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ADP-ribosylation factor 1 (Arf1) interacts with multiple cellular partners and membranes to regulate intracellular traffic, organelle structure and actin dynamics. Defining the dynamic conformational landscape of Arf1 in its active form, when bound to the membrane, is of high functional relevance and key to understanding how Arf1 can alter diverse cellular processes. Through concerted application of nuclear magnetic resonance (NMR), neutron reflectometry (NR) and molecular dynamics (MD) simulations, we show that, while Arf1 is anchored to the membrane through its N-terminal myristoylated amphipathic helix, the G domain explores a large conformational space, existing in a dynamic equilibrium between membrane-associated and membrane-distal conformations. These configurational dynamics expose different interfaces for interaction with effectors. Interaction with the Pleckstrin homology domain of ASAP1, an Arf-GTPase activating protein (ArfGAP), restricts motions of the G domain to lock it in what seems to be a conformation exposing functionally relevant regions. An integrated approach combing solution and solid-state NMR, molecular dynamics simulations and neutron reflectometry is applied to characterize dynamic membrane bound forms of ADP-ribosylation factor 1 (Arf1).

ADP-ribosylation factor (Arf) proteins are small GTPases that regulate intracellular membrane trafficking and actin remodeling through tightly controlled cycles of GTP binding and hydrolysis. Arf1, a central coordinator of Golgi and endosomal transport, and Arf6, which regulates plasma membranes and endosomal dynamics, have both been implicated in late stages of the HIV-1 life cycle. However, the mechanisms by which these GTPases support viral assembly and release remain incompletely defined. Here, we provide direct evidence that both Arf1 and Arf6 are required for efficient trafficking of the HIV-1 Gag polyprotein, assembly, and virion production. Perturbation of Arf1 function using either GTP-locked (Q71L) or GDP-locked (T31N) mutants significantly reduced virus release, impaired Gag association with membrane compartments, and prevented its accumulation at the plasma membrane. Manipulation of Arf1 cycling through the GTPase-activating protein AGAP1 further demonstrated that dynamic transitions between GTP- and GDP-bound states are essential for productive Gag trafficking. Similarly, expression of a constitutively active Arf6 mutant (Q67L) misrouted Gag to intracellular membranes and markedly suppressed virion release. Importantly, disruption of Arf1 or Arf6 activity did not affect the expression, surface levels, or intracellular distribution of the host restriction factor BST-2. Together, these findings identify Arf1- and Arf6-mediated trafficking pathways as critical host determinants of HIV-1 assembly and release and establish that their functions operate independently of BST-2 antagonism.

Treatment of cells with brefeldin A (BFA) blocks secretory vesicle transport and causes a collapse of the Golgi apparatus. To gain more insight into the cellular mechanisms mediating BFA toxicity, we conducted a genome-wide haploid genetic screen that led to the identification of the small G protein ADP-ribosylation factor 4 (ARF4). ARF4 depletion preserves viability, Golgi integrity and cargo trafficking in the presence of BFA, and these effects depend on the guanine nucleotide exchange factor GBF1 and other ARF isoforms including ARF1 and ARF5. ARF4 knockdown cells show increased resistance to several human pathogens including Chlamydia trachomatis and Shigella flexneri . Furthermore, ARF4 expression is induced when cells are exposed to several Golgi-disturbing agents and requires the CREB3 (also known as Luman or LZIP) transcription factor, whose downregulation mimics ARF4 loss. Thus, we have uncovered a CREB3–ARF4 signalling cascade that may be part of a Golgi stress response set in motion by stimuli compromising Golgi capacity. In an insertional mutagenesis screen, Sabatini and colleagues identify the small G protein ARF4 as a mediator of cell death in response to brefeldin A (BFA) treatment. BFA-induced Golgi stress upregulates ARF4, and loss of ARF protects against propagation of pathogens known to induce Golgi fragmentation.

