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) small G-protein subfamily. Their distinct cellular functions must result from specificity of interaction with different effectors and regulators, including guanine nucleotide exchange factors (GEFs). ARF nucleotide-binding site opener (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 membrane; EFA6, like ARF6, is restricted to the plasma membrane. However, depending on conditions, ARNO appears active on ARF6 as well as on ARF1. Here we analyze the origin of these ARF-GEF selectivities. In vitro, in the presence of phospholipid membranes, ARNO activates ARF1 preferentially and ARF6 slightly, whereas EFA6 activates ARF6 exclusively; the stimulation efficiency of EFA6 on ARF6 is comparable with that of ARNO on ARF1. These selectivities are determined by the GEFs Sec7 domains alone, without the pleckstrin homology and N-terminal domains, and by the ARF core domains, without the myristoylated N-terminal helix; they are not modified upon permutation between ARF1 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.
Macia
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Institut de Pharmacologie Moléculaire et Cellulaire, CNRS-UMR 6097, 660 Route des Lucioles, 06560 Valbonne Sophia-Antipolis, France.
Chabre
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Franco
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D020823:ADP-Ribosylation Factor 1 / Q000235:genetics / Q000378:metabolism*; D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors; D006153:Guanosine Diphosphate / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010445:Peptide Elongation Factors / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D017510:Protein Folding; D012995:Solubility; D013329:Structure-Activity Relationship
D000089982:ADP-Ribosylation Factor 6; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D010445:Peptide Elongation Factors; D019269:Phosphatidylinositol 4,5-Diphosphate; C102474:cytohesin-2; D006153:Guanosine Diphosphate; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors