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. 2009 Mar;20(5):1388-99.
doi: 10.1091/mbc.e08-09-0905. Epub 2009 Jan 7.

Oxysterol binding protein-related Protein 9 (ORP9) is a cholesterol transfer protein that regulates Golgi structure and function

Mike Ngo  1 , Neale D Ridgway
Affiliations

Oxysterol binding protein-related Protein 9 (ORP9) is a cholesterol transfer protein that regulates Golgi structure and function

Mike Ngo et al. Mol Biol Cell. 2009 Mar.

Abstract

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) constitute a large gene family that differentially localize to organellar membranes, reflecting a functional role in sterol signaling and/or transport. OSBP partitions between the endoplasmic reticulum (ER) and Golgi apparatus where it imparts sterol-dependent regulation of ceramide transport and sphingomyelin synthesis. ORP9L also is localized to the ER-Golgi, but its role in secretion and lipid transport is unknown. Here we demonstrate that ORP9L partitioning between the trans-Golgi/trans-Golgi network (TGN), and the ER is mediated by a phosphatidylinositol 4-phosphate (PI-4P)-specific PH domain and VAMP-associated protein (VAP), respectively. In vitro, both OSBP and ORP9L mediated PI-4P-dependent cholesterol transport between liposomes, suggesting their primary in vivo function is sterol transfer between the Golgi and ER. Depletion of ORP9L by RNAi caused Golgi fragmentation, inhibition of vesicular somatitus virus glycoprotein transport from the ER and accumulation of cholesterol in endosomes/lysosomes. Complete cessation of protein transport and cell growth inhibition was achieved by inducible overexpression of ORP9S, a dominant negative variant lacking the PH domain. We conclude that ORP9 maintains the integrity of the early secretory pathway by mediating transport of sterols between the ER and trans-Golgi/TGN.

