A Novel Membrane Sensor Controls the Localization and ArfGEF Activity of Bacterial RalF

Abstract

The intracellular bacterial pathogen Legionella pneumophila (Lp) evades destruction in macrophages by camouflaging in a specialized organelle, the Legionella-containing vacuole (LCV), where it replicates. The LCV maturates by incorporating ER vesicles, which are diverted by effectors that Lp injects to take control of host cell membrane transport processes. One of these effectors, RalF, recruits the trafficking small GTPase Arf1 to the LCV. LpRalF has a Sec7 domain related to host ArfGEFs, followed by a capping domain that intimately associates with the Sec7 domain to inhibit GEF activity. How RalF is activated to function as a LCV-specific ArfGEF is unknown. We combined the reconstitution of Arf activation on artificial membranes with cellular expression and Lp infection assays, to analyze how auto-inhibition is relieved for LpRalF to function in vivo. We find that membranes activate LpRalF by about 1000 fold, and identify the membrane-binding region as the region that inhibits the Sec7 active site. It is enriched in aromatic and positively charged residues, which establish a membrane sensor to control the GEF activity in accordance with specific lipid environments. A similar mechanism of activation is found in RalF from Rickettsia prowazekii (Rp), with a different aromatic/charged residues ratio that results in divergent membrane preferences. The membrane sensor is the primary determinant of the localization of LpRalF on the LCV, and drives the timing of Arf activation during infection. Finally, we identify a conserved motif in the capping domain, remote from the membrane sensor, which is critical for RalF activity presumably by organizing its active conformation. These data demonstrate that RalF proteins are regulated by a membrane sensor that functions as a binary switch to derepress ArfGEF activity when RalF encounters a favorable lipid environment, thus establishing a regulatory paradigm to ensure that Arf GTPases are efficiently activated at specific membrane locations.

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

“…67 Both bacterial ArfGEFs are strongly activated by membranes, and the membrane-binding site is identical to the elements in the autoinhibitory domain that blocks the Arf-binding site. 68,69 Thus, activation of Arf GTPases by RalF strictly depends on its recruitment to membranes. Legionella RalF produces Arf-GTP at the surface of the Legionella-containing vacuole where the pathogen hides and replicates; 65 however Rickettsia replicates in the host cytosol and therefore should use RalF for different functions.…”

“…In that regard, it is remarkable that Legionella and Rickettsia RalF respond to different membrane characteristics for their activation. 69 Legionella RalF is activated equally well by membranes that contain acidic lipids or are enriched in unsaturated lipids, suggesting that it is tailored to remain active at the surface of the phagosome-derived vacuole as it matures by incorporating ERderived vesicles. It is also a unique situation in which a membrane-binding domain recognizes 2 membranes with different characteristics.…”

“…These differences in membrane specificities are explained by the ratio between positively charged residues, which bind to acidic lipids, and aromatic residues, which wedge in lipid packing defects, which differs between Legionella and Rickettsia RalF. 69 Thus, both Legionella and Rickettsia have evolved a simplified ArfGEF version to subvert host Arf GTPases, in which the same structural element is responsible for autoinhibition and for membrane binding to support allosteric regulation by membranes. Discussion 50 years after the celebrated Monod-Wyman-Changeux thermodynamic model was first formulated, 70 the concept of allostery experiences a spectacular renaissance.…”

“…Sucrose-loaded liposomes that contained 40% phosphatidylcholine (PC), 35% DOPC, 10% phosphatidylethanolamine (PE), 10% DOPE and 5% phosphatidylinositol-4-phosphate (PI(4)P) were freshly extruded through a 0.2 mm filter (Whatman) and used within 2 days. Co-sedimentation experiments were carried out as described previously 69 after incubation of 1 mM of liposomes with 3 mM of BIG1 DCBHUSSec7 for 15 min at 20 C. Proteins were analyzed by SDS-PAGE on a 4-20% TGX-stain free gel (Bio-Rad) and detected by fluorescence after 2.5 min activation using a ChemiDoc imaging system (Bio-Rad). Quantifications were done with the Bio-Rad Image Lab TM software (v5.2).…”

EFA6 controls Arf1 and Arf6 activation through a negative feedback loop

“…1). These include autoinhibitory elements outside the catalytic GEF domain that block access to the active site (3-7), large conformational changes that release autoinhibition in response to various stimuli (8)(9)(10)(11), positive feedback loops in which freshly produced GTP-bound GTPases stimulate GDP/ GTP exchange (10,(12)(13)(14)(15), and potentiation of nucleotide exchange by colocalization on membranes (11,13,16,17).…”