Fig. 2: ORP9 localizes to the TGN via its N-terminal PH domain and tandem α-helices. | Nature Communications

Fig. 2: ORP9 localizes to the TGN via its N-terminal PH domain and tandem α-helices.

From: Regulation of cellular cholesterol distribution via non-vesicular lipid transport at ER-Golgi contact sites

Fig. 2

a The ribbon diagram of the modeled ORP9 (see Materials and Methods). PH, pleckstrin homology; ORD, OSBP-related domain; N, N-terminus; C, C-terminus. b Domain structure of ORP9 and of various versions of ORP9 analyzed in this study. c Confocal images of live HeLa cells expressing either EGFP-tagged ORP9 full-length (EGFP-ORP9) or one of the indicated versions of EGFP-ORP9 as shown in (b), together with a TGN marker, mCherry-tagged N-terminus of sialyltransferase (SiT-N-mCherry). Insets show at higher magnification the regions around the TGN as indicated by white dashed boxes (green: the indicated version of EGFP-ORP9; magenta: SiT-N-mCherry). Scale bars, 10 µm. d Confocal images of live HeLa cells expressing either EGFP-ORP9 or EGFP-ORP9 carrying the FY/AA mutation in its FFAT motif [EGFP-ORP9 (FY/AA)] together with mCherry-tagged VAPA (mCherry-VAPA). Note the recruitment of EGFP-ORP9, but not EGFP-ORP9 (FY/AA), to the ER labelled by mCherry-VAPA. Scale bars, 10 µm. e Confocal images of live HeLa cells expressing one of the indicated versions of EGFP-ORP9 as shown in (b), together with SiT-N-mCherry. Insets show at higher magnification the regions around the TGN as indicated by white dashed boxes (green: the indicated versions of EGFP-ORP9; magenta: SiT-N-mCherry). Scale bars, 10 µm. f, g Confocal images of live HeLa cells expressing EGFP-ORP9 together with either mCherry-tagged ORP10 (mCherry-ORP10) or mCherry-tagged ORP11 (mCherry-ORP11). Note the extensive colocalization of EGFP-ORP9 with mCherry-ORP10 and mCherry-ORP11. Insets show at higher magnification the regions as indicated by white dashed boxes. Scale bars, 10 µm. h Confocal images of fixed WT HeLa cells and HeLa cells lacking ORP10 and ORP11 [ORP10/ORP11 double knockout (DKO)], in which mNeonGreen was tagged to the C-terminus of ORP9 (endoORP9-mNG). Cells were immunolabeled with antibodies against TGN46. Insets show at higher magnification the regions around the Golgi as indicated by white dashed boxes (green: endoORP9-mNG; magenta: TGN46). Scale bars, 10 μm. i Quantification of endoORP9-mNG fluorescence signals at the Golgi as shown in (h) [mean ± SEM, n = 296 cells (WT), n = 325 cells (DKO); data are pooled from two independent experiments; two-tailed unpaired Student’s t-test, **P < 0.0001]. Source data are provided as a Source Data file.

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