Figure 5. ERK8 regulates COPI-dependent GalNAc-T traffic.

(A) Co-knockdown of ERK8 with Arf1 or GTP exchange factor, GBF1, and staining with Helix pomatia lectin (HPL). NT, non-targeting. Scale bar: 30 μm. (B) Quantification of Tn levels upon ERK8 co-knockdown with Arf proteins and GBF1. Grey bar indicates knockdown of ERK8 only. Blue bars indicate co-knockdowns. (C) SDS-PAGE analysis of total Arf and Arf1-GTP in cells treated with 5 µM Ro-31-8220 (iERK8). (D) Co-staining of Beta-COP (COPB) and GalNAc-T1 in cells treated with 5 µM iERK8 for 15 min. Transient tubular structures emanating from the Golgi appear stained for GalNAc-T1 and beta-COP (arrowhead, second panel). Scale bar: 10 μm. (E) Effect of ERK8 depletion on GalNAc-T1 and KDEL receptor (KDEL-R) subcellular location. (F) Effect of expression of active SRC (SrcE-mcherry, containing the E378G mutation) or inactive SRC (SrcK-mcherry, containing the K295M mutation) on both proteins. Scale bar: 10 μm. (G) Visual scoring of KDEL-R and GalNAc-T1 redistribution from the Golgi in cells subjected to various treatment conditions. Cells were counted in each condition from three independent experiments (NT control: 83 cells; ERK8 KD: 86; SrcE-mcherry: 42; SrcK-mcherry: 32). (H) Temperature-sensitive vesicular stomatitis virus G glycoprotein (VSVG-mcherry) traffic to the Golgi induced by shift from restrictive to permissive temperature for 15 min in KDEL-R expressing cells stained for GalNac-T1. Scale bar: 10 μm. (I) Visual scoring of KDEL-R and GalNAc-T1 relocation in VSVG expressing cells at 0 and 15 min after temperature shift. Cells were counted in each condition from three independent experiments (0 min, 44 cells; 15 min, 63 cells). Values on graphs indicate the mean ± SEM. **p<0.0001, *p<0.05 by two-tailed unpaired t test, relative to NT siRNA-treated (B and G) cells and cells at 0-min timepoint (I).

DOI: http://dx.doi.org/10.7554/eLife.01828.011

Figure 5—figure supplement 1. ERK8 regulated GalNAc-T traffic depends on the activity of COPI regulators.

(A) Tn expression levels, as determined by Helix Pomatia Lectin (HPL) staining in cells expressing dominant-negative mutant Arf1 (Q71L) compared to wild-type Arf1. GRASP55 labels the Golgi apparatus. Scale bar: 10 μm. (B) Quantification of Tn expression in ERK8-depleted cells upon treatment with 50 nM GBF1 inhibitor, Golgicide, for the indicated times. (C) Quantification of Tn expression in cells following co-knockdown with ERK8 and GFP or NT5 siRNA in amounts to equivalent to double (GFP coKD), triple (GFPx2 coKD) and quadruple (GFPx3 coKD) siRNA transfection configurations. (D) SDS-PAGE analysis of protein levels of Arfs and GBF1 in single and double knockdown configurations. (E) Beta-COP (COPB) staining in control and ERK8-depleted cells. Scale bar: 10 μm. (F) Quantification of the relative numbers of COPI transport carriers (COPB) and Golgi fragments (GM130) using the granularity measurement module in MetaXpress software. At least 30 cells (non-targeting (NT) control, 37 cells; ERK8-KD, 34) were quantified for each treatment. (G) Staining of native COPI coatomer in cells treated with 5 μM Ro-31-8220 (iERK8) over the indicated times. (H) Quantification of the relative numbers of COPI transport carriers using the granularity measurement module in MetaXpress software. Twenty-five or more cells (Vehicle, 41 cells; 5 min iERK8 treatment, 25; 25 min iERK8 treatment, 57) were quantified for each treatment. (I) Staining of GalNAc-T1 (left panel) and KDEL receptor (KDELR1) localisation (right panel) in HeLa KDEL-R1-GFP stable cell line depleted of ERK8. (J) Quantification of total intensities of GalNAc-T1 and KDEL-R at the Golgi region demarcated by Giantin (Golgi protein) staining. Values on graphs indicate the mean ± SEM. **p<0.0001, *p<0.05 by two-tailed unpaired t test, relative to vehicle treated (B and H), single ERK8 knockdown (C) and NT siRNA-treated (F and J) cells. NS, not significant.

DOI: http://dx.doi.org/10.7554/eLife.01828.012