Rab1b facilitates lipid droplet growth by ER-to-lipid droplet targeting of DGAT2.

Lipid droplets (LDs) comprise a triglyceride core surrounded by a lipid monolayer enriched with proteins, many of which function in LD homeostasis. How proteins are targeted to the growing LD is still unclear. Rab1b, a GTPase regulating secretory transport, was recently associated with targeting proteins to LDs in a Drosophila RNAi screen. LD formation was prevented in human hepatoma cells overexpressing dominant-negative Rab1b. We thus hypothesized that Rab1b recruits lipid-synthesizing enzymes, facilitating LD growth. Here, FRET between diacylglycerol acyltransferase 2 (DGAT2) and Rab1b and activity mutants of the latter demonstrated that Rab1b promotes DGAT2 ER to the LD surface redistribution. Last, alterations in LD metabolism and DGAT2 redistribution, consistent with Rab1b activity, were caused by mutations in the Rab1b-GTPase activating protein TBC1D20 in Warburg Micro syndrome (WARBM) model mice fibroblasts. These data contribute to our understanding of the mechanism of Rab1b in LD homeostasis and WARBM, a devastating autosomal-recessive disorder caused by mutations in TBC1D20.

SNX32 is a host restriction factor that degrades African swine fever virus CP204L via the RAB1B-dependent autophagy pathway.

African swine fever virus (ASFV) causes a highly contagious and deadly disease in domestic pigs and European wild boars, posing a severe threat to the global pig industry. ASFV CP204L, a highly immunogenic protein, is produced during the early stages of ASFV infection. However, the impact of CP204L protein-interacting partners on the outcome of ASFV infection is poorly understood. To accomplish this, coimmunoprecipitation and mass spectrometry analysis were conducted in ASFV-infected porcine alveolar macrophages (PAMs). We have demonstrated that sorting nexin 32 (SNX32) is a CP204L-binding protein and that CP204L interacted and colocalized with SNX32 in ASFV-infected PAMs. ASFV growth and replication were promoted by silencing SNX32 and suppressed by overexpressing SNX32. SNX32 degraded CP204L by recruiting the autophagy-related protein Ras-related protein Rab-1b (RAB1B). RAB1B overexpression inhibited ASFV replication, while knockdown of RAB1B had the opposite effect. Additionally, RAB1B, SNX32, and CP204L formed a complex upon ASFV infection. Taken together, this study demonstrates that SNX32 antagonizes ASFV growth and replication by recruiting the autophagy-related protein RAB1B. This finding extends our understanding of the interaction between ASFV CP204L and its host and provides new insights into exploring the relationship between ASFV infection and autophagy.IMPORTANCEAfrican swine fever (ASF) is a highly contagious and acute hemorrhagic viral disease with a high mortality near 100% in domestic pigs. ASF virus (ASFV), which is the only member of the family Asfarviridae, is a dsDNA virus of great complexity and size, encoding more than 150 proteins. Currently, there are no available vaccines against ASFV. ASFV CP204L represents the most abundantly expressed viral protein early in infection and plays an important role in regulating ASFV replication. However, the mechanism by which the interaction between ASFV CP204L and host proteins affects ASFV replication remains unclear. In this study, we demonstrated that the cellular protein SNX32 interacted with CP204L and degraded CP204L by upregulating the autophagy-related protein RAB1B. In summary, this study will help us understand the interaction mechanism between CP204L and its host upon infection and provide new insights for the development of vaccines and antiviral drugs.

