ARF1 dimerization is essential for vesicle trafficking and dependent on activation by ARF-GEF dimers in Arabidopsis
Membrane traffic maintains the organization of the eukaryotic cell and delivers cargo proteins to their subcellular destinations such as sites of action or degradation. Membrane vesicle formation requires ARF GTPase activation by the SEC7 domain of ARF guanine-nucleotide exchange factors (ARF-GEFs), resulting in the recruitment of coat proteins by GTP-bound ARFs. In vitro exchange assays were done with monomeric proteins, although ARF-GEFs have been shown to form dimers in vivo. This feature is conserved across the eukaryotes, however its biological significance is unknown. Here we demonstrate ARF1 dimerization in vivo and we show that ARF-GEF dimers mediate ARF1 dimer formation. Mutational disruption of ARF1 dimers interfered with ARF1-dependent trafficking but not coat protein recruitment in Arabidopsis. Mutations disrupting simultaneous binding of two ARF1*GDPs by the two SEC7 domains of GNOM ARF-GEF dimer prevented stable interaction of ARF1 with ARF-GEF and thus, efficient ARF1 activation. Our results suggest a model of activation-dependent dimerization of membrane-inserted ARF1*GTP molecules required for coated membrane vesicle formation. Considering the evolutionary conservation of ARFs and ARF-GEFs, this initial regulatory step of membrane trafficking might well occur in eukaryotes in general.