FIGURE 3.
Characterization of the activity of the prodomain deletion mutants. A, SREBP-2 processing by SKI-1/S1P mutants. SRD12B cells were transfected with the indicated SKI-1/S1P constructs. After 32 h, the SREBP-2 pathway was induced by mevastatin treatment for 18 h, whereas control cells were treated with DMSO. After treatment, total RNA was extracted to analyze HGMCS1 gene induction by quantitative RT-PCR. The results were analyzed by the ΔΔCT method, and the data were normalized against hydroxymethylbilane synthase gene expression. Gene expression is represented as fold induction above levels for control treatment (pIR-HA DMSO) (mean ± S.D.; n = 3). B, expression of SKI-1/S1P variants in A was assessed by Western blot as in Fig. 1B. C and D, LASV and LCMV GPC processing by the SKI-1/S1P mutants. SRD12B cell were co-transfected with either LASV GPC (C) or LCMV GPC (D) and the indicated SKI-1/S1P variants. At 48 h post-transfection, the cell lysates were analyzed by Western blot to assess GPC processing. SKI-1/S1P expression was detected with anti-V5 antibody and tubulin (Tub) detected as loading control. Tubulin, the precursor GPC, mature GP2, and maturation forms of SKI-1/S1P (forms A, B, and C) are indicated. E, schematic of the SKI-1/S1P sensor bearing the cleavage motif of LASV GPC. The signal peptide (SP), the GLuc reporter, the LASV GPC-derived cleavage motif, and the SKI-1/S1P-derived stump region are indicated. F, processing of the SKI-1/S1P sensor by SKI-1/S1P mutants. SRD12B cells were co-transfected with the SKI-1/S1P sensor (SS-LASV) and the indicated SKI-1/S1P variants. At 48 h post-transfection, cell lysates and supernatants were collected and analyzed by Western blot using an anti-GLuc antibody (bottom). Cleaved sensor (cGLuc) and the uncleaved precursor (pGLuc) are indicated. Conditioned media were analyzed for GLuc activity by addition of coelenterazine substrate. The data are shown as relative light units (RLU) (means ± S.D.; n = 3).