Specificities for the small G proteins ARF1 and ARF6 of the guanine nucleotide exchange factors ARNO and EFA6

ARF1 and ARF6 are distant members of the ADP-ribosylation factor (ARF) smal l G-protein subfamily. Their distinct cellular functions must result from s pecificity of interaction with different effecters and regulators, includin g guanine nucleotide exchange factors (GEFs), ARF nucleotide-binding site o pener (ARNO), and EFA6 are analogous ARF-GEFs, both comprising a catalytic "Sec7" domain and a pleckstrin homology domain. In vivo ARNO, like ARF1, is mostly cytosolic, with minor localizations at the Golgi and plasma membran e; EFA6, like ARF6, is restricted to the plasma membrane. However, dependin g on conditions, ARNO appears active on ARF6 as well as on ARF1, Here we an alyze the origin of these ARF-GEF selectivities. In vitro, in the presence of phospholipid membranes, ARNO activates ARF1 preferentially and ARF6 slig htly, whereas EFA6 activates ARF6 exclusively; the stimulation efficiency o f EFA6 on ARF6 is comparable with that of ARNO on ARF1, These selectivities are determined by the GEFs Sec7 domains alone, without the pleckstrin homo logy and N-terminal domains, and by the ARF core domains, without the myris toylated N-terminal helix; they are not modified upon permutation between A RF1 and ARF6 of the few amino acids that differ within the switch regions. Thus selectivity for ARF1 or ARF6 must depend on subtle folding differences between the ARFs switch regions that interact with the Sec7 domains.