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Giardia intestinalis is an enteric pathogen with an extremely modified membrane trafficking system, lacking canonical compartments such as the Golgi, endosomes, and intermediate vesicle carriers. By comparison the fornicate relatives of Giardia possess greater endomembrane system complexity. In eukaryotes, the ADP ribosylation factor (ARF) GTPase regulatory system proteins, which consist of the small GTPase ARF1, and its guanine exchange nucleotide factors (GEFs) and GTPase activating proteins (GAPs), coordinate temporal and directional trafficking of cargo vesicles by recognizing and interacting with heterotetrameric coat complexes at pre-Golgi and post-Golgi interfaces. To understand the evolution of this regulatory system across the fornicate lineage, we have performed comparative genomic and phylogenetic analyses of the ARF GTPases, and their regulatory GAPs and GEFs in fornicate genomes and transcriptomes. Prior to our analysis of the fornicates, we first establish that the ARF GAP sub-family ArfGAP with dual PH domains (ADAP) is sparsely distributed but present in at least four eukaryotic supergroups and thus was likely present in the Last Eukaryotic Common Ancestor (LECA). Next, our collective comparative genomic and phylogenetic investigations into the ARF regulatory proteins in fornicates identify a duplication of ARF1 GTPase yielding two paralogues of ARF1F proteins, ancestral to all fornicates and present in all examined isolates of Giardia. However, the ARF GEF and ARF GAP complement is reduced compared with the LECA. This investigation shows that the system was significantly streamlined prior to the fornicate ancestor but was not further reduced concurrent with a transition into a parasitic lifestyle.

ADP-ribosylation factors (Arfs) are guanosine triphosphate (GTP)-binding proteins that play essential roles in membrane trafficking, and they have been recently reported to be involved in innate immunity in crustaceans. However, little information is available on Arfs in the oriental river prawn Macrobrachium nipponense and their response to ammonia nitrogen stress. In this study, we identified a novel M. nipponense Arfn gene (MnArfn). The full-length cDNA of MnArfn was 1076 bp. It contained a 537 bp open reading frame (ORF) and encoded a 178 amino acid protein with a predicted molecular weight of 19.85 kDa. Sequence and phylogenetic analyses showed that MnArfn was an unidentified Arf, sharing 55-61% identity with other known Arfs. Quantitative real-time PCR (qPCR) indicated that all examined tissues (hepatopancreas, stomach, gill, heart, muscle, and eyestalk) expressed MnArfn. Hepatopancreas and gills, 2 organs involved in environmental stress management, had the highest expression. Under conditions of ammonia nitrogen stress, MnArfn expression in hepatopancreas and gills was significantly up-regulated at 6, 12, and 24 h. Western blotting experiments also revealed that MnArfn was distributed in all examined tissues, with the highest expression in hepatopancreas and gills, consistent with qPCR results. The findings from this study indicate that MnArfn may play an important role in the response of M. nipponense to ammonia nitrogen stress, which provides a new avenue to study the resistance mechanism(s) of crustaceans to ammonia nitrogen and to screen for individuals with resistance to unfavorable environments.

Glioma cells with stem cell-like properties are crucial for tumor initiation, progression and therapeutic resistance. Therefore, identifying specific factors in regulating stem-like traits is critical for the design of novel glioma therapeutics. Herein, we reported that ADP-Ribosylation Factor Like GTPase 4C (ARL4C) was highly expressed in glioma stem-like cells (GSLCs). GSLCs, determined by the efficiency of sphere formation in vitro and tumor growth in vivo, was increased by overexpression of ARL4C. ARL4C induced the tumorigenesis through ALDH1A3. Analyses of 325 patient specimens showed that ARL4C was highly expressed in glioblastoma (GBM) as compared with lower grade gliomas. In addition, higher level ARL4C expression in glioma was correlated with poorer progression-free survival and overall survival of patients. Therefore, ARL4C may act as a novel prognostic marker and a therapeutic target for GBM.

Fusarium graminearum is the main pathogen of Fusarium head blight (FHB) in wheat and related species, which causes serious production decreases and economic losses and produces toxins such as deoxynivalenol (DON), which endangers the health of humans and livestock. Vesicle transport is a basic physiological process required for cell survival in eukaryotes. Many regulators of vesicle transport are reported to be involved in the pathogenicity of fungi. In yeast and mammalian cells, the ADP-ribosylation factor-like small GTPase Arl1 and its orthologs are involved in regulating vesicular trafficking, cytoskeletal reorganization and other significant biological processes. However, the role of Arl1 in F. graminearum is not well understood. In this study, we characterized the Arl1-homologous protein FgArl1 in F. graminearum and showed that FgArl1 is located in the trans-Golgi apparatus. The deletion of FgARL1 resulted in a significant decrease in vegetative growth and pathogenicity. Further analyses of the ΔFgarl1 mutant revealed defects in the production of DON. Taken together, these results indicate that FgArl1 is important in the development and pathogenicity of F. graminearum.

The sodium iodide symporter (NIS) is required for iodide uptake, which facilitates thyroid hormone biosynthesis. NIS has been exploited for over 75 years in ablative radioiodine (RAI) treatment of thyroid cancer, where its ability to transport radioisotopes depends on its localization to the plasma membrane. The advent of NIS-based in vivo imaging and theranostic strategies in other malignancies and disease modalities has recently increased the clinical importance of NIS. However, NIS trafficking remains ill-defined. Here, we used tandem mass spectrometry followed by coimmunoprecipitation and proximity ligation assays to identify and validate two key nodes—ADP-ribosylation factor 4 (ARF4) and valosin-containing protein (VCP)—controlling NIS trafficking. Using cell-surface biotinylation assays and highly inclined and laminated optical sheet microscopy, we demonstrated that ARF4 enhanced NIS vesicular trafficking from the Golgi to the plasma membrane, whereas VCP—a principal component of endoplasmic reticulum (ER)–associated degradation—governed NIS proteolysis. Gene expression analysis indicated VCP expression was particularly induced in aggressive thyroid cancers and in patients who had poorer outcomes following RAI treatment. Two repurposed FDA-approved VCP inhibitors abrogated VCP-mediated repression of NIS function, resulting in significantly increased NIS at the cell-surface and markedly increased RAI uptake in mouse and human thyroid models. Collectively, these discoveries delineate NIS trafficking and highlight the new possibility of systemically enhancing RAI therapy in patients using FDA-approved drugs. Significance: These findings show that ARF4 and VCP are involved in NIS trafficking to the plasma membrane and highlight the possible therapeutic role of VCP inhibitors in enhancing radioiodine effectiveness in radioiodine-refractory thyroid cancer.

