HIV-1 Nefs Are Cargo-Sensitive AP-1 Trimerization Switches in Tetherin Downregulation.

The HIV accessory protein Nef counteracts immune defenses by subverting coated vesicle pathways. The 3.7 Å cryo-EM structure of a closed trimer of the clathrin adaptor AP-1, the small GTPase Arf1, HIV-1 Nef, and the cytosolic tail of the restriction factor tetherin suggested a mechanism for inactivating tetherin by Golgi retention. The 4.3 Å structure of a mutant Nef-induced dimer of AP-1 showed how the closed trimer is regulated by the dileucine loop of Nef. HDX-MS and mutational analysis were used to show how cargo dynamics leads to alternative Arf1 trimerization, directing Nef targets to be either retained at the trans-Golgi or sorted to lysosomes. Phosphorylation of the NL4-3 M-Nef was shown to regulate AP-1 trimerization, explaining how O-Nefs lacking this phosphosite counteract tetherin but most M-Nefs do not. These observations show how the higher-order organization of a vesicular coat can be allosterically modulated to direct cargoes to distinct fates.

Essential role for GABARAP autophagy proteins in interferon-inducible GTPase-mediated host defense.

Mammalian autophagy-related 8 (Atg8) homologs consist of LC3 proteins and GABARAPs, all of which are known to be involved in canonical autophagy. In contrast, the roles of Atg8 homologs in noncanonical autophagic processes are not fully understood. Here we show a unique role of GABARAPs, in particular gamma-aminobutyric acid (GABA)-A-receptor-associated protein-like 2 (Gabarapl2; also known as Gate-16), in interferon-γ (IFN-γ)-mediated antimicrobial responses. Cells that lacked GABARAPs but not LC3 proteins and mice that lacked Gate-16 alone were defective in the IFN-γ-induced clearance of vacuolar pathogens such as Toxoplasma. Gate-16 but not LC3b specifically associated with the small GTPase ADP-ribosylation factor 1 (Arf1) to mediate uniform distribution of interferon-inducible GTPases. The lack of GABARAPs reduced Arf1 activation, which led to formation of interferon-inducible GTPase-containing aggregates and hampered recruitment of interferon-inducible GTPases to vacuolar pathogens. Thus, GABARAPs are uniquely required for antimicrobial host defense through cytosolic distribution of interferon-inducible GTPases.

Structural basis for recruitment and activation of the AP-1 clathrin adaptor complex by Arf1.

AP-1 is a clathrin adaptor complex that sorts cargo between the trans-Golgi network and endosomes. AP-1 recruitment to these compartments requires Arf1-GTP. The crystal structure of the tetrameric core of AP-1 in complex with Arf1-GTP, together with biochemical analyses, shows that Arf1 activates cargo binding by unlocking AP-1. Unlocking is driven by two molecules of Arf1 that bridge two copies of AP-1 at two interaction sites. The GTP-dependent switch I and II regions of Arf1 bind to the N terminus of the ß1 subunit of one AP-1 complex, while the back side of Arf1 binds to the central part of the γ subunit trunk of a second AP-1 complex. A third Arf1 interaction site near the N terminus of the γ subunit is important for recruitment, but not activation. These observations lead to a model for the recruitment and activation of AP-1 by Arf1.

A four-step cycle driven by PI(4)P hydrolysis directs sterol/PI(4)P exchange by the ER-Golgi tether OSBP.

Several proteins at endoplasmic reticulum (ER)-Golgi membrane contact sites contain a PH domain that interacts with the Golgi phosphoinositide PI(4)P, a FFAT motif that interacts with the ER protein VAP-A, and a lipid transfer domain. This architecture suggests the ability to both tether organelles and transport lipids between them. We show that in oxysterol binding protein (OSBP) these two activities are coupled by a four-step cycle. Membrane tethering by the PH domain and the FFAT motif enables sterol transfer by the lipid transfer domain (ORD), followed by back transfer of PI(4)P by the ORD. Finally, PI(4)P is hydrolyzed in cis by the ER protein Sac1. The energy provided by PI(4)P hydrolysis drives sterol transfer and allows negative feedback when PI(4)P becomes limiting. Other lipid transfer proteins are tethered by the same mechanism. Thus, OSBP-mediated back transfer of PI(4)P might coordinate the transfer of other lipid species at the ER-Golgi interface.