ADP ribosylation factor (Arf) 6 anchors to the plasma membrane, where it coordinates membrane trafficking and cytoskeleton remodelling, but how it assembles actin filaments is unknown. By reconstituting membrane-associated actin assembly mediated by the WASP family veroprolin homolog (WAVE) regulatory complex (WRC), we recapitulated an Arf6-driven actin polymerization pathway. We show that Arf6 is divergent from other Arf members, as it was incapable of directly recruiting WRC. We demonstrate that Arf6 triggers actin assembly at the membrane indirectly by recruiting the Arf g uanine nucleotide exchange factor (GEF) ARNO that activates Arf1 to enable WRC-dependent actin assembly. The pathogen Salmonella usurped Arf6 for host cell invasion by recruiting its canonical GEFs EFA6 and BRAG2. Arf6 and its GEFs facilitated membrane ruffling and pathogen invasion via ARNO, and triggered actin assembly by generating an Arf1–WRC signaling hub at the membrane in vitro and in cells. This study reconstitutes Arf6-dependent actin assembly to reveal a mechanism by which related Arf GTPases orchestrate distinct steps in the WRC cytoskeleton remodelling pathway.

An irreversible mechanism of protein demyristoylation catalysed by invasion plasmid antigen J (IpaJ), a Shigella flexneri type III effector protein with cysteine protease activity, is described. N -myristoylated proteins targeted by pathogen Nearly one per cent of eukaryotic proteins are modified with N -myristoyl groups that facilitate dynamic protein–protein and protein–membrane interactions. This means that N -myristoylation is important for cellular signaling but also makes it an inviting target for pathogens seeking to modulate a host cell's signalling landscape. Neal Alto and colleagues describe a previously unrecognized pathogenic mechanism involving irreversible protein demyristoylation catalysed by IpaJ, a Shigella flexneri type III effector protein with cysteine protease activity. IpaJ cleaves an array of N -myristoylated proteins involved in cellular growth, signal transduction, autophagasome maturation and organelle function. Protein N -myristoylation is a 14-carbon fatty-acid modification that is conserved across eukaryotic species and occurs on nearly 1% of the cellular proteome 1 , 2 . The ability of the myristoyl group to facilitate dynamic protein–protein and protein–membrane interactions (known as the myristoyl switch) makes it an essential feature of many signal transduction systems 3 . Thus pathogenic strategies that facilitate protein demyristoylation would markedly alter the signalling landscape of infected host cells. Here we describe an irreversible mechanism of protein demyristoylation catalysed by invasion plasmid antigen J (IpaJ), a previously uncharacterized Shigella flexneri type III effector protein with cysteine protease activity. A yeast genetic screen for IpaJ substrates identified ADP-ribosylation factor (ARF)1p and ARF2p, small molecular mass GTPases that regulate cargo transport through the Golgi apparatus 4 . Mass spectrometry showed that IpaJ cleaved the peptide bond between N -myristoylated glycine-2 and asparagine-3 of human ARF1, thereby providing a new mechanism for host secretory inhibition by a bacterial pathogen 5 , 6 . We further demonstrate that IpaJ cleaves an array of N -myristoylated proteins involved in cellular growth, signal transduction, autophagasome maturation and organelle function. Taken together, these findings show a previously unrecognized pathogenic mechanism for the site-specific elimination of N -myristoyl protein modification.

The mechanisms whereby guanine nucleotide exchange factors (GEFs) coordinate their subcellular targeting to their activation of small GTPases remain poorly understood. Here we analyzed how membranes control the efficiency of human BRAG2, an ArfGEF involved in receptor endocytosis, Wnt signaling, and tumor invasion. The crystal structure of an Arf1-BRAG2 complex that mimics a membrane-bound intermediate revealed an atypical PH domain that is constitutively anchored to the catalytic Sec7 domain and interacts with Arf. Combined with the quantitative analysis of BRAG2 exchange activity reconstituted on membranes, we find that this PH domain potentiates nucleotide exchange by about 2,000-fold by cumulative conformational and membrane-targeting contributions. Furthermore, it restricts BRAG2 activity to negatively charged membranes without phosphoinositide specificity, using a positively charged surface peripheral to but excluding the canonical lipid-binding pocket. This suggests a model of BRAG2 regulation along the early endosomal pathway that expands the repertoire of GEF regulatory mechanisms. Notably, it departs from the auto-inhibitory and feedback loop paradigm emerging from studies of SOS and cytohesins. It also uncovers a novel mechanism of unspecific lipid-sensing by PH domains that may allow sustained binding to maturating membranes.

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.

