Figure 2.

The BORC-ARL8b complex and TRPML3 are required for lysosomal exocytosis in ORF3a-expressing cells

(A–C) Compared with control cells expressing mCherry (A), the number of BORCS6-GFP puncta is dramatically increased in ORF3a-mCherry-expressing cells (B). (C) Shows quantification of the number of BORCS6-GFP puncta per cell. Data are shown as mean ± SEM (n = 31 for each bar). ^∗∗∗p < 0.001.

(D–F) Compared with control cells transfected with control siRNA (E), the level of cell membrane-localized LAMP1 is dramatically decreased in ORF3a-expressing cells transfected with BORCS3 siRNA (F). (D) Shows quantification of the fluorescence intensity of cell membrane-localized LAMP1 per cell. The level in control siRNA-treated cells is set to 1.0. Data are shown as mean ± SEM (n = 23 for control cells, n = 27 for ARL8b KD cells, and n = 22 for BORCS3 KD cells). ^∗∗∗p < 0.001.

(G) In GFP-Trap assays, ORF3a-mCherry is co-immunoprecipitated by BORCS6-GFP. Cell lysates were immunoprecipitated using GFP-Trap and analyzed by immunoblotting with mCherry and GFP antibodies.

(H) The 340/380 nm fluorescence excitation ratio of HeLa cells expressing GFP (control, n = 55) and ORF3a-GFP (ORF3a, n = 49) after loading with fura-2. Data are shown as mean ± SEM ^∗∗∗p < 0.001.

(I) The cytosolic free calcium concentration in HeLa cells expressing mCherry (control, n = 45) and ORF3a-mCherry (ORF3a, n = 9). Data are shown as mean ± SEM ^∗∗∗p < 0.001.

(J–L) Compared with control cells expressing mCherry (J), the number of LAMP1-GCaMP6f puncta is dramatically increased in ORF3a-mCherry-expressing cells (K). (L) shows quantification of the number of LAMP1-GCaMP6f puncta per cell. Data are shown as mean ± SEM (n = 15 for control cells, n = 21 for ORF3a-expressing cells). ^∗∗∗p < 0.001.

(M) The ratio of F/F₀ in control cells expressing mCherry (control) and ORF3a-mCherry (ORF3a) after inducing lysosomal Ca²⁺ release by HBSS.

(N) In GFP-Trap assays, FLAG-TRPML1 and FLAG-TRPML3 but not FLAG-ARL8b are co-precipitated by ORF3a-GFP. Cell lysates were immunoprecipitated using GFP-Trap and analyzed by immunoblotting with FLAG and GFP antibodies.

(O) Shows quantification of ΔF/F₀, related to Figure 2M. Data are shown as mean ± SEM (n = 21 for control, n = 25 for ORF3a). ^∗∗∗p < 0.001.

(P and Q) Compared with ORF3a-expressing cells transfected with control siRNA (Figure 2E), the level of cell membrane-localized LAMP1 is dramatically decreased in ORF3a-expressing cells transfected with TRPML3 siRNA (Q).

(P) Shows quantification of the fluorescence intensity of cell membrane-localized LAMP1 per cell. The level in control siRNA-treated cells is set to 1.0. Data are shown as mean ± SEM (n = 23, 17, 20, and 21 for bars from left to right). ^∗∗∗p < 0.001; n.s., no significant difference. The data for ORF3a-expressing cells transfected with control siRNA are the same as those in Figure 2D.

Scale bars: (A, B, E, F, J, K, and Q) 10 μm.

See also Figures S1 and S2.