A structure-based mechanism for Arf1-dependent recruitment of coatomer to membranes.

Budding of COPI-coated vesicles from Golgi membranes requires an Arf family G protein and the coatomer complex recruited from cytosol. Arf is also required with coatomer-related clathrin adaptor complexes to bud vesicles from the trans-Golgi network and endosomal compartments. To understand the structural basis for Arf-dependent recruitment of a vesicular coat to the membrane, we determined the structure of Arf1 bound to the γζ-COP subcomplex of coatomer. Structure-guided biochemical analysis reveals that a second Arf1-GTP molecule binds to ßδ-COP at a site common to the γ- and ß-COP subunits. The Arf1-binding sites on coatomer are spatially related to PtdIns4,5P(2)-binding sites on the endocytic AP2 complex, providing evidence that the orientation of membrane binding is general for this class of vesicular coat proteins. A bivalent GTP-dependent binding mode has implications for the dynamics of coatomer interaction with the Golgi and for the selection of cargo molecules.

Brefeldin A and M-COPA block the export of RTKs from the endoplasmic reticulum via simultaneous inactivation of ARF1, ARF4, and ARF5.

Normal receptor tyrosine kinases (RTKs) need to reach the plasma membrane (PM) for ligand-induced activation, whereas its cancer-causing mutants can be activated before reaching the PM in organelles, such as the Golgi/trans-Golgi network (TGN). Inhibitors of protein export from the endoplasmic reticulum (ER), such as brefeldin A (BFA) and 2-methylcoprophilinamide (M-COPA), can suppress the activation of mutant RTKs in cancer cells, suggesting that RTK mutants cannot initiate signaling in the ER. BFA and M-COPA block the function of ADP-ribosylation factors (ARFs) that play a crucial role in ER-Golgi protein trafficking. However, among ARF family proteins, the specific ARFs inhibited by BFA or M-COPA, that is, the ARFs involved in RTKs transport from the ER, remain unclear. In this study, we showed that M-COPA blocked the export of not only KIT but also PDGFRA/EGFR/MET RTKs from the ER. ER-retained RTKs could not fully transduce anti-apoptotic signals, thereby leading to cancer cell apoptosis. Moreover, a single knockdown of ARF1, ARF3, ARF4, ARF5, or ARF6 could not block ER export of RTKs, indicating that BFA/M-COPA treatment cannot be mimicked by the knockdown of only one ARF member. Interestingly, simultaneous transfection of ARF1, ARF4, and ARF5 siRNAs mirrored the effect of BFA/M-COPA treatment. Consistent with these results, in vitro pulldown assays showed that BFA/M-COPA blocked the function of ARF1, ARF4, and ARF5. Taken together, these results suggest that BFA/M-COPA targets at least ARF1, ARF4, and ARF5; in other words, RTKs require the simultaneous activation of ARF1, ARF4, and ARF5 for their ER export.

A neurodevelopmental disorder associated with an activating de novo missense variant in ARF1.