Inhibition of Rab1B Impairs Trafficking and Maturation of SARS-CoV-2 Spike Protein.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) utilizes cellular trafficking pathways to process its structural proteins and move them to the site of assembly. Nevertheless, the exact process of assembly and subcellular trafficking of SARS-CoV-2 proteins remains largely unknown. Here, we have identified and characterized Rab1B as an important host factor for the trafficking and maturation of the spike protein (S) after synthesis at the endoplasmic reticulum (ER). Using confocal microscopy, we showed that S and Rab1B substantially colocalized in compartments of the early secretory pathway. Co-expression of dominant-negative (DN) Rab1B N121I leads to an aberrant distribution of S into perinuclear spots after ectopic expression and in SARS-CoV-2-infected cells caused by either structural rearrangement of the ERGIC or Golgi or missing interaction between Rab1B and S. Western blot analyses revealed a complete loss of the mature, cleaved S2 subunit in cell lysates and culture supernatants upon co-expression of DN Rab1B N121I. In sum, our studies indicate that Rab1B is an important regulator of trafficking and maturation of SARS-CoV-2 S, which not only improves our understanding of the coronavirus replication cycle but also may have implications for the development of antiviral strategies.

Dephosphocholination by Legionella effector Lem3 functions through remodelling of the switch II region of Rab1b.

Bacterial pathogens often make use of post-translational modifications to manipulate host cells. Legionella pneumophila, the causative agent of Legionnaires disease, secretes the enzyme AnkX that uses cytidine diphosphate-choline to post-translationally modify the human small G-Protein Rab1 with a phosphocholine moiety at Ser76. Later in the infection, the Legionella enzyme Lem3 acts as a dephosphocholinase, hydrolytically removing the phosphocholine. While the molecular mechanism for Rab1 phosphocholination by AnkX has recently been resolved, structural insights into the activity of Lem3 remained elusive. Here, we stabilise the transient Lem3:Rab1b complex by substrate mediated covalent capture. Through crystal structures of Lem3 in the apo form and in complex with Rab1b, we reveal Lem3's catalytic mechanism, showing that it acts on Rab1 by locally unfolding it. Since Lem3 shares high structural similarity with metal-dependent protein phosphatases, our Lem3:Rab1b complex structure also sheds light on how these phosphatases recognise protein substrates.

Dual function of Rab1A in secretion and autophagy: hypervariable domain dependence.

We currently understand how the different intracellular pathways, secretion, endocytosis, and autophagy are regulated by small GTPases. In contrast, it is unclear how these pathways are coordinated to ensure efficient cellular response to stress. Rab GTPases localize to specific organelles through their hypervariable domain (HVD) to regulate discrete steps of individual pathways. Here, we explored the dual role of Rab1A/B (92% identity) in secretion and autophagy. We show that although either Rab1A or Rab1B is required for secretion, Rab1A, but not Rab1B, localizes to autophagosomes and is required early in stress-induced autophagy. Moreover, replacing the HVD of Rab1B with that of Rab1A enables Rab1B to localize to autophagosomes and regulate autophagy. Therefore, Rab1A-HVD is required for the dual functionality of a single Rab in two different pathways: secretion and autophagy. In addition to this mechanistic insight, these findings are relevant to human health because both the pathways and Rab1A/B were implicated in diseases ranging from cancer to neurodegeneration.

LINC00173 facilitates tumor progression by stimulating RAB1B-mediated PA2G4 and SDF4 secretion in nasopharyngeal carcinoma.

An increasing number of studies have found that long non-coding RNA (lncRNA) play important roles in driving the progression of nasopharyngeal carcinoma (NPC). Our microarray screening revealed that expression of the lncRNA long intergenic non-protein coding RNA 173 (LINC00173) was upregulated in NPC. However, its role and mechanism in NPC have not yet been elucidated. In this study, we demonstrate that high LINC00173 expression indicated a poor prognosis in NPC patients. Knockdown of LINC00173 significantly inhibited NPC cell proliferation, migration and invasion in vitro. Mechanistically, LINC00173 interacted and colocalized with Ras-related protein Rab-1B (RAB1B) in the cytoplasm, but the modulation of LINC00173 expression did not affect the expression of RAB1B at either the mRNA or protein levels. Instead, relying on the stimulation of RAB1B, LINC00173 could facilitate the extracellular secretion of proliferation-associated 2G4 (PA2G4) and stromal cell-derived factor 4 (SDF4; also known as 45-kDa calcium-binding protein) proteins, and knockdown of these proteins could reverse the NPC aggressive phenotype induced by LINC00173 overexpression. Moreover, in vivo LINC00173-knockdown models exhibited a marked slowdown in tumor growth and a significant reduction in lymph node and lung metastases. In summary, LINC00173 serves as a crucial driver for NPC progression, and the LINC00173-RAB1B-PA2G4/SDF4 axis might provide a potential therapeutic target for NPC patients.