Cholera is a major global threat, affecting millions each year. The ADP ribosyltransferase activity of the active cholera toxin catalyses the massive loss of water and electrolytes during cholera infections. The active toxin heterodimer comprises the A1 subunit from Vibrio cholerae and ARF (ADP Ribosylation Factor) from the human host. Although the active toxin is a potential target for drug discovery against cholera, it has been scarcely targeted to date. The A1-ARF interface contains a potential druggable site for small molecule inhibitors. By combining a sequential docking and scoring strategy with molecular dynamics (MD) simulations, this study identified hits against the protein-protein interface (PPI) of the active cholera toxin from an in-house library of 9,175 ADMET-screened alkaloids. The docking algorithms and scoring functions of Glide SP, Glide XP, and AutoDock were employed for initial library screening. Three alkaloids were initially selected by docking-based virtual screening. The stability of the hit-toxin complexes was validated by MD simulations. Two of the three hits, namely, A6225 (7-formyldehydrothalicsimidine) and A16503 (1,2,7,8-tetrahydroxy dibenz[cd,f]indol-4(5H)-one), formed stable complexes with the toxin. Analyses of the hydrogen bond occupancies revealed that the hits formed stable hydrogen bonds with the toxin PPI. The hits identified herein can serve as reference compounds for drug discovery against cholera in the future.

ARF small GTPases in plants serve important cellular functions in subcellular trafficking and developmental functions in auxin-mediated patterning of the plant body. TheArabidopsis thalianaARF regulator ARF-GAP VAN3 has been implicated to act at the plasma membrane (PM) and linked functionally to the clathrin- and dynamin-mediated endocytosis. Here we re-evaluated the localization of VAN3 at the PM and its function in endocytosis. Using Total Internal Reflection Fluorescence microscopy we observed remarkably transient associations of VAN3 to the PM at discrete foci, however, devoid of clathrin, the dynamin isoform DRP1A, or the ARF regulator GNOM, which is also involved in a developmental patterning function mediated from the PM. Clathrin-coated pits are abundant and endocytosis appears to proceed normally invan3-1knockout mutant. In turn, post-translational silencing of clathrin expression indicates that the localization of VAN3 at the PM depends on clathrin function, presumably on clathrin-mediated endocytosis.

Arf GTPase-Activating proteins (ArfGAPs) mediate the hydrolysis of GTP bound to ADP-ribosylation factors (Arfs), which are critical to form transport intermediates. ArfGAPs have been thought to be negative regulators of Arfs; however, accumulating evidence indicates that ArfGAPs are important for cargo sorting and promote membrane traffic. Weibel-Palade bodies (WPBs) are cigar-shaped secretory granules in endothelial cells that contain von Willebrand factor (vWF) as their main cargo. WPB biogenesis at the Golgi was reported to be regulated by Arf and their regulators, but the role of ArfGAPs has been unknown. In this study, we performed siRNA screening of ArfGAPs to investigate the role of ArfGAPs in the biogenesis of WPBs. We found two ArfGAPs, SMAP1 and AGFG2, to be involved in WPB size and vWF exocytosis, respectively. SMAP1 depletion resulted in small-sized WPBs, and the lysosomal inhibitor leupeptin recovered the size of WPBs. The results indicate that SMAP1 functions in preventing the degradation of cigar-shaped WPBs. On the other hand, AGFG2 downregulation resulted in the inhibition of vWF secretion upon Phorbol 12-myristate 13-acetate (PMA) or histamine stimulation, suggesting that AGFG2 plays a role in vWF exocytosis. Our study revealed unexpected roles of ArfGAPs in vWF transport., Summary: The Arf GTPase-activating proteins SMAP1 and AGFG2 regulate the size of Weibel-Palade bodies and exocytosis of von Willebrand factor.

Objectives: To study the effects of ADP ribosylation factor guanylate kinase 1 gene (ASAP1) on the biological behavior of malignant gastric cancer (GC), and explore its possible molecular mechanisms in tumorigenesis and tumor progression.Methods: Quantitative PCR and western blotting (WB) were performed to measure ASAP1 mRNA and protein expression in the GES-1 epithelial cell line, which is derived from normal human mucosa, and three GC cell lines. Molecular biology techniques such as lentivirus packaging, infection, and screening were used to obtain BGC823 GC cells overexpressing ASAP1 and BGC823 and MKN45 cells with ASAP1 knocked down. The Cell Counting Kit-8 assay, colony formation assay, flow cytometry using Annexin V/propidium iodide, Transwell migration and invasion assays, and scratch assay were used to assess the malignant biological behavior of GC cells with ASAP1 overexpression and knockdown. WB was conducted to evaluate the effects of ASAP1 expression on angiogenesis, as well as on the expression of matrix metalloproteinases (MMPs), apoptotic proteins, and epithelial-mesenchymal transition (EMT)-related proteins. Nude mice bearing transplanted tumors were evaluated to determine the effect of ASAP1 knockdown on BGC823 GC cells.Results: ASAP1 expression in GC cells was greater than that in GES-1 normal gastric mucosal epithelial cells. ASAP1 overexpression significantly enhanced the proliferation, invasion, and migration of GC cells and reduced apoptosis; whereas ASAP1 knockdown significantly reduced the proliferation, invasion, and migration of GC cells and promoted apoptosis. In the ASAP1-knockdown group, expression of cleaved-caspase 3, cleaved-poly-ADP-ribose polymerase (PARP), and the epithelial marker E-cadherin increased significantly, whereas the expression of MMP2, MMP9, vascular endothelial growth factor A (VEGFA), and the mesenchymal markers N-cadherin, and vimentin decreased significantly (PASAP1 inhibited the growth of subcutaneously implanted tumors in nude mice.Conclusions: ASAP1 overexpression strongly promotes—whereas knockdown of ASAP1 effectively weakens—the malignant biological behavior of GC cells, possibly by reducing VEGFA expression and thus reducing angiogenesis, upregulating the expression of cleaved-caspase 3 and cleaved-PARP, and reducing the activity of MMPs and EMT.

