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First published online 23 October 2007
doi: 10.1242/jcs.010769

Journal of Cell Science 120, 3929-3940 (2007)
Published by The Company of Biologists 2007
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Dissecting the role of the ARF guanine nucleotide exchange factor GBF1 in Golgi biogenesis and protein trafficking

Tomasz Szul1, Robert Grabski1, Susan Lyons1, Yuichi Morohashi2, Svetlana Shestopal1, Martin Lowe2 and Elizabeth Sztul1,*

1 Department of Cell Biology, University of Alabama at Birmingham, 1918 University Blvd, Birmingham, AL 35924, USA
2 University of Manchester, Faculty of Life Sciences, Manchester M13 9PT, UK


Figure 1
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  		Fig. 1. GBF1 depletion causes COPI dissociation from membranes. (A) Human GBF1 isoforms showing insertions ({triangledown}) and deletions ({Delta}). Shown are the positions of the two siRNAs and the two antibodies against GBF1 used in this study. Both siRNAs target all GBF1 isoforms. Both antibodies recognize epitopes present in all GBF1 isoforms. HeLa cells (B-D,F) or NRK cells (E) were untreated (`con'; control), transfected with reagent alone (`mock'), transfected with siRNA against GBF1 (`siRNA') or transfected with scrambled siRNA (`scr'). After the indicated times (B,C) or after 72 hrs (D,E), cell lysates were western blotted with antibodies against the indicated proteins. GBF1 is efficiently and selectively depleted from HeLa and NRK cells. (F) HeLa cells were transfected with siRNA against GBF1 for 72 hours and then stained with the indicated antibodies. Depletion of GBF1 correlates with dissociation of β-COP. Bars, 16 µm.

 

Figure 2
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  		Fig. 2. GBF1 depletion causes tubulation of the cis-Golgi. (A-G) HeLa cells transfected with #2siRNA against GBF1 for 72 hours were stained with antibodies against the indicated proteins. Depletion of GBF1 does not cause collapse of the ERGIC and the Golgi but leads to extensive tubulation of the cis-Golgi. The level of cis-Golgi tubulation correlates with the level of GBF1 depletion (G). (H) HeLa cells transfected with #1siRNA against GBF1 for 72 hours were stained with antibody against GM130. Analogous extensive tubulation of the cis-Golgi is observed with siRNAs #1 and #2. Bars, 16 µm.

 

Figure 3
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  		Fig. 3. GBF1 co-precipitates with ARF1 and ARF4 in vivo. HeLa cells were incubated with or without 5 µg/ml BFA for 45 minutes. Cell lysates were immunoprecipitated using antibodies against GBF1 and Rab8, and the immunoprecipitates (IPs) were analysed by western blotting with antibodies specific for ARF isoforms. ARF1 and ARF4 co-immunoprecipitate with GBF1 in the presence of BFA.

 

Figure 4
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  		Fig. 4. Golgi tubules connect peripheral ERGIC outposts. (A-C) NRK cells were transfected with siRNA against GBF1 for 72 hours and stained with the indicated antibodies. (A) A series of confocal planes of a GBF1-depleted cell shows the planar arrangement of the tubules. (B,C) A series of focal planes through a single cell shows that the GM130 tubules do not intersect with ERES (ER exit site) but do connect to ERGIC53-containing elements. Bars, 16 µm. (D-G) HeLa cells transfected with siRNA against GBF1 for 48 hours were co-transfected with GFP-tagged GRASP65 and YFP-tagged ERGIC53 and live-imaged 24 hours later. A single frame is presented in (D). A collection of consecutive frames shows stability of the punctate ERGIC53-containing elements (E) and the flow of GRASP65 through the ERGIC (F). Tubules often connect adjacent ERGIC elements (G). In these images, yellow was digitally changed to red for ease of viewing.

 