ADP-ribosylation factor 1 (ARF1) is a small GTPase that regulates membrane traffic at the Golgi apparatus and endosomes through recruitment of several coat proteins and lipid-modifying enzymes. Here, we report a pediatric patient with an ARF1-related disorder because of a monoallelic de novo missense variant (c.296 G > A; p.R99H) in the ARF1 gene, associated with developmental delay, hypotonia, intellectual disability and motor stereotypies. Neuroimaging revealed a hypoplastic corpus callosum and subcortical white matter abnormalities. Notably, this patient did not exhibit periventricular heterotopias previously observed in other patients with ARF1 variants (including p.R99H). Functional analysis of the R99H-ARF1 variant protein revealed that it was expressed at normal levels and properly localized to the Golgi apparatus; however, the expression of this variant caused swelling of the Golgi apparatus, increased the recruitment of coat proteins such as coat protein complex I, adaptor protein complex 1 and GGA3 and altered the morphology of recycling endosomes. In addition, we observed that the expression of R99H-ARF1 prevented dispersal of the Golgi apparatus by the ARF1-inhibitor brefeldin A. Finally, protein interaction analyses showed that R99H-ARF1 bound more tightly to the ARF1-effector GGA3 relative to wild-type ARF1. These properties were similar to those of the well-characterized constitutively active Q71L-ARF1 mutant, indicating that the pathogenetic mechanism of the R99H-ARF1 variant involves constitutive activation with resultant Golgi and endosomal alterations. The absence of periventricular nodular heterotopias in this R99H-ARF1 subject also indicates that this finding may not be a consistent phenotypic expression of all ARF1-related disorders.

The development of resistance to an inhibitor of a cellular protein reveals a critical interaction between the enterovirus protein 2C and a small GTPase Arf1.

The cellular protein GBF1, an activator of Arf GTPases (ArfGEF: Arf guanine nucleotide exchange factor), is recruited to the replication organelles of enteroviruses through interaction with the viral protein 3A, and its ArfGEF activity is required for viral replication, however how GBF1-dependent Arf activation supports the infection remains enigmatic. Here, we investigated the development of resistance of poliovirus, a prototype enterovirus, to increasing concentrations of brefeldin A (BFA), an inhibitor of GBF1. High level of resistance required a gradual accumulation of multiple mutations in the viral protein 2C. The 2C mutations conferred BFA resistance even in the context of a 3A mutant previously shown to be defective in the recruitment of GBF1 to replication organelles, and in cells depleted of GBF1, suggesting a GBF1-independent replication mechanism. Still, activated Arfs accumulated on the replication organelles of this mutant even in the presence of BFA, its replication was inhibited by a pan-ArfGEF inhibitor LM11, and the BFA-resistant phenotype was compromised in Arf1-knockout cells. Importantly, the mutations strongly increased the interaction of 2C with the activated form of Arf1. Analysis of other enteroviruses revealed a particularly strong interaction of 2C of human rhinovirus 1A with activated Arf1. Accordingly, the replication of this virus was significantly less sensitive to BFA than that of poliovirus. Thus, our data demonstrate that enterovirus 2Cs may behave like Arf1 effector proteins and that GBF1 but not Arf activation can be dispensable for enterovirus replication. These findings have important implications for the development of host-targeted anti-viral therapeutics.

THUMPD3-AS1 inhibits ovarian cancer cell apoptosis through the miR-320d/ARF1 axis.

Ovarian cancer is one of the most common gynecologic malignancies that has a poor prognosis. THUMPD3-AS1 is an oncogenic long noncoding RNA (lncRNA) in several cancers. Moreover, miR-320d is downregulated and inhibited proliferation in ovarian cancer cells, whereas ARF1 was upregulated and promoted the malignant progression in epithelial ovarian cancer. Nevertheless, the role of THUMPD3-AS1 in ovarian cancer and the underlying mechanism has yet to be elucidated. Human normal ovarian epithelial cells (IOSE80) and ovarian cancer cell lines (CAVO3, A2780, SKOV3, OVCAR3, and HEY) were adopted for in vitro experiments. The functional roles of THUMPD3-AS1 in cell viability and apoptosis were determined using CCK-8, flow cytometry, and TUNEL assays. Western blot was performed to assess the protein levels of ARF1, Bax, Bcl-2, and caspase 3, whereas RT-qPCR was applied to measure ARF1 mRNA, THUMPD3-AS1, and miR-320d levels. The targeting relationship between miR-320d and THUMPD3-AS1 or ARF1 was validated with dual luciferase assay. THUMPD3-AS1 and ARF1 were highly expressed in ovarian cancer cells, whereas miR-320d level was lowly expressed. THUMPD3-AS1 knockdown was able to repress cell viability and accelerate apoptosis of OVCAR3 and SKOV3 cells. Also, THUMPD3-AS1 acted as a sponge of miR-320d, preventing the degradation of ARF1. MiR-320d downregulation reversed the tumor suppressive function induced by THUMPD3-AS1 depletion. Additionally, miR-320d overexpression inhibited ovarian cancer cell viability and accelerated apoptosis, which was overturned by overexpression of ARF1. THUMPD3-AS1 inhibited ovarian cancer cell apoptosis by modulation of miR-320d/ARF1 axis. The discoveries might provide a prospective target for ovarian cancer treatment.