Propagation of angiosperms mostly relies on sexual reproduction, in which gametophytic development is a pre-requisite. Male gametophytic development requires both gametophytic and sporophytic factors, most importantly early secretion and late programmed cell death of the tapetum. In addition to transcriptional factors, proteins at endomembrane compartments, such as receptor-like kinases and vacuolar proteases, control tapetal function. The cellular machinery that regulates their distribution is beginning to be revealed. We report here that ADP-RIBOSYLATION FACTOR-A1s (ArfA1s) are critical for tapetum-controlled pollen development. All six ArfA1s in the Arabidopsis genome are expressed during anther development, among which ArfA1b is specific to the tapetum and developing microspores. Although the ArfA1b loss-of-function mutant showed no pollen defects, probably due to redundancy, interference with ArfA1s by a dominant negative approach in the tapetum resulted in tapetal dysfunction and pollen abortion. We further showed that all six ArfA1s are associated with the Golgi and the trans-Golgi network/early endosome, suggesting that they have roles in regulating post-Golgi trafficking to the plasma membrane or to vacuoles. Indeed, we demonstrated that the expression of ArfA1bDN interfered with the targeting of proteins critical for tapetal development. The results presented here demonstrate a key role of ArfA1s in tapetum-controlled pollen development by mediating protein targeting through post-Golgi trafficking routes.

The evolution of eukaryotic cellular complexity is interwoven with the extensive diversification of many protein families. One key family is the ARF GTPases that act in eukaryote-specific processes, including membrane traffic, tubulin assembly, actin dynamics, and cilia-related functions. Unfortunately, our understanding of the evolution of this family is limited. Sampling an extensive set of available genome and transcriptome sequences, we have assembled a dataset of over 2,000 manually curated ARF family genes from 114 eukaryotic species, including many deeply diverged protist lineages, and carried out comprehensive molecular phylogenetic analyses. These reconstructed as many as 16 ARF family members present in the last eukaryotic common ancestor (LECA), nearly doubling the previously inferred ancient system complexity. Evidence for the wide occurrence and ancestral origin of Arf6, Arl13 and Arl16 is presented for the first time. Moreover, Arl17, Arl18 and SarB, newly described here, are absent from well-studied model organisms and as a result their function(s) remain unknown. Analyses of our dataset revealed a previously unsuspected diversity of membrane association modes and domain architectures within the ARF family. We detail the step-wise expansion of the ARF family in the metazoan lineage, including discovery of several new animal-specific family members. Delving back to its earliest evolution in eukaryotes, the resolved relationship observed between the ARF family paralogs sets boundaries for scenarios of vesicle coat origins during eukaryogenesis. Altogether, our work fundamentally broadens the understanding of the diversity and evolution of a protein family underpinning the structural and functional complexity of the eukaryote cells.SignificanceARF Family GTPases are crucial regulations of a diversity of cellular compartments and processes and as such the extent of this system in eukaryotes reflects both cellular complexity in modern eukaryotes and its evolution. Strikingly, a comprehensive comparative genomic analysis of the protein family is lacking, leaving its recent and ancient evolution poorly resolved. We performed a comprehensive molecular evolutionary analysis, reconstructing a highly complex ARF family complement in the Last Eukaryotic Common Ancestor, including a number of paralogs never before identified as such, and we find resolved relationships between the paralogs. This work has implications for cellular evolution from eukaryogenesis to cellular complexity in metazoans.

Small GTPases (e.g., Rac1) play key roles in glucose-stimulated insulin secretion (GSIS) in the β-cell. We investigated regulation by RhoGDIβ of glucose-induced activation of Rac1 and insulin secretion. RhoGDIβ is expressed in INS-1 832/13 cells, rodent and human islets. siRNA-mediated knockdown of RhoGDIβ in INS-1 832/13 cells significantly attenuated glucose-induced Rac1 activation without affecting its translocation and membrane association. Further, suppression of RhoGDIβ expression exerted minimal effects on GSIS at the height of inhibition of Rac1 activation, suggesting divergent effects of RhoGDIβ on Rac1 activation and insulin secretion in the glucose-stimulated β-cell. We provide the first evidence for the expression of RhoGDIβ in rodent and human β-cells, and its differential regulatory roles of this protein in G protein activation and GSIS. Abbreviations: Arf6: ADP ribosylation factor; Cdc42: Cell Division Cycle; GAP: GTPase-activating protein; GDI: GDP dissociation inhibitor; GDIα: GDP dissociation inhibitorα; GDIβ: GDP dissociation inhibitorβ; GEF: Guanine nucleotide exchange factor; GSIS: Glucose-stimulated insulin secretion; Rac1: Ras-Related C3 Botulinum Toxin Substrate 1.

