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First published online 7 August 2007
doi: 10.1242/jcs.014225

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Analysis of GTPase-activating proteins: Rab1 and Rab43 are key Rabs required to maintain a functional Golgi complex in human cells

Alexander K. Haas^1,2, Shin-ichiro Yoshimura^1,2, David J. Stephens³, Christian Preisinger^2,*, Evelyn Fuchs¹ and Francis A. Barr^1,2,

¹ Cancer Research Centre, University of Liverpool, 200 London Road, Liverpool, L9 3AT, UK
² Department of Cell Biology, Max-Planck-Institute of Biochemistry, Martinsried 82152, Germany
³ Department of Biochemistry, University of Bristol, Bristol, UK

View larger version (40K): [in this window] [in a new window]   Fig. 1. A screen for TBC domain proteins that alter Golgi structure. (A-C) HeLa and hTERT-RPE1 cells were transfected with the GFP-tagged wild-type and catalytically inactive point mutant TBC-domain proteins listed in the figure. After 24 hours they were fixed, and then stained with antibodies to GM130 or TGN46. (A) The number of cells displaying a fragmented Golgi was then counted (n>100 per condition), expressed as a percentage, and then plotted as a bar graph; grey bars, wild-type (WT) GAPs; open bars, inactive mutants. (B) Cells triply stained for the expressed wild-type (WT) TBC1D20 and RN-tre, TGN46 and GM130 are shown. (C) Cells triply stained for the expressed catalytically inactive mutant (RA) TBC1D20 R105A and RN-tre 150A, TGN46, and GM130 are shown. Bars, 10 µm, in all panels.

View larger version (48K): [in this window] [in a new window]   Fig. 2. A screen for TBC domain proteins that alter ERGIC structure. (A-C) HeLa cells were transfected with the GFP-tagged wild-type and catalytically inactive point mutant TBC-domain proteins listed in the figure. After 24 hours they were fixed, and then stained with antibodies to ERGIC53. (A) The number of cells displaying a fragmented ERGIC was then counted (n>100 per condition), expressed as a percentage, and then plotted as a bar graph: grey bars, wild-type GAPs; open bars, inactive mutants. (B) Cells stained for the expressed wild-type (WT) TBC1D20 and RN-tre, and ERGIC53 are shown. (C) Cells stained for the expressed catalytically inactive mutant (RA) TBC1D20 R105A and RN-tre R150A and ERGIC53 are shown. Bars, 10 µm, in all panels.

View larger version (70K): [in this window] [in a new window]   Fig. 3. TBC1D20 causes a `loss of Golgi' phenotype. HeLa cells transfected with GFP-tagged (A) wild-type TBC1D20 (green), or (B) a catalytically inactive R105A point mutant (green) were fixed after 22 hours and then stained with antibodies to p115, golgin84, golgin97, and EEA1 (all in red) as indicated. (C) HeLa cells expressing GFP-tagged NAGT-I (green) were transfected with Myc-tagged wild-type TBC1D20 or the catalytically inactive R105A mutant (blue), fixed after 12 hours, and then stained with antibodies to GM130 (red) and the Myc-epitope tag. (D) To determine the levels of Golgi proteins, cell extracts were prepared from HeLa cells transfected for 24 hours with Myc-tagged wild-type TBC1D20 (WT) or the catalytically inactive R105A mutant (RA). Of these extracts, 20 µg were western blotted for GM130 and p115, the Myc-epitope to control for equal expression of the TBC1D20 constructs, and -tubulin as marker for equal loading. The asterisk indicates a GM130 breakdown product. (E) HeLa cells left untreated, expressing Myc-tagged TBC1D20 or the dominant-negative Sar1 H79G mutant for 24 hours, or treated with BFA for 30 minutes were fractionated. Equivalent amounts of the total lysate (T), membrane pellet (P), or soluble cytosolic fraction (S) were western blotted for p115, GM130, golgin84 and the Myc epitope. Bars, 10 µm, in all panels.