The small molecule inhibitor NAV-2729 has a complex target profile including multiple ADP-ribosylation factor regulatory proteins.

The ADP-ribosylation factor (Arf) GTPases and their regulatory proteins are implicated in cancer progression. NAV-2729 was previously identified as a specific inhibitor of Arf6 that reduced progression of uveal melanoma in an orthotopic xenograft. Here, our goal was to assess the inhibitory effects of NAV-2729 on the proliferation of additional cell types. We found NAV-2729 inhibited proliferation of multiple cell lines, but Arf6 expression did not correlate with NAV-2729 sensitivity, and knockdown of Arf6 affected neither cell viability nor sensitivity to NAV-2729. Furthermore, binding to native Arf6 was not detected; however, we determined that NAV-2729 inhibited both Arf exchange factors and Arf GTPase-activating proteins. ASAP1, a GTPase-activating protein linked to cancer progression, was further investigated. We demonstrated that NAV-2729 bound to the PH domain of ASAP1 and changed ASAP1 cellular distribution. However, ASAP1 knockdown did not fully recapitulate the cytoskeletal effects of NAV-2729 nor affect cell proliferation. Finally, our screens identified 48 other possible targets of NAV-2729. These results illustrate the complexities of defining targets of small molecules and identify NAV-2729 as a model PH domain-binding inhibitor.

Proximity labeling of endogenous RICTOR identifies mTOR complex 2 regulation by ADP ribosylation factor ARF1.

Mechanistic target of rapamycin (mTOR) complex 2 (mTORC2) regulates metabolism, cell proliferation, and cell survival. mTORC2 activity is stimulated by growth factors, and it phosphorylates the hydrophobic motif site of the AGC kinases AKT, SGK, and PKC. However, the proteins that interact with mTORC2 to control its activity and localization remain poorly defined. To identify mTORC2-interacting proteins in living cells, we tagged endogenous RICTOR, an essential mTORC2 subunit, with the modified BirA biotin ligase BioID2 and performed live-cell proximity labeling. We identified 215 RICTOR-proximal proteins, including proteins with known mTORC2 pathway interactions, and 135 proteins (63%) not previously linked to mTORC2 signaling, including nuclear and cytoplasmic proteins. Our imaging and cell fractionation experiments suggest nearly 30% of RICTOR is in the nucleus, hinting at potential nuclear functions. We also identified 29 interactors containing RICTOR-dependent, insulin-stimulated phosphorylation sites, thus providing insight into mTORC2-dependent insulin signaling dynamics. Finally, we identify the endogenous ADP ribosylation factor 1 (ARF1) GTPase as an mTORC2-interacting protein. Through gain-of-function and loss-of-function studies, we provide functional evidence that ARF1 may negatively regulate mTORC2. In summary, we present a new method of studying endogenous mTORC2, a resource of RICTOR/mTORC2 protein interactions in living cells, and a potential mechanism of mTORC2 regulation by the ARF1 GTPase.

