The Arabidopsis ADP-ribosylation factor (ARF) and ARF-like (ARL) system and its regulation by BIG2, a large ARF–GEF

Abstract

ADP-ribosylation factor (ARF) and ARF-like (ARL) proteins are small GTPases that undergo a GDP/GTP nucleotide exchange cycle. ARF proteins are important regulators of cellular trafficking. Knowledge of plant ARF proteins and their regulators is emerging from genetic and cellular studies. The Arabidopsis BIG2 ARF–guanine nucleotide exchange factor (GEF) gene is expressed in several tissues and the encoded protein is a typical Arabidopsis ARF–GEF. We address here the specificity of BIG2 for different Arabidopsis ARF proteins. The in vitro effect of the catalytic Sec7 domain of BIG2 on the guanine nucleotide exchange rate of five ARF and ARL proteins was measured using real time fluorescence spectroscopy. The Sec7 domain catalyzed nucleotide exchange on ARF1, but had essentially no effect on the exchange rate of ARF8, ARF9, ARL1, and ARL8a. In Western blots with an anti-BIG2 polyclonal antibody, a BIG2 fraction was detected in membranes, especially those deriving from the Golgi apparatus. The activity of the BIG2 Sec7 domain was unaffected by the Sec7 inhibitor brefeldin A, suggesting that BIG2 is a BFA-insensitive GEF for ARF1 or a close homolog. These studies contribute to our understanding of the biochemical and physiological specificity of ARF and ARF–GEF interactions in plants.