ADP-ribosylation factors (ARFs) are a family of small monomeric GTPases comprising six members categorized into three classes: class I (ARF1, 2, and 3), class II (ARF4 and 5), and class III (ARF6). In contrast to class I and III ARFs, which are the key regulators in vesicular membrane trafficking, the cellular function of class II ARFs remains unclear. In the present study, we generated class II ARF-deficient mice and found that ARF4(+/−)/ARF5(−/−) mice exhibited essential tremor (ET)-like behaviors. In vivo electrophysiological recordings revealed that ARF4(+/−)/ARF5(−/−) mice of both sexes exhibited abnormal brain activity when moving, raising the possibility of abnormal cerebellar excitability. Slice patch-clamp experiments demonstrated the reduced excitability of the cerebellar Purkinje cells (PCs) in ARF4(+/−)/ARF5(−/−) mice. Immunohistochemical and electrophysiological analyses revealed a severe and selective decrease of pore-forming voltage-dependent Na(+) channel subunit Nav1.6, important for maintaining repetitive action potential firing, in the axon initial segment (AIS) of PCs. Importantly, this decrease in Nav1.6 protein localized in the AIS and the consequent tremors in ARF4(+/−)/ARF5(−/−) mice could be alleviated by the PC-specific expression of ARF5 using adeno-associated virus vectors. Together, our data demonstrate that the decreased expression of the class II ARF proteins in ARF4(+/−)/ARF5(−/−) mice, leading to a haploinsufficiency of ARF4 in the absence of ARF5, impairs the localization of Nav1.6 to the AIS and hence reduces the membrane excitability in PCs, resulting in the ET-like movement disorder. We suggest that class II ARFs function in localizing specific proteins, such as Nav1.6, to the AIS. SIGNIFICANCE STATEMENT We found that decreasing the expression of class II ARF proteins, through the generation of ARF4(+/−)/ARF5(−/−) mice, impairs Nav1.6 distribution to the axon initial segment (AIS) of cerebellar Purkinje cells (PCs), thereby resulting in the impairment of action potential firing of PCs. The ARF4(+/−)/ARF5(−/−) mutant mice exhibited movement-associated essential tremor (ET)-like behavior with pharmacological profiles similar to those in ET patients. The exogenous expression of ARF5 reduced the tremor phenotype and restored the localization of Nav1.6 immunoreactivity to the AIS in ARF4(+/−)/ARF5(−/−) mice. Thus, our results suggest that class II ARFs are involved in the localization of Nav1.6 to the AISs in cerebellar PCs and that the reduction of class II ARF activity leads to ET-like movement disorder.

Low salinity treatment is proven to be the practical polyploidy inducing method for shellfish with advantages of lower cost, higher operability and reliable food security. However, little is known about the possible molecular mechanism of hypotonic induction. In this study, isobaric tags for relative and absolute quantitation (iTRAQ) based proteomic profiling was pursued to investigate the responses of zygotes of the Pacific oyster Crassostrea gigas to low salinity. A total of 2235 proteins were identified and 87 proteins were considered differentially expressed, of which 14 were up-regulated and 69 were down-regulated. Numerous functional proteins including ADP ribosylation factor 2, DNA repair protein Rad50, splicing factor 3B, tubulin-specific Chaperone D were significantly changed in abundance, and were involved in various biology processes including energy generation, vesicle trafficking, DNA/RNA/protein metabolism and cytoskeleton modification, indicating the prominent modulation of cell division and embryonic development. Parallel reaction monitoring (PRM) analyses were carried out for validation of the expression levels of differentially expressed proteins (DEPs), which indicated high reliability of the proteomic results. Our study not only demonstrated the proteomic alterations in oyster zygotes under low salinity, but also provided, in part, clues to the relatively lower hatching rate and higher mortality of induced larvae. Above all, this study presents a valuable foundation for further studies on mechanisms of hypotonic induction.

Toll-like receptor 7 (TLR7) and type I interferons (IFN-1) are essential for the development of systemic lupus erythematosus (SLE) models such as BXSB.Yaa and 2,6,10,14-tetramethyl-pentadecane (TMPD)-induced experimental lupus. However, the mechanism underlying the development of SLE remains undefined. We report a requirement for ADP-ribosylation factor-like 8b (Arl8b) for TLR7-dependent IFN-1 production in plasmacytoid dendritic cells (pDCs). We analyzed whether Arl8b plays a role in two SLE models by comparing wild-type and Arl8b-deficient Arl8b GeneTrap (Arl8bGt/Gt) mice. We found that BXSB.Yaa Arl8bGt/Gt mice showed none of the abnormalities characterized in BXSB.Yaa mice. TMPD treatment of Arl8bGt/Gt mice significantly inhibited the development of SLE. pDCs were required for TMPD-induced peritonitis. Our data demonstrate that Arl8b contributes to disease pathogenesis in two SLE models via IFN-1-dependent and -independent mechanisms and suggest that Arl8b is an attractive new target for therapeutic intervention in SLE.

ADP-ribosylation factors (Arfs) and Arf-like (Arl) GTPases are key regulators of intracellular vesicle trafficking and Golgi structure. Both Arf and Arl proteins cycle between active GTP-bound and inactive GDP-bound forms, where guanine nucleotide exchange factors (GEFs) regulate the exchange of GDP for GTP, whereas GTPase-activating proteins (GAPs) promote the hydrolysis of bound GTP. Human Arl1 is located at the trans-Golgi network (TGN) and regulates the function and structure of the Golgi complex. However, neither GEFs nor GAPs for human Arl1 have been identified. Here, we report that ArfGAP1, an Arf1 GAP, can promote GTP hydrolysis of Arl1. We show that ArfGAP1 directly interacts with GTP-bound Arl1 and exhibits GAP activity toward Arl1 in vitro. Exogenous expression of ArfGAP1, but not ArfGAP2 and ArfGAP3, causes dissociation of endogenous Arl1 from the TGN. In addition, GAP activity-deficient ArfGAP1 fails to regulate the Golgi localization of Arl1. Using an activity pull-down assay, we demonstrated that ArfGAP1 regulates the levels of Arl1-GTP in cells expressing ArfGAP1-myc or with ArfGAP1 knockdown. Finally, we observed that, similar to expression of putative active Arl1 (Arl1QL), ArfGAP1 knockdown impairs endosome-to-TGN retrograde transport of the Shiga toxin B-subunit. Thus, our findings support the idea that ArfGAP1 acts as an Arl1 GAP to regulate the function of Arl1 in vesicle trafficking at the TGN.

