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.
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Cover Image
Cover Image
Structure of O-acetylserine sulfhydrylase (OASS) from Brucella abortus compared with all known OASS structures. The high degree of variation is observed in N-terminal domain, which determined the size of active site cleft and responsible for interactions with Serine acetyl Transferase. The co-factor Pyridoxal phosphate (PLP) is shown in ball & stick model in the active site. For more information, please see study by Dharavath et al. in this issue, pages 1221–1239. Image provided by Samudrala Gourinath.
Characterization of the activation of small GTPases by their GEFs on membranes using artificial membrane tethering
François Peurois, Simon Veyron, Yann Ferrandez, Ilham Ladid, Sarah Benabdi, Mahel Zeghouf, Gérald Peyroche, Jacqueline Cherfils; Characterization of the activation of small GTPases by their GEFs on membranes using artificial membrane tethering. Biochem J 1 April 2017; 474 (7): 1259–1272. doi: https://doi.org/10.1042/BCJ20170015
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