The Small GTPase FgRab1 Plays Indispensable Roles in the Vegetative Growth, Vesicle Fusion, Autophagy and Pathogenicity of Fusarium graminearum.

Rab GTPases are key regulators of membrane and intracellular vesicle transports. However, the biological functions of FgRab1 are still unclear in the devastating wheat pathogen Fusarium graminearum. In this study, we generated constitutively active (CA) and dominant-negative (DN) forms of FgRAB1 from the wild-type PH-1 background for functional analyses. Phenotypic analyses of these mutants showed that FgRab1 is important for vegetative growth, cell wall integrity and hyphal branching. Compared to the PH-1 strain, the number of spores produced by the Fgrab1DN strain was significantly reduced, with obviously abnormal conidial morphology. The number of septa in the conidia of the Fgrab1DN mutant was fewer than that observed in the PH-1 conidia. Fgrab1DN was dramatically reduced in its ability to cause Fusarium head blight symptoms on wheat heads. GFP-FgRab1 was observed to partly localize to the Golgi apparatus, endoplasmic reticulum and Spitzenkörper. Furthermore, we found that FgRab1 inactivation blocks not only the transport of the v-SNARE protein FgSnc1 from the Golgi to the plasma membrane but also the fusion of endocytic vesicles with their target membranes and general autophagy. In summary, our results indicate that FgRab1 plays vital roles in vegetative growth, conidiogenesis, pathogenicity, autophagy, vesicle fusion and trafficking in F. graminearum.

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.

Focus on the Small GTPase Rab1: A Key Player in the Pathogenesis of Parkinson's Disease.

Parkinson's disease (PD) is the second most frequent neurodegenerative disease. It is characterized by the loss of dopaminergic neurons in the substantia nigra and the formation of large aggregates in the survival neurons called Lewy bodies, which mainly contain α-synuclein (α-syn). The cause of cell death is not known but could be due to mitochondrial dysfunction, protein homeostasis failure, and alterations in the secretory/endolysosomal/autophagic pathways. Survival nigral neurons overexpress the small GTPase Rab1. This protein is considered a housekeeping Rab that is necessary to support the secretory pathway, the maintenance of the Golgi complex structure, and the regulation of macroautophagy from yeast to humans. It is also involved in signaling, carcinogenesis, and infection for some pathogens. It has been shown that it is directly linked to the pathogenesis of PD and other neurodegenerative diseases. It has a protective effect against α-σψν toxicity and has recently been shown to be a substrate of LRRK2, which is the most common cause of familial PD and the risk of sporadic disease. In this review, we analyze the key aspects of Rab1 function in dopamine neurons and its implications in PD neurodegeneration/restauration. The results of the current and former research support the notion that this GTPase is a good candidate for therapeutic strategies.

Establishment and analysis of conditional Rab1- and Rab5-knockout cells using the auxin-inducible degron system.

