Membrane trafficking maintains the organization of the eukaryotic cell and delivers cargo proteins to their subcellular destinations, such as sites of action or degradation. The formation of membrane vesicles requires the activation of the ADP-ribosylation factor ARF GTPase 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 form dimers in vivo. This feature is conserved across eukaryotes, although its biological significance is unknown. Here, we demonstrate the proximity of ARF1•GTPs in vivo by fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy, mediated through coordinated activation by dimers of Arabidopsis (Arabidopsis thaliana) ARF-GEF GNOM, which is involved in polar recycling of the auxin transporter PIN-FORMED1. Mutational disruption of ARF1 spacing interfered with ARF1-dependent trafficking but not with coat protein recruitment. A mutation impairing the interaction of one of the two SEC7 domains of the GNOM ARF-GEF dimer with its ARF1 substrate reduced the efficiency of coordinated ARF1 activation. Our results suggest a model of coordinated activation-dependent membrane insertion of 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.

膜运输维持真核细胞的组织并将货物蛋白递送至其亚细胞目的地，例如作用或降解位点。膜囊泡的形成需要 ARF 鸟嘌呤核苷酸交换因子 (ARF-GEF) 的 SEC7 结构域激活 ADP-核糖基化因子 ARF GTPase，从而导致 GTP 结合的 ARF 募集外壳蛋白。尽管 ARF-GEF 在体内形成二聚体，但体外交换测定是用单体蛋白进行的。这一特征在真核生物中是保守的，尽管其生物学意义尚不清楚。在这里，我们通过荧光共振能量转移-荧光寿命成像显微镜证明了 ARF1•GTP 在体内的接近性，通过拟南芥 ( Arabidopsis thaliana)二聚体的协调激活介导) ARF-GEF GNOM，参与生长素转运蛋白 PIN-FORMED1 的极性回收。ARF1 间距的突变破坏会干扰 ARF1 依赖性运输，但不会干扰外壳蛋白的招募。损害 GNOM ARF-GEF 二聚体的两个 SEC7 结构域之一与其 ARF1 底物相互作用的突变降低了协调 ARF1 激活的效率。我们的结果提出了包被膜囊泡形成所需的ARF1·GTP分子的协调激活依赖性膜插入模型。考虑到 ARF 和 ARF-GEF 的进化保守性，膜运输的这一初始调控步骤很可能普遍发生在真核生物中。