Figure 5
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  		Fig. 5. Trafficking of transmembrane proteins is inhibited in GBF1-depleted cells. (A-C) HeLa cells were transfected with scrambled siRNA (scr) or siRNA against GBF1 for 48 hours. Cells were then transfected with ts045VSV-G-GFP and cultured for an additional ~12 hours at 42°C. Cells were shifted to the permissive temperature of 32°C for 0, 1 or 2 hours. At each time point, cells were processed for immunofluorescence with antibodies against GM130. VSV-G is visualized by green fluorescence. GBF1-depleted cells (indicated by asterisks) are visualized by tubulation of GM130. In GBF1-depleted cells, trafficking of VSV-G is inhibited. (D) The kinetics of VSV-G processing to the Endo-Hf-resistant Rt form were quantitated from fluorographs as described in Materials and Methods and are presented as the percentage of total VSV-G present at each time point. A significant reduction in VSV-G trafficking in GBF1-depleted cells is observed. (E-G) HeLa cells were untreated (E), transfected with scrambled siRNA (F) or siRNA against GBF1 (G) for 72 hours and stained with the indicated antibodies. In control cells, ESL-1 colocalizes with fibronectin in thin connections between cells. Similar surface localization is seen in cells transfected with scrambled siRNA. By contrast, surface expression of ESL-1 is reduced in GBF1-depleted cells. Bars, 16 µm. (H) HeLa cells were untreated (`con'; control) or transfected with scrambled siRNA (`scr') or siRNA against GBF1 (`siRNA') for 72 hours. Cells were lysed, and lysates were western blotted with the indicated antibodies. The level of ESL-1 is decreased in GBF1-depleted cells. (I,J) HeLa cells were transfected with scrambled siRNA (`scr') or with siRNA against GBF1 (`siRNA'). After 72 hours, cells were labelled with 35S-Met/Cys for 30 minutes and chased for the indicated times. At each time point, cells were lysed. Lysates were mock treated or treated with Endo-Hf, and ESL-1 was precipitated and analysed by SDS-PAGE and fluorography. In scr cells, immature ESL-1 (red arrowhead) is processed to a terminally glycosylated Endo-H-resistant form (green arrowhead). In GBF1-depleted cells, ESL-1 remains in the immature form (red arrowhead). The levels of ESL-1 remaining during the chase were quantitated and presented as the ESL-1 present at 1 hour of chase expressed as a percentage of that present at 0 hours of chase. The data show significant degradation of ESL-1 in GBF1-depleted cells.

 

Figure 6
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  		Fig. 6. Soluble proteins are secreted from GBF1-depleted cells. (A-C) NRK cells were transfected with reagent alone (`mock' lanes) or with siRNA against GBF1 (`siRNA' lanes) for 72 hours. Untransfected cells were treated with 5 µg/ml BFA for 30 min (`+BFA' lanes). All cells were pulsed for 30 minutes with 35S-Met/Cys and then chased for 4 hours. At that point, media were collected and cells were lysed. Aliquots of media (B) and lysates (A) were analysed by SDS-PAGE and fluorography. Secretion from BFA-treated cells is inhibited, but proteins are efficiently secreted from control and GBF1-depleted cells. Aliquots of lysates were western blotted to confirm GBF1 depletion (C). (D,E) NRK cells were transfected with scrambled siRNA (`scramble' lanes) or with siRNA against GBF1 (`siRNA' lanes) for 72 hours. Untransfected cells were treated with 5 µg/ml BFA for 30 min (`+BFA' lanes). All cells were pulsed for 30 minutes with 35S-Met/Cys and chased for the indicated times. At each time point, media were collected and cells were lysed. Aliquots of media and lysates were precipitated with acid to obtain total protein-incorporated radioactivity. The amount of secreted protein at each time is represented as a percentage of the radioactivity in the medium and the lysate. Secretion from BFA-treated cells is inhibited, but proteins are efficiently secreted from control and GBF1-depleted cells; n=3 (error bars represent the s.d.). Aliquots of lysates were western blotted to confirm the depletion of GBF1 (E). (F-J) HeLa cells were transfected and treated as in A-C. Secretion from BFA-treated cells is inhibited, but proteins are efficiently secreted from control and GBF1-depleted cells. Aliquots of lysates were western blotted to confirm the depletion of GBF1 (H). Media (from G) were immunoprecipitated with antibodies against fibronectin and laminin, and the precipitates were analysed by SDS-PAGE and fluorography (I,J). Fibronectin and laminin are efficiently secreted from GBF1-depleted cells. (K-N) HeLa cells were transfected and labelled as in D,E. At each time point, media were collected and cells were lysed. Media and lysates were immunoprecipitated with antibodies against fibronectin, and the precipitates analysed by SDS-PAGE and fluorography (K-M). Fibronectin is not secreted from BFA-treated cells but is secreted efficiently from GBF1-depleted cells. Aliquots of lysates were western blotted to confirm depletion of GBF1 (N). (O) HeLa cells were transfected and labelled as in D-E. At each time point, media were collected and cells were lysed. Aliquots of media and lysates were immunoprecipitated with antibodies against fibronectin. The precipitates were analysed by SDS-PAGE and fluorography and quantitated by densitometry. The amount of secreted fibronectin at each time is represented as a percentage of the fibronectin in the medium and the lysate. Proteins are efficiently secreted from control and GBF1-depleted cells; n=2 (error bars represent the s.d.).

 

Figure 7
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  		Fig. 7. A functional secretory pathway is maintained in GBF1-depleted cells. (A) GBF1 depletion inhibits COPI recruitment to membranes and leads to an increased frequency of tubular connections between the ERGIC and the cis-Golgi. Despite the block in COPI recruitment, a functional pathway capable of trafficking soluble cargo proteins is maintained in GBF1-depleted cells. However, trafficking of transmembrane cargo proteins is inhibited without COPI-mediated events. (B) In control cells, tubular connections between the ERGIC and the cis-Golgi facilitate trafficking of soluble proteins by a COPI-independent mechanism. The tubules might also transit transmembrane proteins by a COPI-requiring mechanism.

 

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© The Company of Biologists Ltd 2007