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doi: 10.1242/10.1242/jcs.00093

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Inactivation of G_z causes disassembly of the Golgi apparatus

Masami Nagahama, Shihomi Usui, Takashi Shinohara, Tomohiro Yamaguchi^*, Katsuko Tani and Mitsuo Tagaya

School of Life Science, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo 192-0392, Japan
^* Present address: Department of Life Science, Graduate School and Faculty of Science, Himeji Institute of Technology, Ako-gun, Hyogo 678-1297, Japan

View larger version (43K): [in a new window]   Fig. 1. Dispersal of ß-COP in cells expressing RGSZ1. HeLa cells were transfected with a plasmid for 6xHis-RGSZ1. The cells were allowed to express RGS proteins for 14 hours, fixed, and then processed for ß-COP staining (a,c). Expressed RGSZ1 was detected with the monoclonal anti-tetra-His antibody (b,d). Arrows indicate RGSZ1-expressing cells with dispersed ß-COP staining. Bar, 20 µm.

View larger version (25K): [in a new window]   Fig. 2. Disassembly of the Golgi apparatus is caused by Gz-selective RGSZ1 and RGSZ2, but not by other RGS proteins. (A) HeLa cells were transfected with a plasmid encoding N-terminal FLAG-tagged RGSZ1 (a,b), RGSZ2 (c,d), GAIP (e,f), RGS2 (g,h), RGS3 (i,j), or RGS4 (k,l). At 20 hours after transfection, the cells were fixed, and then double-stained with the anti-ß-COP antibody (a,c,e,g,i,k) and anti-FLAG antibody (b,d,f,h,j,l). Arrows indicate cells expressing RGS proteins. Bar, 20 µm. (B) The percentage of transfected HeLa cells with a dispersed ß-COP staining was determined. The results are expressed as the percentage of the number of cells with a dispersed Golgi apparatus and as the mean±s.e.m. for at least three separate experiments.

View larger version (33K): [in a new window]   Fig. 3. Disassembly of the Golgi apparatus in cells expressing RGSZ1. HeLa cells (a-h) and BHK cells (i-l) were transfected with a plasmid encoding N-terminal FLAG-tagged RGSZ1. At 20 hours after transfection, the cells were fixed, and then double-stained with the anti-FLAG antibody (b,d,f,h,j,l) and antibodies to the various Golgi proteins indicated (a,c,e,g,i,k). Arrows indicate RGSZ1-expressing cells with a dispersed Golgi apparatus. Bar, 20 µm.

View larger version (50K): [in a new window]   Fig. 4. Cellular structures and functions other than the Golgi apparatus are not markedly affected by the expression of RGSZ1. A plasmid for 6xHis-RGSZ1 was transfected into HeLa cells. At 14 hours after transfection, the cells were fixed, and then processed for indirect immunofluorescence. The panels show the localization of tubulin (a), NADH-cytochrome P-450 reductase (FP2; c), and FITC-transferrin (Tf-FITC; e). For the FITC-transferrin uptake experiment, HeLa cells transfected with 6xHis-RGSZ1 were incubated with FITC-transferrin at 37°C for 1 hour. The cells were then processed for immunofluorescence. 6xHis-RGSZ1-expressing cells were detected with the anti-tetra-His antibody (b,d,f). Arrows indicate RGSZ1-expressing cells. Bar, 20 µm.

View larger version (26K): [in a new window]   Fig. 5. Effect of RGSZ1 expression on intracellular trafficking of VSVG-GFP. HeLa cells were transfected with an expression plasmid for VSVG-GFP with a control vector or a FLAG-RGSZ1 expression plasmid. The cells were incubated for 20 hours at 40°C, and then the temperature was changed to 32°C. At the indicated times the cells were fixed for immunofluorescence analysis. RGSZ1-expressing cells were identified by using the anti-FLAG antibody. The compartment to which VSVG-GFP had been transported at the indicated times after the temperature shift was determined. ER, G, FG and PM denote ER, Golgi, fragmented Golgi and plasma membrane, respectively. Values are expressed as a percentage of the total number of cells expressing both VSVG-GFP and RGSZ1. More than 100 cells were scored for each time point.

View larger version (52K): [in a new window]   Fig. 6. Comparison of the distribution of VSVG-GFP with Golgi-resident proteins. BHK cells (a-c) or HeLa cells (d-i) were co-transfected with expression plasmids for VSVG-GFP and FLAG-RGSZ1. The cells were incubated for 20 hours at 40°C, and then the temperature was changed to 32°C. After 1 hour at 32°C, the cells were fixed and then stained with antibodies to Man II (b), ß-COP (e), or ERGIC-53 (h). Merged images are presented on the right (c,f,i). Bar, 10 µm.

View larger version (42K): [in a new window]   Fig. 7. Disruption of the Golgi apparatus by RGSZ1 depends on its Gz binding and GAP function. (A) Lack of the interaction of the mutated RGSZ1 with Gz(QL). The wild-type FLAG-RGSZ1 or E116A/N117A mutant was co-expressed with Gz(QL) in HeLa cells. At 15 hours after transfection, the cells were fixed, and then double-stained with the anti-Gz antibody (a,d) and anti-FLAG antibody (b,e). Merged images are presented on the right (c,f). Bar, 20 µm. (B) HeLa cells transfected with wild-type RGSZ1 (WT; a,b) or mutant RGSZ1 cDNA (E116A/N117A; c,d) were double-stained for FLAG (a,c) and ß-COP (b,d). Arrows indicate RGSZ1-expressing cells. (C) Western blot of extracts of cells expressing the wild-type FLAG-RGSZ1 or E116A/N117A mutant. The cell extracts were subjected to SDS-polyacrylamide gel electrophoresis followed by western blotting using the anti-FLAG antibody. Bands were detected using enhanced chemiluminescence reagents.

View larger version (45K): [in a new window]   Fig. 8. Expression of a dominant-negative Gz results in Golgi disruption. HeLa cells were transfected with wild-type Gz (a,b) or a triple mutant (G204A/E246A/A327S) (c,d). The cells were allowed to express Gz for 20 hours, fixed, and then double-stained with the anti-Gz antibody (a,c) and anti-membrin antibody (b,d). Arrows indicate cells overexpressing the mutated Gz with a dispersed Golgi apparatus. Bar, 20 µm.

View larger version (50K): [in a new window]   Fig. 9. Golgi localization of Gz. BHK cells (a-j) and Clone9 cells (c-l) were fixed and then subjected to fluorescence microscopic analysis. The rabbit polyclonal anti-Gz antibody and mouse monoclonal anti-Man II antibody were used to visualize Gz (a,c,e,g,i,k) and Man II (b,d,f,h,j,l), respectively. In panels e-h, the anti-Gz antibody was preincubated with the peptide used for immunization. In panels i to l, cells were preincubated with 10 µM BFA for 10 minutes (i,j) or 30 minutes (k,l) before fixation.

View larger version (62K): [in a new window]   Fig. 10. Semi-quantitative RT-PCR analysis of Gz mRNA from cultured cells. Total cellular RNA (1 µg) was subjected to the RT-PCR reaction, representative ethidium bromide-stained gels of the Gz and GAPDH RT-PCR products being shown. Size standards are indicated at the left.