View larger version (54K): [in this window] [in a new window]   Fig. 4. TBC1D20 is a GAP for Rab1 and Rab2. Biochemical assays for GTP hydrolysis with the Rabs and GAPs indicated were performed as described previously (Fuchs et al., 2007; Haas et al., 2005). All reactions were carried out for 60 minutes. (A) To determine the specific activity towards a range of GTPases, 0.5 pmoles TBC1D20 were tested against 100 pmoles of the Rab GTPases indicated, as well as ARF1 and Sar1. The basal GTP hydrolysis seen with a buffer control was subtracted for each GTPase. (B) 5 pmoles of wild-type and R105A mutant TBC1D20 full-length or the TBC-domain only (amino acids 1-317) were tested against 100 pmoles wild-type Rab1 or the Rab1 Q67L hydrolysis-defective mutant. Basal GTP hydrolysis seen with buffer is plotted as open bars, and GAP-stimulated GTP hydrolysis as grey bars. (C) Buffer (control) or 0.5 pmoles of the GAP indicated were tested against 100 pmoles Rabs 1 and 2. Basal GTP hydrolysis seen with buffer is plotted as open bars, and GAP-stimulated GTP hydrolysis as grey bars.

View larger version (46K): [in this window] [in a new window]   Fig. 5. Increased p115 staining at the Golgi in TBC1D20-depleted cells. (A) HeLa cells transfected with GFP-tagged dominant-negative Rabs were fixed after 24 hours, and then stained with antibodies to GM130. The number of cells displaying a fragmented Golgi (open bars) or scattered COPII staining (filled bars) was then counted (n>100 per condition), expressed as a percentage, and plotted as a bar graph. (B) HeLa cells transfected with GFP-tagged Rab1, dominant active Rab1Q67L, and dominant-negative Rab1 N121I or Rab43 T32N were fixed after 24 hours, and then stained with antibodies to GM130 or COPII (red). Rabs were visualized by GFP fluorescence (green), and DNA was stained with DAPI. (C) Cell lysates from HeLa cells expressing GFP-tagged TBC1D20 treated with either control or TBC1D20-specific siRNA duplexes were western blotted for GFP to detect TBC1D20, and -tubulin as a loading control. (D) HeLa cells expressing the GFP-tagged R105A inactive form of TBC1D20 (green) as a marker for the efficiency of RNA interference were treated with either control or TBC1D20-specific siRNA duplexes for 72 hours. The cells were then fixed and stained with antibodies to COPII, p115 or GM130 (all in red). DNA was stained with DAPI (blue). Bars, 10 µm.

View larger version (65K): [in this window] [in a new window]   Fig. 6. Rab1 is the only Rab essential for ER to cell surface transport. (A,B) VSV G transport assays were performed as described in the Materials and Methods on HeLa cells expressing the wild-type and catalytically inactive arginine finger point mutant TBC-domain proteins indicated. The cell surface appearance of VSV G was measured for all conditions (A). The bar graph (B) shows the inhibition in transport observed with the various wild-type (grey bars), and catalytically inactive (open bars) TBC-domain proteins. (C) VSV G transport assays were performed in HeLa cells expressing Myc-tagged wild-type (WT) or catalytically inactive arginine finger point mutant (R105A) TBC1D20. After 60 minutes of transport cells were fixed and surface stained for VSV G (red), then permeabilized and stained for the Myc epitope (blue). Total VSV G was observed by GFP fluorescence (green) at all time points. (D) VSV G transport assays were performed in HeLa cells treated with control, Rab1, p115, or GM130 siRNA duplexes for 72 hours. After 60 minutes of transport cells were fixed and surface stained for VSV G (red), and total VSV G was observed by GFP fluorescence (green). (E) VSV G transport assays were performed in HeLa cells expressing Myc-tagged wild-type (WT), dominant-negative (N121I) or constitutive active (Q67L) point mutants of Rab1. After 60 minutes of transport the cells were fixed and surface stained for VSV G (red), then permeabilized and stained for the Myc epitope (blue). Total VSV G was observed by GFP fluorescence (green). Bars, 10 µm.

View larger version (47K): [in this window] [in a new window]   Fig. 7. Loss of perinuclear ERES in TBC1D20-expressing cells. (A) HeLa cells transfected with GFP-tagged wild-type TBC1D20 or a catalytically inactive R105A point mutant (green) were fixed after 22 hours and then stained with antibodies to the COPII subunit Sec31 (red). DNA was stained with DAPI (blue). The clustering of COPII structures in the perinuclear region, or TBC1D20-induced scattering was counted and the results are show in the bar graph below (100 cells per experiments, n=2). Bar, 10 µm, in all panels. (B) FRAP was performed on cells expressing Myc-TBC1D20 and Venus-Sec16 using a Leica TCS SP3 AOBS scanning confocal microscope with a pinhole size of 2 Airy units, eightfold line averaging, at 1 frame per second, and region-of-interest bleaching with 10 iterations of the 514 nm laser at 100% AOTF power. Cells used for photobleaching were confirmed to express Myc-TBC1D20 by subsequent immunofluorescence; 95% of all cell transfected with Venus-Sec16 were found to be transfected with Myc-TBC1D20. Images taken from the time points indicated were quantified using ImageJ. Plot of the average intensity of individual structures over time, error bars indicate the standard deviation (five cells per experiment, n=3).