The signalling pathways underpinning cell growth and invasion use overlapping components, yet how mutually exclusive cellular responses occur is unclear. Here, we report development of 3-Dimensional culture analyses to separately quantify growth and invasion. We identify that alternate variants of IQSEC1, an ARF GTPase Exchange Factor, act as switches to promote invasion over growth by controlling phosphoinositide metabolism. All IQSEC1 variants activate ARF5- and ARF6-dependent PIP5-kinase to promote PI(3,4,5)P3-AKT signalling and growth. In contrast, select pro-invasive IQSEC1 variants promote PI(3,4,5)P3 production to form invasion-driving protrusions. Inhibition of IQSEC1 attenuates invasion in vitro and metastasis in vivo. Induction of pro-invasive IQSEC1 variants and elevated IQSEC1 expression occurs in a number of tumour types and is associated with higher-grade metastatic cancer, activation of PI(3,4,5)P3 signalling, and predicts long-term poor outcome across multiple cancers. IQSEC1-regulated phosphoinositide metabolism therefore is a switch to induce invasion over growth in response to the same external signal. Targeting IQSEC1 as the central regulator of this switch may represent a therapeutic vulnerability to stop metastasis., The signalling pathways underpinning cell growth and invasion use overlapping components, yet how mutually exclusive responses occur is unclear. Here, the authors show that alternate isoforms of the ARF GTPase exchange factor IQSEC1 direct phosphoinositide metabolism to control this switch.

Small guanosine triphosphatases (GTPases) of the Rab and Arf families are key regulators of vesicle formation and membrane trafficking. Membrane transport plays an important role in the central nervous system. In this regard, neurons require a constant flow of membranes for the correct distribution of receptors, for the precise composition of proteins and organelles in dendrites and axons, for the continuous exocytosis/endocytosis of synaptic vesicles and for the elimination of dysfunctional proteins. Thus, it is not surprising that Rab and Arf GTPases have been associated with neurodegenerative diseases such as Alzheimer’s and Parkinson’s. Both pathologies share characteristics such as the presence of protein aggregates and/or the fragmentation of the Golgi apparatus, hallmarks that have been related to both Rab and Arf GTPases functions. Despite their relationship with neurodegenerative disorders, very few studies have focused on the role of these GTPases in the pathogenesis of neurodegeneration. In this review, we summarize their importance in the onset and progression of Alzheimer’s and Parkinson’s diseases, as well as their emergence as potential therapeutical targets for neurodegeneration. A.A.S. is a recipient of a predoctoral fellowship (PRE_2017_1_0016) from the Basque Government. M.L.M. is a recipient of a fellowship from Foundation “Jesús de Gangoiti y Barrera”. J.L.Z. was supported by the Instituto de Salud Carlos III (PI18/00207) and the University of Basque Country Grant (US19/04).

PURPOSE: ADP ribosylation factor guanine nucleotide exchange factors (ARFGEFs) are a family of proteins implicated in cellular trafficking between the Golgi apparatus and the plasma membrane through vesicle formation. Among them is ARFGEF1/BIG1, a protein involved in axon elongation, neurite development, and polarization processes. ARFGEF1 has been previously suggested as a candidate gene for different types of epilepsies, although its implication in human disease has not been well characterized.METHODS: International data sharing, in silico predictions, and in vitro assays with minigene study, western blot analyses, and RNA sequencing.RESULTS: We identified 13 individuals with heterozygous likely pathogenic variants in ARFGEF1. These individuals displayed congruent clinical features of developmental delay, behavioral problems, abnormal findings on brain magnetic resonance image (MRI), and epilepsy for almost half of them. While nearly half of the cohort carried de novo variants, at least 40% of variants were inherited from mildly affected parents who were clinically re-evaluated by reverse phenotyping. Our in silico predictions and in vitro assays support the contention that ARFGEF1-related conditions are caused by haploinsufficiency, and are transmitted in an autosomal dominant fashion with variable expressivity.CONCLUSION: We provide evidence that loss-of-function variants in ARFGEF1 are implicated in sporadic and familial cases of developmental delay with or without epilepsy.

The ARL15 gene (ADP ribosylation factor like protein 15) encodes for an uncharacterized small GTP-binding protein. Its exact role in human physiology remains unknown, but a number of genetic association studies have recognised different variants in this gene to be statistically associated with numerous traits and complex diseases. We have previously reported a novel association of ARL15 with rheumatoid arthritis (RA) based on a genome-wide association study in a north Indian cohort. Subsequent investigations have provided leads for its involvement in RA pathophysiology, especially its potential as a novel therapeutic target. However, the absence of an experimentally determined tertiary structure for ARL15 significantly hinders the understanding of its biochemical and physiological functions, as well as development of potential lead molecules. We, therefore, aimed to derive a high quality, refined model of the three dimensional structure of human ARL15 protein using two different computational protein structure prediction methods – template-based threading and ab initio modelling. The best model each from among the five each derived from both the approaches was selected based on stringent quality assessment and refinement. Molecular dynamics simulations over long timescales revealed the ab initio model to be relatively more stable, and it marginally outperformed the template-based model in the quality assessment as well. A putative GTP-binding site was also predicted using homology for the ARL15 protein, where potential competitive inhibitors can be targeted. This high quality predicted model may provide insights to the biological role(s) of ARL15 and inform and guide further experimental, structural and biochemical characterization efforts. Communicated by Ramaswamy H. Sarma

Arf GTPase activating (ArfGAP) proteins are critical regulatory and effector proteins in membrane trafficking pathways. Budding yeast contain two ArfGAP proteins (Gcs1 and Glo3) implicated in COPI coat function at the Golgi, and yeast require Glo3 catalytic function for viability. A new X-ray crystal structure of the Glo3 GAP domain was determined at 2.1 A resolution using molecular replacement methods. The structure reveals a Cys4-family zinc finger motif with an invariant residue (R59) positioned to act as an “arginine finger” during catalysis. Comparisons among eukaryotic GAP domains show a key difference between ArfGAP1 and ArfGAP2/3 family members in the final helix located within the domain. Conservation at both the sequence and structural levels suggest the Glo3 GAP domain interacts with yeast Arf1 switch I and II regions to promote catalysis. Together, the structural data presented here provide additional evidence for placing Glo3 near Arf1 triads within membrane-assembled COPI coats and further support the molecular niche model for COPI coat regulation by ArfGAPs.

