Figure 8.

Effect of Secdin on ARF1 Activation.

(A) Inhibitory efficiency of BFA (50 µM) and Secdin (50 µM) on purified Arabidopsis BIG5 Sec7 protein construct.

(B) Spontaneous (EDTA) and human GEF-stimulated ARF1 activation in the presence of Secdin. Recombinant human protein constructs comprising the Sec7 domain of BIG1 (BIG^Sec7), ARNO (ARNO^Sec7), and BRAG2 (BRAG2^Sec7), the Sec7 domain of BRAG2 followed by phospholipid binding PH domain (BRAG2^Sec7-PH), as well as the full-length ARNO protein (ARNO^FL) were assayed. The chemical impact was analyzed in vitro by monitoring the change in tryptophan fluorescence that follows the conformational alteration from ARF-GDP to ARF-GTP.

All experiments in (A) and (B) were done with N-terminally truncated bovine ∆17ARF1 (identical to the human form). The spontaneous GDP/GTP exchange in Δ17ARF1 in the absence of a GEF is examined in (B) by addition of EDTA, which displaces the bound Mg²⁺ ion. Values are means from three independent experiments. Error bars indicate sd. *P < 0.05 (Student’s t test).