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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.
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