Pathogenic Vibrio species use many different approaches to subvert, attack, and undermine the host response. The toxins they produce are often responsible for the devastating effects associated with their diseases. These toxins target a variety of host proteins, which leads to deleterious effects, including dissolution of cell organelle integrity and inhibition of protein secretion. Becoming increasingly prevalent as cofactors for Vibrio toxins are proteins of the small GTPase families. ADP-ribosylation factor small GTPases (ARFs) in particular are emerging as a common host cofactor necessary for full activation of Vibrio toxins. While ARFs are not the direct target of Vibrio cholerae cholera toxin (CT), ARF binding is required for its optimal activity as an ADP-ribosyltransferase. The makes caterpillars floppy (MCF)-like and the domain X (DmX) effectors of the Vibrio vulnificus multifunctional autoprocessing repeats-in-toxin (MARTX) toxin also both require ARFs to initiate autoprocessing and activation as independent effectors. ARFs are ubiquitously expressed in eukaryotes and are key regulators of many cellular processes, and as such they are ideal cofactors for Vibrio pathogens that infect many host species. In this review, we cover in detail the known Vibrio toxins that use ARFs as cross-kingdom activators to both stimulate and optimize their activity. We further discuss how these contrast to toxins and effectors from other bacterial species that coactivate, stimulate, or directly modify host ARFs as their mechanisms of action.

Arf-family small GTPases are essential protein components for membrane trafficking in all eukaryotic endomembrane systems, particularly during the formation of membrane-bound, coat protein complex-coated transport carriers. In addition to their roles in the transport carrier formation, a number of Arf-family GTPases have been reported to physically associate with coiled-coil tethering proteins and multisubunit tethering complexes, which are responsible for membrane tethering, a process of the initial contact between transport carriers and their target subcellular compartments. Nevertheless, whether and how indeed Arf GTPases are involved in the tethering process remain unclear. Here, using a chemically-defined reconstitution approach with purified proteins of two representative Arf isoforms in humans (Arf1, Arf6) and synthetic liposomes for model membranes, we discovered that Arf6 can function as a bona fide membrane tether, directly and physically linking two distinct lipid bilayers even in the absence of any other tethering factors, whereas Arf1 retained little potency to trigger membrane tethering under the current experimental conditions. Arf6-mediated membrane tethering reactions require trans-assembly of membrane-anchored Arf6 proteins and can be reversibly controlled by the membrane attachment and detachment cycle of Arf6. The intrinsic membrane tethering activity of Arf6 was further found to be significantly inhibited by the presence of membrane-anchored Arf1, suggesting that the tethering-competent Arf6-Arf6 assembly in trans can be prevented by the heterotypic Arf1-Arf6 association in a cis configuration. Taken together, these findings lead us to postulate that self-assemblies of Arf-family small GTPases on lipid bilayers contribute to driving and regulating the tethering events of intracellular membrane trafficking.

The ADP ribosylation factor (ARF) GTPase activation protein ASAP1 possesses multiple biological functions, including regulation of cytoskeletal dynamics, small GTP-binding protein receptor recycling, and intracellular vesicle trafficking. Recently, ASAP1 polymorphisms have been reported to be associated with human susceptibility to tuberculosis (TB) according to a large-scale genome-wide association study (GWAS); ASAP1 expression affects dendritic cell migration, which may be involved in TB predisposition. However, it remains unclear whether ASAP1 affects TB in vivo. To address this issue, we used zebrafish as a model system to examine the effects of Asap1 against Mycobacterium marinum, an organism closely related to Mycobacterium tuberculosis. Two zebrafish asap1 homologs (asap1a and asap1b) were identified and characterized. By morpholino knockdown of asap1a and asap1b as a whole, we found that the asap1 morphants showed a higher mycobacterial load than the controls, which was almost rescued by injecting asap1 mRNA that confers resistance to mycobacterial infection. These Asap1-depleted zebrafish also exhibited decreased macrophage migration in response to tail injury or upon infection with M. marinum in the hindbrain ventricle, which was also proved in THP1-derived macrophages of knockdown ASAP1. Together, these findings represent a new perspective on the role of Asap1 in resistance to mycobacterial infection.

Ras-related C3 botulinum toxin substrate 1 (Rac1) is a member of the Rho family of GTPases that functions as a molecular switch to regulate many important cellular events including actin cytoskeleton remodeling during neurite outgrowth. Engulfment and cell motility 1 (ELMO1)-dedicator of cytokinesis 1 (DOCK180) is a bipartite guanine nucleotide exchange factor (GEF) complex that has been reported to activate Rac1 on the plasma membrane (PM). Emerging evidence suggests that the small GTPase ADP ribosylation factor 6 (ARF6) activates Rac1 via the ELMO1/DOCK180 complex. However, the exact mechanism by which ARF6 triggers ELMO1/DOCK180-mediated Rac1 signaling remains unclear. Here, we report that the neuronal scaffold protein FE65 serves as a functional link between ARF6 and ELMO1, allowing the formation of a multimeric signaling complex. Interfering with formation of this complex by transfecting either FE65-binding-defective mutants or FE65 siRNA attenuates both ARF6-ELMO1-mediated Rac1 activation and neurite elongation. Notably, the PM trafficking of ELMO1 is markedly decreased in cells with suppressed expression of either FE65 or ARF6. Likewise, this process is attenuated in the FE65-binding-defective mutants transfected cells. Moreover, overexpression of FE65 increases the amount of ELMO1 in the recycling endosome, an organelle responsible for returning proteins to the PM, whereas knockout of FE65 shows opposite effect. Together, our data indicates that FE65 potentiates ARF6-Rac1 signaling by orchestrating ARF6 and ELMO1 to promote the PM trafficking of ELMO1 via the endosomal recycling pathway, and thus, promotes Rac1-mediated neurite outgrowth.

ADP ribosylation factor 6 (Arf6) is a small GTP-binding protein implicated in neuronal morphogenesis through endosomal trafficking and actin remodeling. In this study, we identified Vps52, a core subunit of the Golgi-associated retrograde protein (GARP) and endosome-associated recycling protein (EARP) complexes, as a novel Arf6-binding protein by yeast two-hybrid screening. Vps52 interacted specifically with GTP-bound Arf6 among the Arf family. Immunohistochemical analyses of hippocampal pyramidal cells revealed that fine punctate immunolabeling for Vps52 was distributed throughout neuronal compartments, most densely in the cell body and dendritic shafts, and was largely associated with trans-Golgi network and vesicular endomembranes. In cultured hippocampal neurons, knockdown of Vps52 increased total length of axons and dendrites; these phenotypes were completely restored by co-expression of shRNA-resistant full-length Vps52. However, co-expression of a Vps52 mutant lacking the ability to interact with Arf6 restored only the Vps52-knockdown phenotype of the dendritic length. The present findings suggest that Vps52 is a novel Arf6-interacting protein that regulates neurite outgrowth in hippocampal neurons.

