Figure 2.
Newly synthesized GLUT4 is delayed in the early secretory pathway compared with GLUT1. (A) Representative stills extracted from Video 1 showing a HeLa cell expressing the ER Ii-hook fused to streptavidin along with HA-GLUT1-SBP-mCherry (GLUT1, red) and HA-GLUT4-SBP-GFP (GLUT4, green). The intracellular traffic of GLUT1-mCherry and GLUT4-GFP was simultaneously tracked for 1 h after biotin addition released them from the ER. Upon ER exit, both GLUT1 and GLUT4 accumulated in the perinuclear region of the cell (yellow). From 26 min onward, highly mobile GLUT1 vesicles (arrowheads) were visible (red), while GLUT4 remained perinuclear. Scale bar: 10 µm. (B, D, F, H, and J) Representative IF staining for GLUT1-SBP-GFP or GLUT4-SBP-GFP (detected with anti-GFP antibody, green), CHC22 (red), and CNX (blue; B), ERGIC-53 (blue; D), p115 (blue; F), GM130 (blue, H), or TGN46 (blue; J) in HeLa cells expressing HA-GLUT1-SBP-GFP or HA-GLUT4-SBP-GFP along with the ER Ii-hook. Traffic of GLUT4 and GLUT1 was tracked at 0, 15, 30, and 60 min after release from the ER by biotin. Arrows point to GLUT1 detected at the plasma membrane and arrowheads point to GLUT1-positive endosomal structures. Merged images show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in cyan, and red/green/blue overlap in white. Scale bars: 10 µm. (C, E, G, I, K, and L) Pearson’s overlap between GLUT1 or GLUT4 and CNX, ERGIC-53, p115, GM130, TGN46, or CHC22 at different time points after ER release. Data expressed as mean ± SEM, n = 3–4, 10–46 cells per experiment. One-way ANOVA followed by Sidak’s multiple comparison post hoc test, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 to test differences between GLUT1 and GLUT4 overlap with markers at each time point. (M) Pearson’s overlap between CHC22 and GLUT4, ER marker calreticulin, ERGIC markers p115 and ERGIC-53, cis-Golgi marker GM130, or trans-Golgi marker TGN46 in HeLa-GLUT4 from images taken by confocal microscopy (corresponding representative IF staining in Fig. S1). Data expressed as mean ± SEM, n = 3, 4–10 cells across three independent samples.

Newly synthesized GLUT4 is delayed in the early secretory pathway compared with GLUT1. (A) Representative stills extracted from Video 1 showing a HeLa cell expressing the ER Ii-hook fused to streptavidin along with HA-GLUT1-SBP-mCherry (GLUT1, red) and HA-GLUT4-SBP-GFP (GLUT4, green). The intracellular traffic of GLUT1-mCherry and GLUT4-GFP was simultaneously tracked for 1 h after biotin addition released them from the ER. Upon ER exit, both GLUT1 and GLUT4 accumulated in the perinuclear region of the cell (yellow). From 26 min onward, highly mobile GLUT1 vesicles (arrowheads) were visible (red), while GLUT4 remained perinuclear. Scale bar: 10 µm. (B, D, F, H, and J) Representative IF staining for GLUT1-SBP-GFP or GLUT4-SBP-GFP (detected with anti-GFP antibody, green), CHC22 (red), and CNX (blue; B), ERGIC-53 (blue; D), p115 (blue; F), GM130 (blue, H), or TGN46 (blue; J) in HeLa cells expressing HA-GLUT1-SBP-GFP or HA-GLUT4-SBP-GFP along with the ER Ii-hook. Traffic of GLUT4 and GLUT1 was tracked at 0, 15, 30, and 60 min after release from the ER by biotin. Arrows point to GLUT1 detected at the plasma membrane and arrowheads point to GLUT1-positive endosomal structures. Merged images show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in cyan, and red/green/blue overlap in white. Scale bars: 10 µm. (C, E, G, I, K, and L) Pearson’s overlap between GLUT1 or GLUT4 and CNX, ERGIC-53, p115, GM130, TGN46, or CHC22 at different time points after ER release. Data expressed as mean ± SEM, n = 3–4, 10–46 cells per experiment. One-way ANOVA followed by Sidak’s multiple comparison post hoc test, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 to test differences between GLUT1 and GLUT4 overlap with markers at each time point. (M) Pearson’s overlap between CHC22 and GLUT4, ER marker calreticulin, ERGIC markers p115 and ERGIC-53, cis-Golgi marker GM130, or trans-Golgi marker TGN46 in HeLa-GLUT4 from images taken by confocal microscopy (corresponding representative IF staining in Fig. S1). Data expressed as mean ± SEM, n = 3, 4–10 cells across three independent samples.

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