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First published online 22 August 2006
doi: 10.1242/jcs.03173

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GBF1, a cis-Golgi and VTCs-localized ARF-GEF, is implicated in ER-to-Golgi protein traffic

Xinhua Zhao¹, Alejandro Claude^1,*, Justin Chun¹, David J. Shields^1,, John F. Presley² and Paul Melançon^1,

¹ Department of Cell Biology, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada
² Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, H3A 2B2, Canada

View larger version (140K): [in a new window]   Fig. 1. Endogenous GBF1 localizes to ß-COP positive peripheral VTCs at steady state. (A) Images a and b. NRK cells were fixed and processed for confocal IF using affinity-purified antibodies against GBF1 [9D5 (a) or 9D2 (b)]. Arrows mark peripheral 9D2-positive puncta. Bar, 5 µm. Images c-e. NRK cells were fixed and processed for standard IF in the presence of 1 µg GBF1 antigen using monoclonal anti-ß-COP antibody and either 9D5 (c,d) or 9D2 (e,f). Bar, 10 µm. (B) NRK cells were fixed and processed for standard IF using affinity purified 9D2 and ß-COP antibody. Bar, 5 µm.

View larger version (143K): [in a new window]   Fig. 2. GFP-GBF1, like endogenous GBF1, is recruited to cis-Golgi membranes and peripheral VTCs membranes. (A) Schematic representation of GFP-GBF1: EGFP (green) was fused in frame with full length CHO-derived GBF1 (orange) at its N terminus. Additional residues encoded by plasmid-derived and UTR sequences shown in blue. (B) NRK cells stably expressing GFP-GBF1 were fixed and processed for either double-label confocal IF by staining with affinity purified polyclonal antibody against GFP (a) and monoclonal antibody against p115 (3A10) (c) or single-label IF by staining with polyclonal antibody against BIG1 (9D3) (f) or monoclonal antibody against ß-COP (M3A5) (i). Arrowheads mark puncta revealed by GFP fluorescence (d,g) or GFP antibodies (a). Middle panels (b,e,h) show merged left and right images. Insets show threefold magnification of the area indicated by arrow. Bars, 10 µm.

View larger version (72K): [in a new window]   Fig. 3. Kinetics of GBF1 binding to and dissociation from Golgi and VTCs membranes in NRK cells stably expressing GFP-GBF1. Live NRK cells expressing GFP-GBF1 were examined by confocal microscopy at 37°C as described in Materials and Methods. (A) Cytosol FRAP was performed as described in Materials and Methods. Shown are still images at the indicated times. (B) Golgi FRAP. Experiment similar to that shown in 3A except that the ROI includes the Golgi complex. (C) Quantification of the Golgi FRAP experiment presented in 3B. The curve was obtained by fitting FRAP data to a single exponential corresponds to the equation: y=0.520 * (1-e-0.055t) + 0.381 with an R value of 0.991. (D) Golgi FRAP in absence of microtubules. FRAP analysis was performed as in 3B, except that cells were chilled on ice for 15 minutes and warmed to 37°C in the presence of 1 µg ml-1 NOZ. (E) VTCs FRAP. Similar to experiment shown in 3B except that the ROI was located in the cell periphery and contained GBF1-positive VTCs. Bars, 5 µm.

View larger version (105K): [in a new window]   Fig. 4. GBF1 accumulates on membranes of the Golgi complex and peripheral VTCs upon treatment with BFA. (A) Images of NRK cells expressing GFP-GBF1 were captured every 2 seconds for 10 minutes immediately after BFA addition (1 µg ml-1). Still images from indicated time points illustrate the transient accumulation of GBF1 onto peripheral VTCs and Golgi complex. (supplementary material Movie 1). Bar, 5 µm. (B) NRK cells treated with BFA (5 µg ml-1) for 30 seconds were fixed and processed for standard IF using Alexa488-conjugated anti-GBF1 rabbit antibody (H-154) and rabbit anti-p58 antibodies. Merged image shows threefold magnification of boxed area in red and green channels. Bar, 5 µm. (C) NRK cells treated with 5 µg ml-1 BFA for 1 minute were fixed and processed for confocal IF using polyclonal antibodies against GBF1 (H154) and BIG1 (Alexa488-conjugated 9D3). Bar, 10 µm. (D) Quantification of experiment 4A showing Golgi/total ratio plotted against treatment length. (E) NRK cells were washed in ice-cold buffer containing either DMSO vehicle control (0), or 0.5 µg ml-1 (0.5) or 5 µg ml-1 (5.0) BFA. Cytosol (C) and microsomes (M) fractions were prepared and analyzed for GBF1 content by immunoblot (see Materials and Methods).