Membrane trafficking maintains the organization of the eukaryotic cell and delivers cargo proteins to their subcellular destinations, such as sites of action or degradation. The formation of membrane vesicles requires the activation of the ADP-ribosylation factor ARF GTPase by the SEC7 domain of ARF guanine-nucleotide exchange factors (ARF-GEFs), resulting in the recruitment of coat proteins by GTP-bound ARFs. In vitro exchange assays were done with monomeric proteins, although ARF-GEFs form dimers in vivo. This feature is conserved across eukaryotes, although its biological significance is unknown. Here, we demonstrate the proximity of ARF1•GTPs in vivo by fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy, mediated through coordinated activation by dimers of Arabidopsis (Arabidopsis thaliana) ARF-GEF GNOM, which is involved in polar recycling of the auxin transporter PIN-FORMED1. Mutational disruption of ARF1 spacing interfered with ARF1-dependent trafficking but not with coat protein recruitment. A mutation impairing the interaction of one of the two SEC7 domains of the GNOM ARF-GEF dimer with its ARF1 substrate reduced the efficiency of coordinated ARF1 activation. Our results suggest a model of coordinated activation-dependent membrane insertion of ARF1•GTP molecules required for coated membrane vesicle formation. Considering the evolutionary conservation of ARFs and ARF-GEFs, this initial regulatory step of membrane trafficking might well occur in eukaryotes in general.

Membrane traffic maintains the organization of the eukaryotic cell and delivers cargo proteins to their subcellular destinations such as sites of action or degradation. Membrane vesicle formation requires ARF GTPase activation by the SEC7 domain of ARF guanine-nucleotide exchange factors (ARF-GEFs), resulting in the recruitment of coat proteins by GTP-bound ARFs.In vitroexchange assays were done with monomeric proteins, although ARF-GEFs have been shown to form dimersin vivo. This feature is conserved across the eukaryotes, however its biological significance is unknown. Here we demonstrate ARF1 dimerizationin vivoand we show that ARF-GEF dimers mediate ARF1 dimer formation. Mutational disruption of ARF1 dimers interfered with ARF1-dependent trafficking but not coat protein recruitment in Arabidopsis. Mutations disrupting simultaneous binding of two ARF1•GDPs by the two SEC7 domains of GNOM ARF-GEF dimer prevented stable interaction of ARF1 with ARF-GEF and thus, efficient ARF1 activation. Our results suggest a model of activation-dependent dimerization of membrane-inserted ARF1•GTP molecules required for coated membrane vesicle formation. Considering the evolutionary conservation of ARFs and ARF-GEFs, this initial regulatory step of membrane trafficking might well occur in eukaryotes in general.

The ADP-ribosylation factors (Arfs) comprise a family of regulatory GTP binding proteins. The Arfs regulate membrane trafficking and cytoskeleton remodeling, processes critical for eukaryotes and which have been the focus of most studies on Arfs. A more limited literature describes a role in signaling and in integrating several signaling pathways to bring about specific cell behaviors. Here, we will highlight work describing function of Arf1, Arf6 and several effectors and regulators of Arfs in signaling.

VAN3 is a plant ACAP-type ADP-ribosylation factor-GTPase activating protein (ARF-GAP) that regulates auxin transport-mediated plant morphogenesis such as continuous venation and lateral root development in Arabidopsis. Previous studies suggested that VAN3 localizes at the plasma membrane (PM) and intracellular structures. However, the role of PM localization in mediating the van3 mutant phenotype is not clear. Here we performed subcellular localization analysis of VAN3 and its regulators CVP2 and VAB to determine their endogenous functions. We found that GFP-tagged CVP2 and VAB preferentially localize at the PM in stably transformed plants. We determined that transgenic plants with lower expression levels of GFP- or mRFP-tagged VAN3 displayed PM localization, which was sufficient to rescue the van3 mutant. Functional VAN3-mRFP and VAB-GFP colocalized at PMs. The van3 mutant phenotype was suppressed by mutation of VAN7/GNOM, which encodes an ARF-GEF that localizes at the PM and Golgi apparatus. These combined results suggest that ARF-GTPase machinery at the PM regulates auxin transport-mediated plant growth and development.

Summary Membrane dynamic processes require Arf GTPase activation by guanine nucleotide exchange factors (GEFs) with a Sec7 domain. Cytohesin family Arf GEFs function in signaling and cell migration through Arf GTPase activation on the plasma membrane and endosomes. In this study, the structural organization of two cytohesins (Grp1 and ARNO) was investigated in solution by size exclusion-small angle X-ray scattering and negative stain-electron microscopy and on membranes by dynamic light scattering, hydrogen-deuterium exchange-mass spectrometry and guanosine diphosphate (GDP)/guanosine triphosphate (GTP) exchange assays. The results suggest that cytohesins form elongated dimers with a central coiled coil and membrane-binding pleckstrin-homology (PH) domains at opposite ends. The dimers display significant conformational heterogeneity, with a preference for compact to intermediate conformations. Phosphoinositide-dependent membrane recruitment is mediated by one PH domain at a time and alters the conformational dynamics to prime allosteric activation by Arf-GTP. A structural model for membrane targeting and allosteric activation of full-length cytohesin dimers is discussed., Graphical Abstract, Highlights • Structural organization of cytohesins investigated by SEC-SAXS, NS-EM, DLS, and HDX-MS • Cytohesin dimers have an elongated and conformationally dynamic quaternary structure • Cytohesin dimers bind membranes using one PH domain at a time • Membrane binding perturbs the structural organization to prime allosteric activation, Arf GEFs activate Arf GTPases to control membrane dynamics. Das et al. investigate the structural organization of cytohesin dimers in solution and on membranes. The dimers adopt elongated, dynamic conformations and use one PH domain at a time to bind membranes, which perturbs the structural organization to prime allosteric activation.

