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First published online May 4, 2004
doi: 10.1242/10.1242/jcs.01090

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The GDP-bound form of Arf6 is located at the plasma membrane

¹ Institut de Pharmacologie Moléculaire et Cellulaire, CNRS-UMR 6097, 660 route des Lucioles, 06560 Valbonne Sophia-Antipolis, France
² Laboratoire d'Enzymologie et de Biochimie Structurales, CNRS, 1, avenue de la Terrasse, 91198 Gif sur Yvette, France
³ Institut de Pharmacologie Moléculaire et Cellulaire, CNRS-UMR 6097, 660 route des Lucioles, 06560 Valbonne-Sophia-Antipolis, France

View larger version (32K): [in a new window]   Fig. 1. Representation of the structural positions of the Thr27, Thr44 and Gln67 residues of Arf6 in both GDP and GTP conformations based on crystallographic studies of Arf6-GDP (PDB entry 1EOS) (Menetrey et al., 2000) and Arf6-GTPS (PDB entry 1HFV) (Pasqualato et al., 2001). Thr27 residue (in green) is involved in the binding of the magnesium ion (in copper) and the ß phosphate of the two nucleotides. Gln67 (in brown) from the switch II region and Thr44 (in purple) from the switch I region undergo a large conformational change between the two states that repositions them near the phosphate in the GTP state. Thr44 is involved in the binding of the phosphate and the magnesium ion.

View larger version (21K): [in a new window]   Fig. 2. T27N mutation decreases the affinity of Arf6 for both GDP and GTPS and destabilizes the protein. T44N mutation affects the binding of GTPS but not of GDP. (A,B) Arf6 wt(), Arf6-T27N() or Arf6-T44N() were loaded for 40 minutes at 37°C with [3H]-GDP (A) or [35S]-GTPS (B) in the presence of 1 µM free Mg2+. Then, the free Mg2+ concentration was raised to 1 mM and [3H]-GDP and [35S]-GTPS dissociations were initiated by adding 1 mM unlabeled GDP or GTPS, respectively. The curves are best fits assuming first-order kinetics for nucleotide dissociation; nucleotide dissociation rates are shown on the left. (C) Arf6 T27N () or T44N () were loaded at 37°C with [3H]-GDP in the presence of 1 µM free Mg2+. Then, the free Mg2+ concentration was raised to 1 mM and, without isotopic dilution, the radioactivity bound to the proteins was measured over time. (D) Arf6-T27N or Arf6-T44N (5 µM) was incubated in the presence of 50 µM GDP and 1 mM free Mg2+ for 2 hours at 37°C. After ultracentrifugation, pellet (P) and supernatant (S) were analysed by SDS-PAGE and Coomassie Blue staining. The distribution (as percentages) of Arf between pellet and supernatant is indicated.

View larger version (55K): [in a new window]   Fig. 3. Arf6(T27N) localizes to detergent-insoluble structures reminiscent of aggresomes. (A,B) When expressed in cells, Arf6(T27N) is essentially insoluble even in the presence of detergent. Cells were transfected with expression plasmids encoding HA-tagged Arf6-wt and the T27N, T44N and Q67L mutants. (A) The cells were homogenized and the post-nuclear supernatants were centrifuged at 100,000 g to give a membrane (m) and a cytosolic (c) fraction. (B) BHK-21 cells were lysed at 4°C in Triton-X-100-containing buffer and centrifuged to separate Triton-X-100-soluble (s) and -insoluble (i) fractions. The different fractions were immunoblotted using anti-HA mouse monoclonal antibody. (C) Expression of Arf6(T27N)-EGFP induces the formation of punctate structures that are regrouped near the nucleus over time in a microtubule-dependent manner. BHK-21 cells were transfected with Arf6(T27N)-EGFP constructs and fixed at different time points. Cells were treated for 2 hours with 30 µM nocodazole before fixation (d). 48 hours after transfection, cells were probed with anti-vimentin antibodies (e-g). At high expression levels, perinuclear Arf6(T27N)-EGFP structures (e) are surrounded with a vimentin ring (f, arrowheads). A merged image is presented (g).