Rab1b-GBF1-ARF1 Secretory Pathway Axis Is Required for Birnavirus Replication.

Birnaviruses are members of the Birnaviridae family, responsible for major economic losses to poultry and aquaculture. The family is composed of nonenveloped viruses with a segmented double-stranded RNA (dsRNA) genome. Infectious bursal disease virus (IBDV), the prototypic family member, is the etiological agent of Gumboro disease, a highly contagious immunosuppressive disease in the poultry industry worldwide. We previously demonstrated that IBDV hijacks the endocytic pathway for establishing the viral replication complexes on endosomes associated with the Golgi complex (GC). Here, we report that IBDV reorganizes the GC to localize the endosome-associated replication complexes without affecting its secretory functionality. By analyzing crucial proteins involved in the secretory pathway, we showed the essential requirement of Rab1b for viral replication. Rab1b comprises a key regulator of GC transport and we demonstrate that transfecting the negative mutant Rab1b N121I or knocking down Rab1b expression by RNA interference significantly reduces the yield of infectious viral progeny. Furthermore, we showed that the Rab1b downstream effector Golgi-specific BFA resistance factor 1 (GBF1), which activates the small GTPase ADP ribosylation factor 1 (ARF1), is required for IBDV replication, since inhibiting its activity by treatment with brefeldin A (BFA) or golgicide A (GCA) significantly reduces the yield of infectious viral progeny. Finally, we show that ARF1 dominant negative mutant T31N overexpression hampered IBDV infection. Taken together, these results demonstrate that IBDV requires the function of the Rab1b-GBF1-ARF1 axis to promote its replication, making a substantial contribution to the field of birnavirus-host cell interactions. IMPORTANCE Birnaviruses are unconventional members of the dsRNA viruses, with the lack of a transcriptionally active core being the main differential feature. This structural trait, among others that resemble those of the plus single-stranded (+ssRNA) viruses features, suggests that birnaviruses might follow a different replication program from that conducted by prototypical dsRNA members and the hypothesis that birnaviruses could be evolutionary links between +ssRNA and dsRNA viruses has been argued. Here, we present original data showing that IBDV-induced GC reorganization and the cross talk between IBDV and the Rab1b-GBF1-ARF1 mediate the intracellular trafficking pathway. The replication of several +ssRNA viruses depends on the cellular protein GBF1, but its role in the replication process is not clear. Thus, our findings make a substantial contribution to the field of birnavirus-host cell interactions and provide further evidence supporting the proposed evolutionary connection role of birnaviruses, an aspect which we consider especially relevant for researchers working in the virology field.

ADP-ribosylation factor D1 modulates Golgi morphology, cell plate formation, and plant growth in Arabidopsis.

ADP-ribosylation factor (ARF) family proteins, one type of small guanine-nucleotide-binding (G) proteins, play a central role in regulating vesicular traffic and organelle structures in eukaryotes. The Arabidopsis (Arabidopsis thaliana) genome contains more than 21 ARF proteins, but relatively little is known about the functional heterogeneity of ARF homologs in plants. Here, we characterized the function of a unique ARF protein, ARFD1B, in Arabidopsis. ARFD1B exhibited both cytosol and punctate localization patterns, colocalizing with a Golgi marker in protoplasts and transgenic plants. Distinct from other ARF1 homologs, overexpression of a dominant-negative mutant form of ARFD1B did not alter the localization of the Golgi marker mannosidase I (ManI)-RFP in Arabidopsis cells. Interestingly, the ARFD1 artificial microRNA knockdown mutant arfd1 displayed a deleterious growth phenotype, while this phenotype was restored in complemented plants. Further, confocal imaging and transmission electron microscopy analyses of the arfd1 mutant revealed defective cell plate formation and abnormal Golgi morphology. Pull-down and liquid chromatography-tandem mass spectrometry analyses identified Coat Protein I (COPI) components as interacting partners of ARFD1B, and subsequent bimolecular fluorescence complementation, yeast (Saccharomyces cerevisiae) two-hybrid, and co-immunoprecipitation assays further confirmed these interactions. These results demonstrate that ARFD1 is required for cell plate formation, maintenance of Golgi morphology, and plant growth in Arabidopsis.