Approximately one-third of all eukaryotic proteins are delivered to their destination by trafficking within the endomembrane system. Such cargo proteins are incorporated into forming membrane vesicles on donor compartments and delivered to acceptor compartments by vesicle fusion. How cargo proteins are sorted into forming vesicles is still largely unknown. Here we review the roles of small GTPases of the ARF/SAR1 family, their regulators designated ARF guanine-nucleotide exchange factors (ARF-GEFs) and ARF GTPase-activating proteins (ARF-GAPs) as well as coat protein complexes during membrane vesicle formation. Although conserved across eukaryotes, these four functional groups of proteins display plant-specific modifications in composition, structure and function.

Arf6/ARF-6 is a crucial regulator of the endosomal PI(4,5)P2 pool in endocytic recycling. Chen et al. show that SAC-1 is important for epithelial cell recycling in C. elegans. SAC-1 acts as a novel interactor for ARF-6, curbing ARF-6 activity by limiting the access of ARF-6(GDP) to its GEF BRIS-1., Arf6/ARF-6 is a crucial regulator of the endosomal phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) pool in endocytic recycling. To further characterize ARF-6 regulation, we performed an ARF-6 interactor screen in Caenorhabditis elegans and identified SAC-1, the homologue of the phosphoinositide phosphatase Sac1p in yeast, as a novel ARF-6 partner. In the absence of ARF-6, basolateral endosomes show a loss of SAC-1 staining in epithelial cells. Steady-state cargo distribution assays revealed that loss of SAC-1 specifically affected apical secretory delivery and basolateral recycling. PI(4,5)P2 levels and the endosomal labeling of the ARF-6 effector UNC-16 were significantly elevated in sac-1 mutants, suggesting that SAC-1 functions as a negative regulator of ARF-6. Further analyses revealed an interaction between SAC-1 and the ARF-6-GEF BRIS-1. This interaction outcompeted ARF-6(guanosine diphosphate [GDP]) for binding to BRIS-1 in a concentration-dependent manner. Consequently, loss of SAC-1 promotes the intracellular overlap between ARF-6 and BRIS-1. BRIS-1 knockdown resulted in a significant reduction in PI(4,5)P2 levels in SAC-1-depleted cells. Interestingly, the action of SAC-1 in sequestering BRIS-1 is independent of SAC-1’s catalytic activity. Our results suggest that the interaction of SAC-1 with ARF-6 curbs ARF-6 activity by limiting the access of ARF-6(GDP) to its guanine nucleotide exchange factor, BRIS-1.

Mucor circinelloides is a dimorphic fungus used to study cell differentiation that has emerged as a model to characterize mucormycosis. In this work, we identified four ADP-ribosylation factor (Arf)-encoding genes (arf1-arf4) and study their role in the morphogenesis and virulence. Arfs are key regulators of the vesicular trafficking process and are associated with both growth and virulence in fungi. Arf1 and Arf2 share 96% identity and Arf3 and Arf4 share 89% identity, which suggests that the genes arose through gene-duplication events in M. circinelloides. Transcription analysis revealed that certain arf genes are affected by dimorphism of M. circinelloides, such as the arf2 transcript, which was accumulated during yeast development. Therefore, we created knockout mutants of four arf genes to evaluate their function in dimorphism and virulence. We found that both arf1 and arf2 are required for sporulation, but these genes also perform distinct functions; arf2 participates in yeast development, whereas arf1 is involved in aerobic growth. Conversely, arf3 and arf4 play only minor roles during aerobic growth. Moreover, we observed that all single arf-mutant strains are more virulent than the wild-type strain in mouse and nematode models, with the arf3 mutant being most virulent. Lastly, arf1/arf2 and arf3/arf4 double mutations produced heterokaryon strains that did not reach the homokaryotic state, indicating that these genes participate in essential and redundant functions. Overall, this work reveals that Arfs proteins regulate important cellular processes in M. circinelloides such as morphogenesis and virulence, laying the foundation to characterize the molecular networks underlying this regulation.

Group I metabotropic glutamate receptors (mGluRs), mGluR1 and mGluR5, in the spinal cord are implicated in nociceptive transmission and plasticity through G protein-mediated second messenger cascades leading to the activation of various protein kinases such as extracellular signal-regulated kinase (ERK). In this study, we demonstrated that cytohesin-2, a guanine nucleotide exchange factor for ADP ribosylation factors (Arfs), is abundantly expressed in subsets of excitatory interneurons and projection neurons in the superficial dorsal horn. Cytohesin-2 is enriched in the perisynapse on the postsynaptic membrane of dorsal horn neurons and forms a protein complex with mGluR5 in the spinal cord. Central nervous system-specific cytohesin-2 conditional knockout mice exhibited reduced mechanical allodynia in inflammatory and neuropathic pain models. Pharmacological blockade of cytohesin catalytic activity with SecinH3 similarly reduced mechanical allodynia and inhibited the spinal activation of Arf6, but not Arf1, in both pain models. Furthermore, cytohesin-2 conditional knockout mice exhibited reduced mechanical allodynia and ERK1/2 activation following the pharmacological activation of spinal mGluR1/5 with 3,5-dihydroxylphenylglycine (DHPG). The present study suggests that cytothesin-2 is functionally associated with mGluR5 during the development of mechanical allodynia through the activation of Arf6 in spinal dorsal horn neurons.

Tuberculosis is initiated by the entry of Mycobacterium tuberculosis (Mtb) into macrophages in the lungs. A study of the cellular factors responsible for the entry of Mtb into host cells will potentially benefit the development of therapeutic treatments or preventive agents against Mtb infection. Using human THP1-derived macrophages as a model, we found that infection of Mtb H37Ra transiently reduced the level of ASAP1, an ADP ribosylation factor (Arf)-GTPase activating protein. Furthermore, knockdown of ASAP1 increased the efficiency of H37Ra entry into the cell and altered the status of actin remodeling as indicated by the enhanced aggregation of F-actin and the increased numbers of vinculin- and paxillin-rich puncta. Collectively, the results in this report identified ASAP1 as a regulator controlling the entry of Mtb H37Ra into macrophage by remodeling actin cytoskeleton.