ◽  
Ovary Cell ◽  
Cell Fractionation ◽  
Marker Enzyme ◽  
Ovary Cells

We used Chinese hamster ovary cells, a cell line of fibroblastic origin, to investigate whether lysosomes are an exocytic compartment. To label lysosomal contents, Chinese hamster ovary cells were incubated with the solute marker horseradish peroxidase. After an 18-h uptake period, horseradish peroxidase was found in lysosomes by cell fractionation in Percoll gradients and by electron microscope cytochemistry. Over a 24-h period, lysosomal horseradish peroxidase was quantitatively retained by Chinese hamster ovary cells and inactivated with a t 1/2 of 6 to 8 h. Lysosomes were radioiodinated in situ by soluble lactoperoxidase internalized over an 18-h uptake period. About 70% of the radioiodine incorporation was pelleted at 100,000 X g under conditions in which greater than 80% of the lysosomal marker enzyme beta-hexosaminidase was released into the supernatant. By one-dimensional electrophoresis, about 18 protein species were present in the lysosomal membrane fraction, with radioiodine incorporation being most pronounced into species of 70,000 to 75,000 daltons. After a 30-min or 2-h chase at 37 degrees C, radioiodine that was incorporated into lysosomal membranes and contents was retained in lysosomes. These observations indicate that lysosomes labeled by fluid-phase pinocytosis are a terminal component of endocytic pathways in fibroblasts.

Sign in / Sign up

Export Citation Format

Share Document