EFA6 controls Arf1 and Arf6 activation through a negative feedback loop

Abstract

Guanine nucleotide exchange factors (GEFs) of the exchange factor for Arf6 (EFA6), brefeldin A-resistant Arf guanine nucleotide exchange factor (BRAG), and cytohesin subfamilies activate small GTPases of the Arf family in endocytic events. These ArfGEFs carry a pleckstrin homology (PH) domain in tandem with their catalytic Sec7 domain, which is autoinhibitory and supports a positive feedback loop in cytohesins but not in BRAGs, and has an as-yet unknown role in EFA6 regulation. In this study, we analyzed how EFA6A is regulated by its PH and C terminus (Ct) domains by reconstituting its GDP/GTP exchange activity on membranes. We found that EFA6 has a previously unappreciated high efficiency toward Arf1 on membranes and that, similar to BRAGs, its PH domain is not autoinhibitory and strongly potentiates nucleotide exchange on anionic liposomes. However, in striking contrast to both cytohesins and BRAGs, EFA6 is regulated by a negative feedback loop, which is mediated by an allosteric interaction of Arf6-GTP with the PH-Ct domain of EFA6 and monitors the activation of Arf1 and Arf6 differentially. These observations reveal that EFA6, BRAG, and cytohesins have unanticipated commonalities associated with divergent regulatory regimes. An important implication is that EFA6 and cytohesins may combine in a mixed negative-positive feedback loop. By allowing EFA6 to sustain a pool of dormant Arf6-GTP, such a circuit would fulfill the absolute requirement of cytohesins for activation by Arf-GTP before amplification of their GEF activity by their positive feedback loop.endocytosis | membrane traffic | kinetics | autoinhibition

Allosteric regulation of Arf GTPases and their GEFs at the membrane interface

“…Remarkably, although EFA6 is strictly specific for Arf6 in solution, it activates both Arf1 and Arf6 efficiently on membranes, highlighting the fact that membranes can modulate the specificity of GEFs for their small GTPase substrates. 49 The regulatory regime of EFA6 is however strikingly different from that of the other 2 subfamilies. EFA6 activity decreases as more Arf-GTP is produced on membranes, suggesting that it might be regulated by negative feedback.…”

“…While the function of N-terminal regions has remained poorly understood, the regulatory mechanisms of PH domains have been extensively studied and this revealed an unexpected diversity of regulatory regimes. Although they display minimal homology to each other, the PH domains of cytohesins, 5 BRAG 29,46,47 and EFA6 48,49 all bind PI(4,5)P 2 phosphoinositides. Cytohesins also interact with PI(3,4,5)P 3 , with splice variants with a di-glycine instead of a tri-glycine in the b1-b2 loop of the PH domain showing a marked preference for this rare phosphoinositide.…”

“…54,55 Whereas the activities and specificities of these 3 subfamilies differ broadly in solution, all are highly potent at activating both Arf1 and Arf6 in the presence of membranes, including EFA6 which is not active on Arf1 in solution. 29,46,49,[56][57][58] These stark differences highlight the importance to assess specificity using membranes and myristoylated Arf GTPases, as this is often the starting point for building models of the biology of the proteins being studied. k cat /K m toward Arf1 in solution and on membranes determined using a range of GEF concentrations are given in Table 1 for Sec-PH constructs of representative members of the cytohesin (ref.…”

“…56 Neither the PH nor the PH-Ct domains are autoinhibitory; yet, PIP 2 -containing membranes increase activity by 2 orders of magnitude 48,49 (Table 1). Remarkably, although EFA6 is strictly specific for Arf6 in solution, it activates both Arf1 and Arf6 efficiently on membranes, highlighting the fact that membranes can modulate the specificity of GEFs for their small GTPase substrates.…”

“…EFA6 activity decreases as more Arf-GTP is produced on membranes, suggesting that it might be regulated by negative feedback. 49 Although the structural details of this effect remain to be investigated, titration by the PH-Ct domains suggests that Arf-GTP regulates EFA6 by interacting with this domain.…”