View larger version (91K): [in this window] [in a new window]   Fig. 8. Loss of recycling ERGIC markers from ERES in TBC1D20-expressing cells. HeLa cells expressing GFP-tagged p24 (green) were transfected with wild-type (WT) or catalytically inactive (RA) Myc-tagged TBC1D20 (blue). After 18 hours cells were fixed and then stained with antibodies to either (A) the ERGIC marker ERGIC53 or (B) the Sec31 subunit of the COPII vesicle coat (red), the Myc epitope (blue). GFP was visualized directly (green). The enlargements correspond to 10x10 µm regions of the merged images. Bar indicates 10 µm in all panels.

View larger version (52K): [in this window] [in a new window]   Fig. 9. TBC1D20 is an ER membrane protein. (A) A schematic showing the proposed topology of TBC1D20. Carbonate extraction experiments were performed as described in the Materials and Methods to investigate the membrane association of TBC1D20. Golgin84 and GM130 were taken as representative integral and peripheral membrane proteins, respectively. The topology of TBC1D20 was investigated using proteinase K (PK) digestion experiments in the presence or absence of Triton X-100 (TX). TGN46 and Golgin84 were taken as controls for proteins with large lumenal or cytosolic domains, respectively. (B) HeLa cells transfected with Myc-tagged TBC1D20 and a series of C-terminal deletions were fixed after 24 hours, and then stained with antibodies to GM130 (green) and the Myc epitope (red). The reticular or cytosolic nature of TBC1D20 constructs is more clearly visible in the enlargements. (C) HeLa cells expressing the GFP-tagged R105A mutant of TBC1D20, or a series of N-terminal deletions were fixed after 24 hours, and then stained with antibodies to GM130 (red). TBC1D20 constructs were visualized by GFP fluorescence (green). DNA was stained with DAPI (blue). Bars, 10 µm in all panels. (D) A summary of the TBC1D20 constructs used and results obtained in B and C. Activity refers to the ability to cause the `loss of Golgi' phenotype; localization of the different constructs is scored as ER, including nuclear envelope, and Golgi. The TBC domain is marked in red, and the putative transmembrane domain in green.

View larger version (56K): [in this window] [in a new window]   Fig. 10. Reticulon interacts with and modulates TBC1D20 function at the ER. (A) A schematic of reticulon 1 variant 2 (reticulon 1v2) showing the two hydrophobic regions (HR), which by analogy with other reticulons may form transmembrane or membrane anchoring sequences (Voeltz et al., 2006). The minimal binding fragment identified by yeast two-hybrid (Y2H) screening is shown in blue. Directed Y2H analysis was performed using reticulon 1v2 as the prey and full-length TBC1D20 and the various deletion constructs indicated. All combinations grow on non-selective media (–LW), whereas only a subset can grow on the selective media (QDO). GFP-immunoprecipitations were performed from HeLa S3 cells cotransfected with Myc-tagged reticulon, and the GFP-tagged TBC1D20 constructs indicated, using a published method (Voeltz et al., 2006). The immunoprecipitates were blotted with GFP and Myc antibodies to detect TBC1D20 and reticulon, respectively. (B) HeLa cells transfected with reticulon 1v2 (reticulon; green) were fixed after 24 hours, and then stained with antibodies to calnexin or GM130 (red). (C,D) HeLa cells expressing Myc-tagged wild-type, R105A catalytically inactive mutant TBC1D20, or the minimal TBC domain (amino acids 1-317) in the absence or presence of GFP-tagged reticulon 1v2 (reticulon) were fixed, and then stained for GM130 and antibodies to the Myc epitope. In some panels DNA was stained with DAPI. Bars, 10 µm. (D) Quantification of the cells showing intact Golgi, the `loss of Golgi' phenotype, or Golgi fragmentation in cells expressing TBC1D20 constructs and reticulon as indicated. (E) Western blots were performed to control for the expression levels of TBC1D20 and reticulon, with tubulin as a loading control.

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