Two small GTPases, Rab1 and Rab5, are key membrane trafficking regulators that are conserved in all eukaryotes. They have recently been found to be essential for cell survival and/or growth in cultured mammalian cells, thereby precluding the establishment of Rab1-knockout (KO) and Rab5-KO cells, making it extremely difficult to assess the impact of complete Rab1 or Rab5 protein depletion on cellular functions. Here, we generated and analyzed cell lines with conditional KO (CKO) of either Rab1 (Rab1A and Rab1B) or Rab5 (Rab5A, Rab5B and Rab5C) by using the auxin-inducible protein degradation system. Rab1 CKO and Rab5 CKO led to eventual cell death from 18 h and 48 h, respectively, after auxin exposure. After acute Rab1 protein depletion, the Golgi stack and ribbon structures were completely disrupted, and endoplasmic reticulum (ER)-to-Golgi trafficking was severely inhibited. Moreover, we discovered a novel Rab1-depletion phenotype: perinuclear clustering of early endosomes and delayed transferrin recycling. In contrast, acute Rab5 protein depletion resulted in loss of early endosomes and late endosomes, but lysosomes appeared to be normal. We also observed a dramatic reduction in the intracellular signals of endocytic cargos via receptor-mediated or fluid-phase endocytosis in Rab5-depleted cells.

Rab1A promotes cell proliferation and migration by upregulating Gli1 in colorectal cancer.

Rab1A, as a highly conserved small guanosine triphosphatase (GTPase), plays contentious roles in different types of cancers. The role of Rab1A in colorectal cancer (CRC) has been described in previous studies, but the molecular mechanisms of Rab1A in CRC remain far from being addressed. In the present study, we found that Rab1A expression was significantly upregulated in CRC tissues and increased Rab1A expression correlated with tumor size, lymph node metastasis (LNM) and tumor-node-metastasis (TNM) stage of CRC patients. We also found that Rab1A exerts its promotive effect on CRC cell proliferation, migration and EMT progress. Further mechanistic experiments showed that glioma-associated oncogene-1 (Gli1), as a key transcriptional factor of the Hedgehog pathway, was implicated in Rab1A-mediated regulation of CRC cell proliferation and migration. In addition, Rab1A upregulated Gli1 expression through Smoothened homolog (SMO)-independent pathway. Finally, Rab1A activated mechanistic target of rapamycin (mTOR) signaling in CRC cells. Collectively, our results define Rab1A as a novel regulator of Gli1 to promote CRC cell proliferation and migration, and suggest that the Rab1A/mTOR/Gli1 axis may serve as a promising therapeutic target for the treatment of CRC.

Mycobacterium tuberculosis PE_PGRS20 and PE_PGRS47 Proteins Inhibit Autophagy by Interaction with Rab1A.

Autophagy is a fundamental cellular process that has important roles in innate and adaptive immunity against a broad range of microbes. Many pathogenic microbes have evolved mechanisms to evade or exploit autophagy. It has been previously demonstrated that induction of autophagy can suppress the intracellular survival of mycobacteria, and several PE_PGRS family proteins of Mycobacterium tuberculosis have been proposed to act as inhibitors of autophagy to promote mycobacterial survival. However, the mechanisms by which these effectors inhibit autophagy have not been defined. Here, we report detailed studies of M. tuberculosis deletion mutants of two genes, pe_pgrs20 and pe_pgrs47, that we previously reported as having a role in preventing autophagy of infected host cells. These mutants resulted in increased autophagy and reduced intracellular survival of M. tuberculosis in macrophages. This phenotype was accompanied by increased cytokine production and antigen presentation by infected cells. We further demonstrated that autophagy inhibition by PE_PGRS20 and PE_PGRS47 resulted from canonical autophagy rather than autophagy flux inhibition. Using macrophages transfected to express PE_PGRS20 or PE_PGRS47, we showed that these proteins inhibited autophagy initiation directly by interacting with Ras-related protein Rab1A. Silencing of Rab1A in mammalian cells rescued the survival defects of the pe_pgrs20 and pe_pgrs47 deletion mutant strains and reduced cytokine secretion. To our knowledge, this is the first study to identify mycobacterial effectors that directly interact with host proteins responsible for autophagy initiation. IMPORTANCE Tuberculosis is a significant global infectious disease caused by infection of the lungs with Mycobacterium tuberculosis, which then resides and replicates mainly within host phagocytic cells. Autophagy is a complex host cellular process that helps control intracellular infections and enhance innate and adaptive immune responses. During coevolution with humans, M. tuberculosis has acquired various mechanisms to inhibit host cellular processes, including autophagy. We identified two related M. tuberculosis proteins, PE_PGRS20 and PE_PGRS47, as the first reported examples of specific mycobacterial effectors interfering with the initiation stage of autophagy. Autophagy regulation by these PE_PGRS proteins leads to increased bacterial survival in phagocytic cells and increased autophagic degradation of mycobacterial antigens to stimulate adaptive immune responses. A better understanding of how M. tuberculosis regulates autophagy in host cells could facilitate the design of new and more effective therapeutics or vaccines against tuberculosis.

