Fig 2: Effects of knockdown of OSBP, CERT/OSBP, or SCAP on trafficking of GPI-anchored protein or VSV-G. (A) mKate2-FM4-GPI transport from the ER to the PM via the Golgi complex in control (Cont), OSBP, and CERT/OSBP knockdown cells. The cells were incubated at 37°C with D/D solubilizer and cycloheximide and fixed at the indicated times. The graph shows the percentages of cells with mKate2-FM4-GPI at the ER, ER/Golgi, Golgi/PM, or PM at the indicated times. The data shown are for a single representative experiment out of three performed (n = 229–268 cells per condition). (B) mKate2-FM4-GPI transport in the presence or absence of the Myc-OSBP PH-FFAT mutant. Asterisks denote cells coexpressing mKate2-FM4-GPI and Myc-OSBP PH-FFAT. (C) VSV-G–GFP transport from the ER to the PM via the Golgi complex in control and SCAP knockdown cells. The cells were incubated at 32°C with cycloheximide and fixed at the indicated times. The graph shows the percentages of cells with VSV-G–GFP at the ER, ER/Golgi, Golgi/PM, or PM at the indicated times. The data shown are for a single representative experiment out of three performed (n = 203–224 cells per condition). Scale bars, 10 µm.

Fig 3: SCAP is important for PI4P turnover and VAP-A–OSBP complex distribution at ER–Golgi MCSs. (A) Filipin staining in parental HeLa and shSCAP HeLa cells. High magnifications of the boxed areas are shown in the right panels. (B and C) PI4P staining in parental HeLa and shSCAP HeLa cells with (top two rows in C) or without (B and bottom two rows in C) sialyltransferase (ST)–mRFP expression. The graph shows determination of the Golgi PI4P levels in parental HeLa (control) and shSCAP HeLa cells. Data are means ± SEM (n = 139 cells per condition; ****, P < 0.0001; unpaired two-tailed Student’s t test). N, nucleus. (D) BiFC visualization of the OSBP–VAP-A interaction in control (Cont) and SCAP knockdown cells. HeLa cells stably coexpressing Vn-OSBP and Vc-VAP-A were transfected with siRNA. After 72 h, the cells were treated with or without 2 µg/ml 25-HC for 2.5 h. High magnifications of the boxed areas are shown in the insets where brightness/contrast enhancement was applied. The graph shows the percentage of cells with the peripheral BiFC signal of Vn-OSBP–Vc-VAP-A. Data are means ± SEM (n = 3 independent experiments; 100–131 cells per condition; ***, P < 0.005; unpaired two-tailed Student’s t test). (E) Close apposition of CD63- but not of TGN46-positive membranes to the peripheral BiFC signal of Vn-OSBP–Vc-VAP-A in SCAP knockdown cells treated with 2 µg/ml 25-HC for 2.5 h. High magnifications of the boxed areas are shown in the insets. Scale bars, 10 µm (large panels), 5 µm (insets).

Fig 4: Sterol-dependent localization of SCAP. (A and B) Localization of stably expressed GFP-SCAP in HeLa cells with or without cholesterol depletion (Chol Depl) for 3 h or treated with 2 µg/ml 25-HC for 2.5 h. (B) Accumulation of GFP-SCAP in GM130 (cis-Golgi matrix protein)– and mannosidase II (cis/medial-Golgi marker enzyme)–positive membranes upon cholesterol depletion. N, nucleus. (C) Proteolytic activation of SREBP2 upon cholesterol depletion. Lysates of HeLa cells with or without cholesterol depletion for 3 h or treated with 2 µg/ml 25-HC for 3 h were analyzed by Western blotting with an anti-SREBP2 antibody. Arrowheads indicate the precursor (P) and mature (M) forms of SREBP2. Asterisk denotes nonspecific bands. (D) Localization of the stably expressed hamster SCAP WT and mutants in shSCAP HeLa cells with or without cholesterol depletion for 3 h. Merged images for the hamster SCAP WT or mutants (green) and the cis/medial-Golgi marker GPP130 (magenta) of the boxed areas are shown in the insets. Scale bars, 10 µm.

Fig 5: SCAP regulates CARTS biogenesis in a cholesterol-dependent manner. (A) Schematic representation of the SCAP topology. SSD is highlighted in light blue. N, N-terminus; C, C-terminus. (B) Establishment of shSCAP HeLa cells stably expressing the hamster SCAP WT and mutants. IB, immunoblotted. (C) Recovery of PI4P turnover on expression of hamster SCAP WT or D451A/L452A, but not of Y234A, Y298C, or L315F. The graph shows determination of the Golgi PI4P levels in the indicated cells. Data are means ± SEM (n = 69–96 cells per condition; ****, P < 0.0001; one-way ANOVA multiple comparison test). (D) Recovery of the biogenesis of mKate2-FM4-PAUF–containing CARTS on expression of hamster SCAP WT or D451A/L452A, but not of Y234A, Y298C, or L315F. The graph shows the number of mKate2-FM4-PAUF–containing CARTS in the indicated cells at 15 min after the temperature shift to 37°C. Data are means ± SEM (n = 20 cells per condition; ****, P < 0.0001; one-way ANOVA multiple comparison test). Scale bars, 10 µm.