Arf1 and Arf6 Synergistically Maintain Survival of T Cells during Activation.

ADP-ribosylation factor (Arf) family consisting of six family members, Arf1-Arf6, belongs to Ras superfamily and orchestrates vesicle trafficking under the control of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins. It is well established that brefeldin A, a potent inhibitor of ArfGEFs, blocks cytokine secretion from activated T cells, suggesting that the Arf pathway plays important roles in T cell functions. In this study, because Arf1 and Arf6 are the best-characterized members among Arf family, we established T lineage-specific Arf1-deficient, Arf6-deficient, and Arf1/6 double-deficient mice to understand physiological roles of the Arf pathway in the immune system. Contrary to our expectation, Arf deficiency had little or no impact on cytokine secretion from the activated T cells. In contrast, the lack of both Arf1 and Arf6, but neither Arf1 nor Arf6 deficiency alone, rendered naive T cells susceptible to apoptosis upon TCR stimulation because of imbalanced expression of Bcl-2 family members. We further demonstrate that Arf1/6 deficiency in T cells alleviates autoimmune diseases like colitis and experimental autoimmune encephalomyelitis, whereas Ab response under Th2-polarizing conditions is seemingly normal. Our findings reveal an unexpected role for the Arf pathway in the survival of T cells during TCR-induced activation and its potential as a therapeutic target in the autoimmune diseases.

Myristoylome profiling reveals a concerted mechanism of ARF GTPase deacylation by the bacterial protease IpaJ.

N-myristoylation is an essential fatty acid modification that governs the localization and activity of cell signaling enzymes, architectural proteins, and immune regulatory factors. Despite its importance in health and disease, there are currently no methods for reversing protein myristoylation in vivo. Recently, the Shigella flexneri protease IpaJ was found to cleave myristoylated glycine of eukaryotic proteins, yet the discriminatory mechanisms of substrate selection required for targeted demyristoylation have not yet been evaluated. Here, we performed global myristoylome profiling of cells treated with IpaJ under distinct physiological conditions. The protease is highly promiscuous among diverse N-myristoylated proteins in vitro but is remarkably specific to Golgi-associated ARF/ARL family GTPases during Shigella infection. Reconstitution studies revealed a mechanistic framework for substrate discrimination based on IpaJ's function as a GTPase "effector" of bacterial origin. We now propose a concerted model for IpaJ function that highlights its potential for programmable demyristoylation in vivo.

Gßγ translocation to the Golgi apparatus activates ARF1 to spatiotemporally regulate G protein-coupled receptor signaling to MAPK.

After activation of G protein-coupled receptors, G protein ßγ dimers may translocate from the plasma membrane to the Golgi apparatus (GA). We recently report that this translocation activates extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) via PI3Kγ; however, how Gßγ-PI3Kγ activates the ERK1/2 pathway is unclear. Here, we demonstrate that chemokine receptor CXCR4 activates ADP-ribosylation factor 1 (ARF1), a small GTPase important for vesicle-mediated membrane trafficking. This activation is blocked by CRISPR-Cas9-mediated knockout of the GA-translocating Gγ9 subunit. Inducible targeting of different Gßγ dimers to the GA can directly activate ARF1. CXCR4 activation and constitutive Gßγ recruitment to the GA also enhance ARF1 translocation to the GA. We further demonstrate that pharmacological inhibition and CRISPR-Cas9-mediated knockout of PI3Kγ markedly inhibit CXCR4-mediated and Gßγ translocation-mediated ARF1 activation. We also show that depletion of ARF1 by siRNA and CRISPR-Cas9 and inhibition of GA-localized ARF1 activation abolish ERK1/2 activation by CXCR4 and Gßγ translocation to the GA and suppress prostate cancer PC3 cell migration and invasion. Collectively, our data reveal a novel function for Gßγ translocation to the GA to activate ARF1 and identify GA-localized ARF1 as an effector acting downstream of Gßγ-PI3Kγ to spatiotemporally regulate G protein-coupled receptor signaling to mitogen-activated protein kinases.

