N-glycosylation-deficient E protein inhibits the ER-Golgi transition of SVPs.

(A) Primary structure of a prME expression construct with GFP11 insertion (S275-G11 (E)). The GFP11 tag sequence (green) and GS linker sequence (black) are indicated. (B) Confirmation of SVP formation of GFP11-inserted prME protein. Cell lysates from the indicated construct-expressing 293T cells were fractionated using 10–40% sucrose density gradient ultracentrifugation and then E protein in each fraction was detected using immunoblotting with anti-E protein antibody. (C) GFP complementation in cells expressing prME (S275-G11 (E)) and GFP1-10. HeLa cells expressing GFP1-10 were transfected with prME WT (left panel) or prME (S275-G11 (E)) (right panel). GFP fluorescence of cells was analyzed using flow cytometry at 24 hours post-transfection. Black and green line in the panels indicates empty and prME vector transfected cell, respectively. (D) Subcellular localization of complemented GFP in cells expressing prME (S275-G11 (E)) and GFP1-10. HeLa cells expressing GFP1-10 were transfected with prME (S275-G11 (E)). At 24 hours post-transfection., cells were fixed and stained with anti-E antibody (red) and Hoechst (Cyan). GFP fluorescence (Green) was observed. Scale bar: 10 μm. (E) Outline of the strategy for the visualization of synchronized transport of SVPs by the RUSH system. In the absence of biotin, ss-Streptavidin-Binding Peptide (SBP) -GFP1-10 reporter with prME (S275-G11 (E)) is retained in the ER by the streptavidin-KDEL hook. After biotin addition, the reconstituted GFP-labeled SVPs leave the ER and translocate to the Golgi apparatus. (F) Timelapse imaging analysis of HeLa cells expressing GFP-labeled SVPs under the RUSH system. Images of GFP fluorescence are shown, and elapsed times after addition of biotin are indicated at the top of each panel. Scale bar: 10 μm. (G) Golgi transport of GFP-labeled SVPs. HeLa cells expressing RUSH-GFP-SVPs WT (top panels) or N154A (bottom panels) were fixed and stained with anti-GM130 (red) at 0 min (left panels) or 30 min (right panels) after biotin addition, and GFP fluorescence (green) was observed. Scale bar: 10 μm. (H) Colocalization of GFP and GM130 signals (G) was determined by calculating Pearson’s correlation coefficient. *p < 0.0001; NS, not significant. Error bars represent standard deviation. Means and SDs from three independent experiments are presented.