title: Characterization of the activation of small GTPases by their GEFs on membranes using artificial membrane tethering.
abstract: Active, GTP-bound small GTPases need to be attached to membranes by post-translational lipid modifications in order to process and propagate information in cells. However, generating and manipulating lipidated GTPases has remained difficult, which has limited our quantitative understanding of their activation by guanine nucleotide exchange factors (GEFs) and their termination by GTPase-activating proteins. Here, we replaced the lipid modification by a histidine tag in 11 full-length, human small GTPases belonging to the Arf, Rho and Rab families, which allowed to tether them to nickel-lipid-containing membranes and characterize the kinetics of their activation by GEFs. Remarkably, this strategy uncovered large effects of membranes on the efficiency and/or specificity in all systems studied. Notably, it recapitulated the release of autoinhibition of Arf1, Arf3, Arf4, Arf5 and Arf6 GTPases by membranes and revealed that all isoforms are efficiently activated by two GEFs with different regulatory regimes, ARNO and Brag2. It demonstrated that membranes stimulate the GEF activity of Trio toward RhoG by ∼30 fold and Rac1 by ∼10 fold, and uncovered a previously unknown broader specificity toward RhoA and Cdc42 that was undetectable in solution. Finally, it demonstrated that the exceptional affinity of the bacterial RabGEF DrrA for the phosphoinositide PI(4)P delimits the activation of Rab1 to the immediate vicinity of the membrane-bound GEF. Our study thus validates the histidine-tag strategy as a potent and simple means to mimic small GTPase lipidation, which opens a variety of applications to uncover regulations brought about by membranes.
authors.affiliation: Laboratoire de Biologie et Pharmacologie Appliquée, CNRS and Ecole Normale Supérieure Paris-Saclay, 61 Avenue du Président Wilson, Cachan 94235, France.
authors.forename: François
authors.initials: F
authors.lastname: Peurois
authors.affiliation: Laboratoire de Biologie et Pharmacologie Appliquée, CNRS and Ecole Normale Supérieure Paris-Saclay, 61 Avenue du Président Wilson, Cachan 94235, France.
authors.forename: Simon
authors.initials: S
authors.lastname: Veyron
authors.affiliation: Laboratoire de Biologie et Pharmacologie Appliquée, CNRS and Ecole Normale Supérieure Paris-Saclay, 61 Avenue du Président Wilson, Cachan 94235, France.
authors.forename: Yann
authors.initials: Y
authors.lastname: Ferrandez
authors.affiliation: Laboratoire de Biologie et Pharmacologie Appliquée, CNRS and Ecole Normale Supérieure Paris-Saclay, 61 Avenue du Président Wilson, Cachan 94235, France.
authors.forename: Ilham
authors.initials: I
authors.lastname: Ladid
authors.affiliation: Laboratoire de Biologie et Pharmacologie Appliquée, CNRS and Ecole Normale Supérieure Paris-Saclay, 61 Avenue du Président Wilson, Cachan 94235, France.
authors.forename: Sarah
authors.initials: S
authors.lastname: Benabdi
authors.affiliation: Laboratoire de Biologie et Pharmacologie Appliquée, CNRS and Ecole Normale Supérieure Paris-Saclay, 61 Avenue du Président Wilson, Cachan 94235, France.
authors.forename: Mahel
authors.initials: M
authors.lastname: Zeghouf
authors.affiliation: Laboratoire de Biologie et Pharmacologie Appliquée, CNRS and Ecole Normale Supérieure Paris-Saclay, 61 Avenue du Président Wilson, Cachan 94235, France.
authors.forename: Gérald
authors.initials: G
authors.lastname: Peyroche
authors.affiliation: Laboratoire de Biologie et Pharmacologie Appliquée, CNRS and Ecole Normale Supérieure Paris-Saclay, 61 Avenue du Président Wilson, Cachan 94235, France jacqueline.cherfils@ens-paris-saclay.fr.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Cherfils
mesh_terms: D020823:ADP-Ribosylation Factor 1 / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D020690:GTPase-Activating Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016952:Legionella pneumophila / Q000737:chemistry; D008567:Membranes, Artificial; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C101895:DrrA protein, Bacteria; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; C471213:His-His-His-His-His-His; C504483:IQSEC1 protein, human; D008567:Membranes, Artificial; D009842:Oligopeptides; D010716:Phosphatidylinositols; D020033:Protein Isoforms; C107953:RAC1 protein, human; D011994:Recombinant Proteins; C102474:cytohesin-2; C066737:RHOA protein, human; C075397:RHOG protein, human; D006639:Histidine; D020823:ADP-Ribosylation Factor 1; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein
keywords: enzyme kinetics; guanine nucleotide exchange factors; membranes; post-translational lipid modification; small GTPases