Exomer complex regulates protein traffic at the TGN through differential interactions with cargos and clathrin adaptor complexes.

Protein sorting at the trans-Golgi network (TGN) usually requires the assistance of cargo adaptors. However, it remains to be examined how the same complex can mediate both the export and retention of different proteins or how sorting complexes interact among themselves. In Saccharomyces cerevisiae, the exomer complex is involved in the polarized transport of some proteins from the TGN to the plasma membrane (PM). Intriguingly, exomer and its cargos also show a sort of functional relationship with TGN clathrin adaptors that is still unsolved. Here, using a wide range of techniques, including time-lapse and BIFC microscopy, we describe new molecular implications of the exomer complex in protein sorting and address its different layers of functional interaction with clathrin adaptor complexes. Exomer mutants show impaired amino acid uptake because it facilitates not only the polarized delivery of amino acid permeases to the PM but also participates in their endosomal traffic. We propose a model for exomer where it modulates the recruitment of TGN clathrin adaptors directly or indirectly through the Arf1 function. Moreover, we describe an in vivo competitive relationship between the exomer and AP-1 complexes for the model cargo Chs3. These results highlight a broad role for exomer in regulating protein sorting at the TGN that is complementary to its role as cargo adaptor and present a model to understand the complexity of TGN protein sorting.

Cytosolic pH regulates cell growth through distinct GTPases, Arf1 and Gtr1, to promote Ras/PKA and TORC1 activity.

Regulation of cell growth by nutrients is governed by highly conserved signaling pathways, yet mechanisms of nutrient sensing are still poorly understood. In yeast, glucose activates both the Ras/PKA pathway and TORC1, which coordinately regulate growth through enhancing translation and ribosome biogenesis and suppressing autophagy. Here, we show that cytosolic pH acts as a cellular signal to activate Ras and TORC1 in response to glucose availability. We demonstrate that cytosolic pH is sensitive to the quality and quantity of the available carbon source (C-source). Interestingly, Ras/PKA and TORC1 are both activated through the vacuolar ATPase (V-ATPase), which was previously identified as a sensor for cytosolic pH in vivo. V-ATPase interacts with two distinct GTPases, Arf1 and Gtr1, which are required for Ras and TORC1 activation, respectively. Together, these data provide a molecular mechanism for how cytosolic pH links C-source availability to the activity of signaling networks promoting cell growth.

Glutamine and asparagine activate mTORC1 independently of Rag GTPases.

Nutrient sensing by cells is crucial, and when this sensing mechanism is disturbed, human disease can occur. mTOR complex 1 (mTORC1) senses amino acids to control cell growth, metabolism, and autophagy. Leucine, arginine, and methionine signal to mTORC1 through the well-characterized Rag GTPase signaling pathway. In contrast, glutamine activates mTORC1 through a Rag GTPase-independent mechanism that requires ADP-ribosylation factor 1 (Arf1). Here, using several biochemical and genetic approaches, we show that eight amino acids filter through the Rag GTPase pathway. Like glutamine, asparagine signals to mTORC1 through Arf1 in the absence of the Rag GTPases. Both the Rag-dependent and Rag-independent pathways required the lysosome and lysosomal function for mTORC1 activation. Our results show that mTORC1 is differentially regulated by amino acids through two distinct pathways.

Cytohesin-2 mediates group I metabotropic glutamate receptor-dependent mechanical allodynia through the activation of ADP ribosylation factor 6 in the spinal cord.

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.