The intracellular bacterium Chlamydia trachomatis develops in a parasitic compartment called the inclusion. Posttranslationally modified microtubules encase the inclusion, controlling the positioning of Golgi complex fragments around the inclusion. The molecular mechanisms by which Chlamydia coopts the host cytoskeleton and the Golgi complex to sustain its infectious compartment are unknown. Here, using a genetically modified Chlamydia strain, we discovered that both posttranslationally modified microtubules and Golgi complex positioning around the inclusion are controlled by the chlamydial inclusion protein CT813/CTL0184/InaC and host ARF GTPases. CT813 recruits ARF1 and ARF4 to the inclusion membrane, where they induce posttranslationally modified microtubules. Similarly, both ARF isoforms are required for the repositioning of Golgi complex fragments around the inclusion. We demonstrate that CT813 directly recruits ARF GTPases on the inclusion membrane and plays a pivotal role in their activation. Together, these results reveal that Chlamydia uses CT813 to hijack ARF GTPases to couple posttranslationally modified microtubules and Golgi complex repositioning at the inclusion. IMPORTANCE Chlamydia trachomatis is an important cause of morbidity and a significant economic burden in the world. However, how Chlamydia develops its intracellular compartment, the so-called inclusion, is poorly understood. Using genetically engineered Chlamydia mutants, we discovered that the effector protein CT813 recruits and activates host ADP-ribosylation factor 1 (ARF1) and ARF4 to regulate microtubules. In this context, CT813 acts as a molecular platform that induces the posttranslational modification of microtubules around the inclusion. These cages are then used to reposition the Golgi complex during infection and promote the development of the inclusion. This study provides the first evidence that ARF1 and ARF4 play critical roles in controlling posttranslationally modified microtubules around the inclusion and that Chlamydia trachomatis hijacks this novel function of ARF to reposition the Golgi complex. Chlamydia trachomatis is an important cause of morbidity and a significant economic burden in the world. However, how Chlamydia develops its intracellular compartment, the so-called inclusion, is poorly understood. Using genetically engineered Chlamydia mutants, we discovered that the effector protein CT813 recruits and activates host ADP-ribosylation factor 1 (ARF1) and ARF4 to regulate microtubules. In this context, CT813 acts as a molecular platform that induces the posttranslational modification of microtubules around the inclusion. These cages are then used to reposition the Golgi complex during infection and promote the development of the inclusion. This study provides the first evidence that ARF1 and ARF4 play critical roles in controlling posttranslationally modified microtubules around the inclusion and that Chlamydia trachomatis hijacks this novel function of ARF to reposition the Golgi complex.

It has been shown that inhibition of GTPase-activating protein of ADP-ribosylation factor (Arf), ArfGAP, with a small molecule (QS11) results in synergistic activation of Wnt/β-catenin signaling. However, the role of Arf in Wnt/β-catenin signaling has not yet been elucidated. Here, we show that activation of Arf is essential for Wnt/β-catenin signaling. The level of the active form of Arf (Arf-GTP) transiently increased in the presence of Wnt, and this induction event was abrogated by blocking the interaction between Wnt and Frizzled (Fzd). In addition, knockdown of Fzds, Dvls or LRP6 blocked the Wnt-mediated activation of Arf. Consistently, depletion of Arf led to inhibition of Wnt-mediated membrane PtdIns (4,5)P 2 (phosphatidylinositol 4, 5-bisphosphate) synthesis and LRP6 phosphorylation. Overall, our data suggest that transient activation of Arf modulates LRP6 phosphorylation for the transduction of Wnt/β-catenin signaling.

Enterovirus 71 is one of the major causative agents of hand, foot, and mouth disease in infants and children. Replication of enterovirus 71 depends on host cellular factors. The viral replication complex is formed in novel, cytoplasmic, vesicular compartments. It has not been elucidated which cellular pathways are hijacked by the virus to create these vesicles. Here, we investigated whether proteins associated with the cellular secretory pathway were involved in enterovirus 71 replication. We used a loss-of-function assay, based on small interfering RNA. We showed that enterovirus 71 RNA replication was dependent on the activity of Class I ADP-ribosylation factors. Simultaneous depletion of ADP-ribosylation factors 1 and 3, but not three others, inhibited viral replication in cells. We also demonstrated with various techniques that the brefeldin-A-sensitive guanidine nucleotide exchange factor, GBF1, was critically important for enterovirus 71 replication. Our results suggested that enterovirus 71 replication depended on GBF1-mediated activation of Class I ADP-ribosylation factors. These results revealed a connection between enterovirus 71 replication and the cellular secretory pathway; this pathway may represent a novel target for antiviral therapies.