Figure 5. Phosphorylation at Y876 and Y898 regulates GEF activity of GBF1.

(A) SDS-PAGE analysis of GTP-loaded Arf1 at 0 min (-) and 10 min (+) imidazole treatment in HEK-IS cells expressing wild-type GBF1 and phospho-defective mutants GBF1-Y876F, GBF1-Y898F, or GBF1-Y876.898F. (B) Quantification of Arf1-GTP loading levels in (A) from three independent experiments. Values were normalized to untreated cells (-) expressing wild-type GBF1. (C) Representative images of Helix pomatia lectin (HPL) staining in HeLa cells coexpressing wild-type GBF1 or GBF1-Y876.898F mutant with active SrcEG. Scale bar: 50 μm. (D) Quantification of HPL staining levels of cells coexpressing wild-type or mutant GBF1 with inactive SrcKM (blue bars) or active SrcEG (pink bars). Values were from three replicates. (E) SDS-PAGE analysis of the levels of recombinant Arf1-His bound to wild-type or phospho-defective GBF1 mutants immunoprecipitated (IP) from cells expressing inactive SrcKM or active SrcEG in an in vitro binding assay. (F) Quantification of the levels of bound Arf1-His. Values were from four experimental replicates and normalized to wild-type GBF1 IP from cells expressing active SrcEG from each experiment. IP’ed GFP protein used as a negative control for nonspecific binding with GFP (gray bar). (G) Still images from time-lapse imaging of GALNT2-mCherry in HeLa cells that were either transfected with wild-type GBF1 (GBF1 WT) or phosphomimetic mutant (GBF1 Y876.898E). Arrows indicate tubule formation. Scale bar: 5 μm. (H) Quantification of the number of tubules per minute of acquisition. (I) Representative images of HPL staining in HeLa cells expressing phosphomimetic (Y-to-E) mutants 4 hr post transfection. (J) Quantification of HPL staining levels in (I). Values on graphs indicate the mean ± SD. Statistical significance (p) was measured by two-tailed paired t-test. *p<0.05, **p<0.01, ***p<0.001 relative to untreated cells or to 10 min imidazole (imdz)-treated cells expressing wild-type GBF1. NS, nonsignificant.

Figure 5—figure supplement 1. Phosphomimetic mutants at Y876 and Y898 recapitulate the effects of active Src.

(A) Representative images of Helix pomatia lectin (HPL) staining in HeLa cells coexpressing wild-type GBF1 or phospho-defective mutants with active SrcEG or inactive SrcKM shown in Figure 5C. Scale bar: 50 μm. (B) Quantification of HPL staining levels of HeLa-IS cells coexpressing with wild-type or phospho-defective mutant GBF1 without (blue bars) or with 4 hr imidazole (imdz) treatment (pink bars). Values were from three experimental replicates. (C) SDS-PAGE analysis of total Arf1 and GTP-loaded Arf1 in HEK293T cells expressing phospho-defective and phosphomimetic mutants of GBF1. Two experimental replicates for each condition are shown in the blot. (D) Quantification of Arf1-GTP loading in (C). (E) An extension of images from time-lapse imaging of GALNT2-mCherry in HeLa cells that were either transfected with wild-type GBF1 (GBF1 WT) or phosphomimetic mutant (GBF1 Y876.898E) shown in Figure 5G. Arrows indicate tubule formation. Scale bar: 5 μm. (F) Quantification of the number of tubules observed in the first 30 min of acquisition. Values on graphs indicate the mean ± SD. Statistical significance (p) was measured by two-tailed paired t-test. *p<0.05 and **p<0.001 relative to untreated (0 hr) or GFP-expressing cells. NS, nonsignificant.

Figure 5—video 1. Video of tubule formation in GALNT2-mCherry-expressing HeLa cells transfected with wild-type GBF1 (GBF1 WT).

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Scale bar: 5 μm.

Figure 5—video 2. Video of tubule formation in GALNT2-mCherry-expressing HeLa cells transfected with phosphomimetic GBF1 mutant (GBF1 Y876.898E).

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Scale bar: 5 μm.