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

Abstract

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.

FIGURE 1:

OSBP depletion mislocalizes intra-Golgi v-SNAREs (GS28 and GS15) throughout the cytoplasm and reduces the level of Golgi enzyme Man II. HeLa cells were transfected with either control or OSBP siRNA (OSBP siRNA#1). All experiments were performed 72 h after siRNA transfections. (A) Cell lysates were subjected to SDS–PAGE and immunoblotted with the indicated antibodies. (B) Cells were fixed and stained with the indicated antibodies. Scale bar, 10 μm. (C) GS28 localization was examined by immunofluorescence and classified into three categories: 1) perinuclear Golgi localization, 2) Golgi plus cytoplasmic localization, and 3) cytoplasmic localization. More than 40 cells were examined per sample. The data (mean ± SEM) are calculated based on three independent experiments. (D) Coimmunoprecipitation between GS28 and Syn5. Cell lysates were immunoprecipitated with anti-GS28 antibody, and the precipitates were immunoblotted with the indicated antibodies.

FIGURE 2:

CERT depletion induces Golgi fragmentation. HeLa cells were transfected with the indicated siRNAs for 72 h. (A) Cell lysates were subjected to SDS–PAGE and immunoblotted with the indicated antibodies. (B) Cells were fixed and stained with anti-GM130 (green) and anti-TGN46 (red) antibodies, or anti-GS28 (green) and anti-Syn5 (red) antibodies. Scale bar, 10 μm. (C) GM130 localization was examined by immunofluorescence, and percentage of cells with partially or totally fragmented GM130 staining was determined. More than 40 cells were examined per sample. The data (mean ± SEM) are calculated based on three independent experiments. (D) HeLa cells were transfected with OSBP siRNA#1 for 48 h, followed by transfection with the FLAG-tagged CERT plasmid. Seventy-two hours after siRNA transfection, cells were fixed and stained with anti-GS28 (green) and anti-FLAG (red) antibodies. DNA was stained with DAPI (blue). Scale bar, 10 μm.

FIGURE 3:

PH domain, FFAT motif, and sterol-binding domain of OSBP are necessary for perinuclear localization of GS28. (A) Schematic representations of the wild-type (WT) OSBP and OSBP mutants. EQVSHHPP, OSBP signature motif; FFAT, two phenylalanines in an acidic tract motif; SBD, sterol-binding domain. Stars indicate the site of the mutation introduced. (B) HeLa cells were transfected with control or OSBP#1 siRNA for 48 h, followed by transfection with the indicated plasmids encoding Myc-tagged OSBP or OSBP mutants that contain silent mutations within the siRNA targeting sequence. Seventy-two hours after siRNA transfection, cell lysates were subjected to SDS–PAGE and immunoblotted with anti-OSBP antibody. Transfection with the siRNA-resistant OSBP construct (WT*) increased OSBP expression close to the normal level. (C, D) Cells were transfected with OSBP#1 siRNA for 48 h, followed by transfection with the indicated plasmids encoding siRNA-resistant OSBP mutants. Seventy-two hours after siRNA transfection, cells were fixed and stained with anti-GS28 (green) and anti-Myc (red) antibodies. DNA was stained with DAPI (blue). GS28 localization was quantified (C) as in Figure 1C, and representative images are shown (D). Arrows indicate cells with restored perinuclear localization of GS28 by the expression of WT*, S381A, or S381/384/387E mutants. Scale bar, 10 μm.

FIGURE 4:

Effect of OSBP depletion on Cav1-GFP localization and ACAT activity. (A) HeLa cells were transfected with control or OSBP#1 siRNA for 48 h, followed by transfection with the Cav1-GFP plasmid. Seventy-two hours after siRNA transfection, cells were fixed and stained with anti-GM130 (red) and anti-OSBP (blue) antibodies. Arrows indicate Cav1-GFP accumulation in the Golgi of OSBP-depleted cells. Scale bar, 10 μm. (B) HeLa cells were transfected with control or CERT siRNA#1 for 48 h, followed by transfection with the Cav1-GFP plasmid. Seventy-two hours after siRNA transfection, cells were fixed and stained with anti-GM130 (red) and anti-OSBP (blue) antibodies. Scale bar, 10 μm. (C) Cells were transfected with control or OSBP#1 siRNA. Seventy-two hours after transfection, the rate of cholesterol esterification in the cells was determined. Data represent mean ± SEM (n = 4). *p < 0.05 vs. control RNA interference.

FIGURE 5:

Cellular cholesterol depletion mislocalizes intra-Golgi v-SNAREs (GS28 and GS15) throughout the cytoplasm. HeLa cells were grown in medium containing 10% FBS or 5% LPDS plus 5 μM AY9944 for 48 h. (A) Lipids extracted from the cells were analyzed by gas chromatography. The solid and open arrowheads indicate the peak position of cholesterol and 7-DHC, respectively. Total cholesterol (black), 7-DHC (gray), and other lipids (white) in the cells were quantified. Note the accumulation of 7-DHC in LPDS/AY9944-treated cells, which is the immediate precursor of cholesterol. (B) Cells were fixed and stained with the indicated antibodies. Scale bar, 10 μm. (C) Cell lysates were subjected to SDS–PAGE and immunoblotted with the indicated antibodies. (D) Cell lysates were immunoprecipitated with anti-GS28 antibody, and the precipitates were immunoblotted with the indicated antibodies.

FIGURE 6:

Involvement of COP-I–mediated transport in GS28 mislocalization. (A) HeLa cells were transfected with the indicated siRNAs for 72 h. Cells were fixed and stained with anti-GM130 (green) and anti-γ-COP (red) antibodies. Scale bar, 10 μm. (B–D) Simultaneous depletion of ArfGAP1 and OSBP suppresses the GS28 dispersion induced by depletion of OSBP alone. HeLa cells were transfected with the indicated siRNAs twice. The subsequent experiments were performed 5 d after the first siRNA transfection. (B) Cell lysates were subjected to SDS–PAGE and immunoblotted with the indicated antibodies. (C, D) Cells were fixed and stained with anti-GS28 (green) and anti-Syn5 (red) antibodies (C), and the GS28 localization was quantified (D) as in Figure 1C. Scale bar, 10 μm.