Arabidopsis RAB8A, RAB8B and RAB8D Proteins Interact with Several RTNLB Proteins and are Involved in the Agrobacterium tumefaciens Infection Process.

Arabidopsis thaliana small GTP-binding proteins, AtRAB8s, associate with the endomembrane system and modulate tubulovesicular trafficking between compartments of the biosynthetic and endocytic pathways. There are five members in Arabidopsis, namely AtRAB8A-8E. Yeast two-hybrid assays, bimolecular fluorescence complementation assays and glutathione-S-transferase pull-down assays showed that RAB8A, 8B and 8D interacted with several membrane-associated reticulon-like (AtRTNLB) proteins in yeast, plant cells and in vitro. Furthermore, RAB8A, 8B and 8D proteins showed interactions with the Agrobacterium tumefaciens virulence protein, VirB2, a component of a type IV secretion system (T4SS). A. tumefaciens uses a T4SS to transfer T-DNA and Virulence proteins to plants, which causes crown gall disease in plants. The Arabidopsis rab8A, rab8B and rab8D single mutants showed decreased levels of Agrobacterium-mediated root and seedling transformation, while the RAB8A, 8B and 8D overexpression transgenic Arabidopsis plants were hypersusceptible to A. tumefaciens and Pseudomonas syringae infections. RAB8A-8E transcripts accumulated differently in roots, rosette leaves, cauline leaves, inflorescence and flowers of wild-type plants. In summary, RAB8A, 8B and 8D interacted with several RTNLB proteins and participated in A. tumefaciens and P. syringae infection processes.

RAB1A regulates glioma cellular proliferation and invasion via the mTOR signaling pathway and epithelial-mesenchymal transition.

Aim: We aimed at investigating the mechanism of RAB1A proliferation and invasion in gliomas. Materials & methods: Genome-wide expression profile data and immunohistochemistry were analyzed to assess RAB1A expression in gliomas. The Transwell assay, wound healing assay, brain slice coculture model, cellular fluorescence and intracranial xenograft model of nude mice were used to determine the proliferation and invasion of glioma cells. Results & conclusion: RAB1A was highly expressed in gliomas compared with normal brain tissue. The overall survival time of glioma patients with high RAB1A expression was significantly shortened. RAB1A regulated the activity of RAC1 by inhibiting the mTOR signaling pathway, affecting actin polymerization, cell morphology and cell polarity. RAB1A downregulation inhibited the epithelial-mesenchymal transition, proliferation and invasion of glioma cells.

Electroacupuncture Inhibits Autophagy of Neuron Cells in Postherpetic Neuralgia by Increasing the Expression of miR-223-3p.