View larger version (33K): [in a new window]   Fig. 4. Arf6(T27N) acts as a dominant-negative mutant both in vitro and in vivo by trapping the exchange factor EFA6. (A) In vitro Arf6(T27N) inhibits EFA6-catalysed activation of Arf6-wt. Kinetics of GDP to GTP exchange on Arf6 were monitored by the correlated variation in tryptophan fluorescence. Purified Arf6 was preloaded with GDP and injected at 0.5 µM in the fluorescence cuvette containing azolectin vesicles (0.4 g l–1) in HKM buffer at 30°C. When indicated, purified EFA6 (100 nM) and increasing concentration of Arf6(T27N) (0-1 µM) were injected, followed by the addition of GTP (200 µM). For better clarity, we have only represented the curve at 1 µM of Arf6(T27N). For each experiments the value of the fold stimulation is plotted. The fold stimulation is the ratio of the EFA6-catalysed over the spontaneous exchange rate. (B) Co-expression with VSV-G-EFA6 induces the redistribution of Arf6(T27N) to the plasma membrane. BHK-21 cells were transfected with VSV-G-EFA6 (a) or Arf6(T27N)-HA (b) or both (c,d). After fixation, the cells were probed with anti-VSV-G antibody to detect EFA6 (a,c) and with anti-HA antibody to detect Arf6 (b,d). The co-expression of Arf6(T27N) and EFA6 induces plasma membrane extensions where they colocalize. Arrows indicate some zones of costaining. (C) EFA6 co-immunoprecipitates with Arf6(T27N). Lysates of BHK-21 cells untransfected or transiently expressing Arf6(T27N)-Myc, EGFP-EFA6 or both were immunoprecipitated (IP) with an anti-Myc antibody. Immunoprecipitates were resolved on SDS-PAGE, blotted on nitrocellulose membranes and probed with an anti-GFP antibody to detect EGFP-EFA6. 5% of the input (cell lysates) was also immunoblotted with anti-GFP and anti-Myc antibodies to ensure that EGFP-EFA6 and Arf6(T27N) respectively were expressed.

View larger version (22K): [in a new window]   Fig. 5. In vivo, expression of EFA6 leads to the activation of Arf6-wt but not of Arf6(T44N). (A) BHK-21 cells were transfected with expression plasmids encoding Myc-tagged Arf6-wt, or Arf6(T44N) alone or in combination with a plasmid encoding EGFP-EFA6 and grown in the presence of [32P]-orthophosphate. After immunoprecipitation with the anti-Myc monoclonal antibody, the bound nucleotides were determined by thin-layer chromatography and subjected to autoradiography. The position of GDP and GTP are indicated. (bottom) Quantification of the results from two independent experiments (mean ± SD). (B) BHK-21 cells were transfected with different Arf6 constructs alone or in combination with EGFP-EFA6. Arf6-GTP was isolated by incubation with a GST fusion containing an ARF6 effector domain.

View larger version (82K): [in a new window]   Fig. 6. Arf6-GDP localizes with EFA6 and ezrin in actin- and PIP2-enriched structures of the plasma membrane. BHK-21 cells were transfected with Arf6(T44N)-HA alone or with GFP-Rab5, GFP-Rab7, PH-PLC-GFP or PH-EFA6-GFP. After fixation, the cells were probed with anti-HA antibody to detect Arf6, in combination with phalloidin, anti-Giantin, anti-ezrin, anti-Tfn-R or anti-clathrin-heavy-chain antibodies and analysed by xy or xz (for WGA and anti-Giantin labelling) confocal sectioning. The cell surface was stained using Texas-red-conjugated WGA. Arrows indicate some zones of co-staining. Scale bars, 15 µm.

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