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Figures

Figure 1.
Figure 1.
Phosphatidylinositol phosphate–binding specificity of the ORP9 PH domain. (A) Purified GST fusion proteins of the OSBP, ORP9, and ORP9 R22E PH domains (2 μg) were separated by SDS-12% PAGE and stained with Coomassie Blue. (B) His-tagged ORP9L and OSBP (2 μg) purified from Sf21 cells were resolved on SDS-8% PAGE and stained with Coomassie Blue. (C) GST-PH domain fusion proteins, ORP9L and OSBP (300 pmol each), were incubated with the indicated phospholipids (100 pmol) immobilized on Hybond C filters. GST-fusion protein binding to phospholipids was detected using an anti-GST monoclonal, whereas ORP9 and OSBP were detected with monospecific polyclonal antibodies. (D) OSBP and ORP9 (100 pmol) were incubated with PC liposomes containing the indicated mol% of PI4P for 20 min at 20°C and sedimented at 100,000 × g for 30 min, and the pellet (P) and supernatant (S) fractions were resolved by SDS-8% PAGE and stained with Coomassie Blue.
Figure 2.
Figure 2.
ORP9 preferentially binds and transfers liposomal cholesterol. (A) [3H]25-hydroxycholesterol (100 nM) binding by OSBP and ORP9L (100 pmol) was assayed using a charcoal-dextran method (Ridgway et al., 1992). (B) OSBP and ORP9L (100 pmol) were incubated with 600 nM [3H]cholesterol, and bound sterol was quantified after binding to Talon nickel affinity resin. (C) Extraction of [3H]cholesterol from liposomes was measured in the presence of increasing amounts of ORP9L and quantified as described in Materials in Methods. Results are the mean of two separate experiments. (D) Extraction of cholesterol from PC liposomes by OSBP, ORP9L, and ORP9L ΔSB (100 pmol each). (E) Distribution of ORP9L, OSBP, and ORP9L ΔSB in the supernatant (S) and pellet (P) fraction of a cholesterol extraction assay in the presence and absence of 1 mol% cholesterol liposomes as determined by SDS-PAGE. (F) [3H]Cholesterol transfer from donor to acceptor liposomes by OSBP and ORP9L was assayed as described in the Material and Methods. Liposomes were prepared with or without 10 mol% PI-4P. (G) The sterol transfer activity of ORP9L, OSBP, and OSBP PH-RR/EE (100 pmol) was determined with donor and acceptor vesicles each containing no addition or 10 mol% PI-3P, PI-4P, or PI-5P. Unless indicated, results are the mean and SEM of 3–6 separate experiments.
Figure 3.
Figure 3.
ORP9L localizes to the trans-Golgi/TGN. CHO cells cultured in medium A were treated with nocodazole (2 μg/ml) or solvent control for 1 h and immediately fixed and immunostained with an ORP9 polyclonal antibody and a goat anti-rabbit AlexaFluor594-conjugated secondary antibody. This was followed by a giantin antibody coupled to AlexaFluor488, a PI-4K IIIβ monoclonal and goat anti-mouse AlexaFluor488-conjugated secondary or a γ-adaptin monoclonal antibody, and a goat anti-mouse AlexaFluor488-conjugated secondary antibody. Images are single confocal sections (0.8 μm) taken through the midplane of the cells. Selected high-magnification fields are boxed and are shown in the bottom three panels.
Figure 4.
Figure 4.
The ORP9L sterol-binding and PH domains regulate interaction with the Golgi apparatus. CHO cells stably expressing ORP9L (A), OPR9L-FY/AA (B), ORP9L- R22E (C), and ORP9L-ΔSB (D) under the control of the TET promoter were cultured in medium A and induced with 1 μg/ml doxycycline. After 24 h, cells were fixed and immunostained with primary antibodies against V5 (ORP9L), VAP, and/or giantin-conjugated AlexaFluor488, followed by goat anti-rabbit or goat anti-mouse secondary antibodies conjugated to AlexaFluor488 (giantin and VAP) or AlexaFluor594 (V5-tagged ORP9L). Images are single confocal scans (0.8 μm) though the middle plane of the cells.
Figure 5.
Figure 5.
Knockdown of ORP9L expression does not affect oxysterol-regulated SM synthesis. (A) CHO cells cultured in medium A were transiently transfected with siOSBP (75 nM), siORP9L (75 nM), siOSBP and siORP9L (75 nM each), or a nontargeting control siNT (75 nM) for 48 h. Cells then received serine-free medium A containing no addition (formula image) or 25-hydroxycholesterol (2.5 μg/ml; ■) for 2 h followed by pulse-labeling with [3H]serine (10 μCi/ml) for 2 h. [3H]Serine-labeled SM, GlcCer and ceramide were extracted from cells, separated by thin-layer chromatography, and quantified as previously described (Perry and Ridgway, 2006). Results are the mean and SE for three separate experiments. (B) Expression of OSBP and ORP9L in CHO cells transiently transfected with control and targeting siRNAs. (C) CHO cells expressing ORP9L under the control of the Tet repressor were cultured in medium A without (−Dox) or with doxycycline (+Dox, 1 μg/ml) for 24 h. Cells then received solvent control (formula image) or 25OH (■) and were pulse-labeled with [3H]serine as described in A. Results are the mean and SEM for three separate experiments.
Figure 6.
Figure 6.
Knockdown of ORP9L expression results in Golgi fragmentation. CHO cells were transfected with siORP9 for 48 h in medium A and processed for immunostaining of ORP9L and giantin or γ-adaptin as described in the legend to Figure 4. Fields were identified in which ORP9L expression displayed variability between individual cells. ORP9L expression was almost undetectable in cells indicated by arrows. Images are single confocal sections (0.8 μm) through the midplane of the cells.
Figure 7.
Figure 7.
Knockdown of ORP9L expression partially suppresses VSVG transport to the Golgi apparatus. (A) CHO cells cultured in medium A were transfected with siORP9 (75 nM) or siNT (75 nM). After 48 h, cells were shifted to 40°C and transiently transfected with pcDNA3.1-VSV-G-tsO45-(myc)3 for a further 16 h. Cells were then maintained at 40°C or shifted to 33°C for 120 min to initiate folding and export of 045-VSVG to the Golgi apparatus. Cells were harvested, total extracts incubated with and without endoH, and sensitive and resistant forms of VSVG were detected by SDS-10%PAGE and immunoblotting as described in Materials and Methods. (B) The distribution of endoH-resistant and -sensitive forms of VSVG was quantified by densitometry. Results are the mean and SEM of three independent experiments.
Figure 8.
Figure 8.
Silencing of ORP9L expression promotes cholesterol retention in the endosomal/lysosomal compartment. CHO cells cultured in medium A were transfected with siORP9 (75 nM) or siNT (75 nM) for 32 h. Cells then received medium containing 5% FCS or lipoprotein deficient serum (LPDS) for an additional 16 h. Cells were fixed and incubated with an ORP9L polyclonal antibody, and goat anti-rabbit AlexaFluor594-conjugated secondary antibody and cholesterol was detected with filipin. (A) A representative field of siNT- and siORP9L-transfected cells cultured in FCS and stained with an ORP9L antibody and filipin (cholesterol). (B) Fluorescent intensity of filipin staining was quantified in control and ORP9L knockdown cells cultured in FCS or LPDS for 16 h as described in Material and Methods. Results are the mean and SEM of three independent experiments that quantified 10–12 fields of 20–30 cells. (C) Total (T) and unesterified cholesterol (U) mass was quantified in control and ORP9L-depleted CHO cells cultured in medium with 5% FCS or LPDS. Results are the mean and SEM of three separate experiments.
Figure 9.
Figure 9.
Inducible expression of ORP9S fragments the Golgi apparatus. CHO cells stably transfected with plasmids encoding the indicated ORP9S proteins were cultured in medium A with or without doxycycline (1 μg/ml) for 24 h. (A) CHO cells expressing ORP9S were induced with doxycycline and localization with VAP was determined. (B) CHO cells expressing ORP9S from the Tet promoter were cultured in the absence (−Dox) or presence of doxycycline (+Dox) and localization of ORP9S and giantin was determined by immunofluorescence as described in the legend to Figure 3. (C) After induction with doxycycline, localization of ORP9S-FF/AA with VAP and giantin was determined as described above. (D) After induction with doxycycline, localization of ORP9S-ΔSB with VAP and giantin was determined as described in above. Images are single confocal sections (0.8 μm) through the midplane of the field of cells.
Figure 10.
Figure 10.
Enforced expression of ORP9S prevents ER–Golgi transport of 045-VSVG. (A) CHO cells stably expressing ORP9L and ORP9S under the control the Tet-repressor were transfected with pcDNA3.1-VSV-G-tsO45-(myc)3 for 6 h at 40°C. Cells then received medium A with or without doxycycline (Dox, 1 μg/ml) for 10 h at 40°C. Cells were shifted to 33°C and at the indicated times harvested for analysis of VSVG glycosylation as described in Materials and Methods. (B) CHO cells expressing ORP9L, ORP9S, ORP9S-FF/AA, and ORP9S-ΔSB under the control of the Tet-repressor were transfected with pcDNA3.1-VSV-G-tsO45-(myc)3 for 6 h and then incubated in the presence or absence or doxycycline for 10 h at 33°C before harvesting and analysis of VSVG endoH sensitivity. (C) Quantification of data in B showing the relative proportion of endoH-sensitive O45-VSVG in control and overexpressing cells. Results are the mean and SEM of three experiments.
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