View larger version (85K): [in a new window]   Fig. 5. BFA treatment traps GBF1 onto membranes. (A) NRK cells expressing GFP-GBF1 were incubated on ice for 15 minutes with 5 µg ml-1 NOZ prior to transfer onto microscope stage (37°C). Following equilibration, BFA (5 µg ml-1) was added and FRAP performed as for 3B. Bar, 5 µm. (B) FRAP was performed as for 3A except that cells were treated with 5 µg ml-1 BFA for 10 minutes prior to bleach. ROI size was similar to that used for untreated cells in Fig. 3A. Bar, 5 µm. (C) Quantification of panels 5A (+BFA) and 3D (-BFA) showing relative Golgi/total ratio plotted against treatment length. (D) Quantification of panels 5B (+BFA) and 3A (-BFA) showing relative Golgi/total ratio plotted against treatment length.

View larger version (120K): [in a new window]   Fig. 6. BFA-induced accumulation of GBF1 coincides with loss of COPI from peripheral VTCs that lie in close proximity to Sec31p-positive structures. (A) NRK cells treated with BFA (5 µg ml-1) for various times (0; 10 seconds; 30 seconds and 1 minute) were fixed and processed for standard IF using anti-GBF1 rabbit antibody (9D2) and anti-ß-COP mouse antibodies (M3A5). White arrowheads mark puncta containing both GBF1 and ß-COP. (B) NRK cells treated with BFA (5 µg ml-1) for 30 seconds were fixed and processed for standard IF using Alexa488-conjugated anti-GBF1 rabbit antibody (H-154) and rabbit anti-sec31p antibodies. Merged image shows a threefold magnification of boxed area in red and green channels. Bars, 5 µm.

View larger version (33K): [in a new window]   Fig. 7. Nocodazole (NOZ) treatment stabilizes GBF1-positive peripheral VTCs. NRK cells expressing GFP-GBF1 were treated on ice for 15 minutes with either 20 µg ml-1 NOZ or 0.5% DMSO. Following a 2 minute warm-up at 37°C, live cells were examined at 37°C on a Zeiss Axiovert 200M spinning disk microscope. Z-stacks of 6 slices each 1 µm thick were acquired continuously after addition of BFA (5 µg ml-1). See supplementary material Movie 2. Still images from projected stacks at indicated times are shown. Bar, 20 µm.

View larger version (143K): [in a new window]   Fig. 8. GBF1 decorates matured VTCs labeled by VSVGtsO45. COS-1 cells infected with VSV tsO45 were incubated at 40.5°C for 3 hours (0 min) (a-c) and then shifted to permissive temperature 32°C for either 1 min (d-i) or 6 minutes (j-l). Cells were fixed and then processed for standard IF using monoclonal antibodies against VSVG (a,d,g,j) and polyclonal antibodies against either COPII (c,f) or GBF1 (i,l). Middle panels (b,e,h,k) show merged left and right images. Bar, 5 µm.

View larger version (118K): [in a new window]   Fig. 9. Microinjection of anti-GBF1 antibodies specifically causes membrane dissociation of the COPI but not the clathrin coat. HeLa cells were microinjected with affinity-purified polyclonal anti-GBF1 antibodies (9D2), and fixed 2 hours post-injection. Microinjected cells, identified using goat anti-rabbit antibody, are indicated by white stars. COPI or clathrin coats were revealed using anti-ß-COP (M3A5) or anti-clathrin (X22) monoclonal antibodies. Bars, 10 µm.

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