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Mitochondrial plasticity is a key regulator of cell fate decisions. Mitochondrial division involves Dynamin-related protein-1 (Drp1) oligomerization, which constricts membranes at endoplasmic reticulum (ER) contact sites. The mechanisms driving the final steps of mitochondrial division are still unclear. Here, we found that microdomains of phosphatidylinositol 4-phosphate [PI(4)P] on trans-Golgi network (TGN) vesicles were recruited to mitochondria–ER contact sites and could drive mitochondrial division downstream of Drp1. The loss of the small guanosine triphosphatase ADP-ribosylation factor 1 (Arf1) or its effector, phosphatidylinositol 4-kinase IIIβ [PI(4)KIIIβ], in different mammalian cell lines prevented PI(4)P generation and led to a hyperfused and branched mitochondrial network marked with extended mitochondrial constriction sites. Thus, recruitment of TGN-PI(4)P–containing vesicles at mitochondria–ER contact sites may trigger final events leading to mitochondrial scission.

Type I interferon (IFN) signalling is tightly controlled. Upon recognition of DNA by cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING) translocates along the endoplasmic reticulum (ER)-Golgi axis to induce IFN signalling. Termination is achieved through autophagic degradation or recycling of STING by retrograde Golgi-to-ER transport. Here, we identify the GTPase ADP-ribosylation factor 1 (ARF1) as a crucial negative regulator of cGAS-STING signalling. Heterozygous ARF1 missense mutations cause a previously unrecognized type I interferonopathy associated with enhanced IFN-stimulated gene expression. Disease-associated, GTPase-defective ARF1 increases cGAS-STING dependent type I IFN signalling in cell lines and primary patient cells. Mechanistically, mutated ARF1 perturbs mitochondrial morphology, causing cGAS activation by aberrant mitochondrial DNA release, and leads to accumulation of active STING at the Golgi/ERGIC due to defective retrograde transport. Our data show an unexpected dual role of ARF1 in maintaining cGAS-STING homeostasis, through promotion of mitochondrial integrity and STING recycling. Self-derived DNA may trigger interferon-driven autoinflammation mediated by the cGAS-STING axis. Here, the authors find that mutations in the GTPase ARF1 cause an interferonopathy by promoting aberrant mitochondrial DNA release and impairing STING recycling.

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.

Background: The ADP‐ribosylation factor 1 (ARF1) gene encodes a protein which plays a critical role in intra‐Golgi transport. Clinical evidence suggests that individuals harbouring variants in the ARF1 gene display a consistent set of phenotypic features, including intellectual disability, microcephaly, epilepsy, and periventricular nodular heterotopia (PVNH). Methods: This study describes the case of a 6‐year and 5‐month‐old female presenting with focal seizures on a fixed side that were resistant to various anti‐seizure medications. The genetic aetiology was elucidated through exome sequencing of the pedigree. The pathogenicity of the identified variant was subsequently assessed using molecular dynamics structural analysis, western blotting, and co‐immunoprecipitation techniques. Results: A de novo variant, c.509T > C (p.Leu170Pro), was detected in the ARF1 gene, and functional analysis demonstrated that this modification is anticipated to hinder its association with the Golgi‐localising, γ‐adaptin ear homology domain and ARF‐binding protein, thereby playing a role in the pathogenesis of the disease. Conclusion: This study introduces the initial instance of ARF1‐related disease in China, wherein the patient is without the presence of PVNH. The findings add novel clinical phenotypes to the range of ARF1‐related diseases, and an investigation into the potential pathogenic mechanisms of this condition was conducted by confirming the deleterious impacts of the variant.

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.

Cancer stem cells (CSCs) may be responsible for treatment resistance, tumor metastasis, and disease recurrence. Here we demonstrate that the Arf1-mediated lipid metabolism sustains cells enriched with CSCs and its ablation induces anti-tumor immune responses in mice. Notably, Arf1 ablation in cancer cells induces mitochondrial defects, endoplasmic-reticulum stress, and the release of damage-associated molecular patterns (DAMPs), which recruit and activate dendritic cells (DCs) at tumor sites. The activated immune system finally elicits antitumor immune surveillance by stimulating T-cell infiltration and activation. Furthermore, TCGA data analysis shows an inverse correlation between Arf1 expression and T-cell infiltration and activation along with patient survival in various human cancers. Our results reveal that Arf1-pathway knockdown not only kills CSCs but also elicits a tumor-specific immune response that converts dying CSCs into a therapeutic vaccine, leading to durable benefits. Cancer stem cells (CSC) have been shown as the origin for therapeutic resistance and patient relapse. Here, the authors show that targeting Arf1-mediated lipid metabolism in CSC induces cell death but also an immunogenic anti-cancer response.

The ADP-ribosylation factors (Arfs) constitute a family of small GTPases within the Ras superfamily, with a distinguishing structural feature of a hypervariable N-terminal extension of the G domain modified with myristate. Arf proteins, including Arf1, have roles in membrane trafficking and cytoskeletal dynamics. While screening for Arf1:small molecule co-crystals, we serendipitously solved the crystal structure of the non-myristoylated engineered mutation [L8K]Arf1 in complex with a GDP analogue. Like wild-type (WT) non-myristoylated Arf1•GDP, we observed that [L8K]Arf1 exhibited an N-terminal helix that occludes the hydrophobic cavity that is occupied by the myristoyl group in the GDP-bound state of the native protein. However, the helices were offset from one another due to the L8K mutation, with a significant change in position of the hinge region connecting the N-terminus to the G domain. Hypothesizing that the observed effects on behavior of the N-terminus affects interaction with regulatory proteins, we mutated two hydrophobic residues to examine the role of the N-terminal extension for interaction with guanine nucleotide exchange factors (GEFs) and GTPase Activating Proteins (GAPs. Different than previous studies, all mutations were examined in the context of myristoylated Arf. Mutations had little or no effect on spontaneous or GEF-catalyzed guanine nucleotide exchange but did affect interaction with GAPs. [F13A]myrArf1 was less than 1/2500, 1/1500, and 1/200 efficient as substrate for the GAPs ASAP1, ARAP1 and AGAP1; however, [L8A/F13A]myrArf1 was similar to WT myrArf1. Using molecular dynamics simulations, the effect of the mutations on forming alpha helices adjacent to a membrane surface was examined, yet no differences were detected. The results indicate that lipid modifications of GTPases and consequent anchoring to a membrane influences protein function beyond simple membrane localization. Hypothetical mechanisms are discussed.

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.

The discovery that endoplasmic reticulum (ER) luminal chaperones such as GRP78/BiP can escape to the cell surface upon ER stress where they regulate cell signaling, proliferation, apoptosis, and immunity represents a paradigm shift. Toward deciphering the mechanisms, we report here that, upon ER stress, IRE1α binds to and triggers tyrosine kinase SRC activation, leading to ASAP1 phosphorylation and Golgi accumulation of ASAP1 and Arf1-GTP, resulting in KDEL receptor dispersion from the Golgi and suppression of retrograde transport. At the cell surface, GRP78 binds to and acts in concert with a glycosylphosphatidylinositol-anchored protein, CD109, in blocking TGF-β signaling by promoting the routing of the TGF-β receptor to the caveolae, thereby disrupting its binding to and activation of Smad2. Collectively, we uncover a SRC-mediated signaling cascade that leads to the relocalization of ER chaperones to the cell surface and a mechanism whereby GRP78 counteracts the tumor-suppressor effect of TGF-β.