Postherpetic neuralgia (PHN) is a complication of herpes zoster viral infection. Its main manifestations are continuous or intermittent burning-like and electroshock-like pain in the affected nerves. Electroacupuncture (EA) is widely used in clinical treatment and exerts effects in alleviating neuropathic pain. In this study, we investigated the effect and underlying mechanism of EA on PHN. Sprague-Dawley rats were treated with resiniferatoxin (RTX) to establish a PHN model and subjected to EA and/or miR-223-3p overexpression (OV) or interference. Mechanical withdrawal latency was measured as an indication of pain sensitivity. Hematoxylin-eosin staining and transmission electron microscopy were performed to observe neuron cell morphology and autophagic vacuoles, respectively. ELISA was performed to detect reactive oxygen species (ROS) production and the levels of tumor necrosis factor- (TNF-) α, inducible nitric oxide synthase (iNOS), interleukin- (IL-) 6, and IL-10. Changes in autophagy and apoptosis-related miRNAs were detected by immunofluorescence and qRT-PCR, respectively. In RTX-treated rats, OV and EA reduced pain sensitivity, decreased the number of eosinophils, and increased that of nerve cells. ROS generation and the levels of TNF-α and iNOS were significantly reduced, while those of IL-6 and IL-10 were increased. OV and EA induced fewer autophagic vacuoles than those in the model group. The expression of autophagy-related protein microtubule-associated protein 1 light chain 3-II, ATG9, and Rab1 was decreased by OV and EA, whereas that of P62 was increased. qRT-PCR revealed that miR-223-3p expression in the model group decreased but was increased by EA. EA inhibits neuron cell autophagy in PHN by increasing miR-223-3p expression.

The fungal RABOME: RAB GTPases acting in the endocytic and exocytic pathways of Aspergillus nidulans (with excursions to other filamentous fungi).

RAB GTPases are major determinants of membrane identity that have been exploited as highly specific reporters to study intracellular traffic in vivo. A score of fungal papers have considered individual RABs, but systematic, integrated studies on the localization and physiological role of these regulators and their effectors have been performed only with Aspergillus nidulans. These studies have influenced the intracellular trafficking field beyond fungal specialists, leading to findings such as the maturation of trans-Golgi (TGN) cisternae into post-Golgi RAB11 secretory vesicles, the concept that these RAB11 secretory carriers are loaded with three molecular nanomotors, the understanding of the role of endocytic recycling mediated by RAB6 and RAB11 in determining the hyphal mode of life, the discovery that early endosome maturation and the ESCRT pathway are essential, the identification of specific adaptors of dynein-dynactin to RAB5 endosomes, the exquisite dependence that autophagy displays on RAB1 activity, the role of TRAPPII as a GEF for RAB11, or the conclusion that the RAB1-to-RAB11 transition is not mediated by TRAPP maturation. A remarkable finding was that the A. nidulans Spitzenkörper contains four RABs: RAB11, Sec4, RAB6, and RAB1. How these RABs cooperate during exocytosis represents an as yet outstanding question.

Structural basis for VPS34 kinase activation by Rab1 and Rab5 on membranes.

The lipid phosphatidylinositol-3-phosphate (PI3P) is a regulator of two fundamental but distinct cellular processes, endocytosis and autophagy, so its generation needs to be under precise temporal and spatial control. PI3P is generated by two complexes that both contain the lipid kinase VPS34: complex II on endosomes (VPS34/VPS15/Beclin 1/UVRAG), and complex I on autophagosomes (VPS34/VPS15/Beclin 1/ATG14L). The endosomal GTPase Rab5 binds complex II, but the mechanism of VPS34 activation by Rab5 has remained elusive, and no GTPase is known to bind complex I. Here we show that Rab5a-GTP recruits endocytic complex II to membranes and activates it by binding between the VPS34 C2 and VPS15 WD40 domains. Electron cryotomography of complex II on Rab5a-decorated vesicles shows that the VPS34 kinase domain is released from inhibition by VPS15 and hovers over the lipid bilayer, poised for catalysis. We also show that the GTPase Rab1a, which is known to be involved in autophagy, recruits and activates the autophagy-specific complex I, but not complex II. Both Rabs bind to the same VPS34 interface but in a manner unique for each. These findings reveal how VPS34 complexes are activated on membranes by specific Rab GTPases and how they are recruited to unique cellular locations.

Amino acids-Rab1A-mTORC1 signaling controls whole-body glucose homeostasis.

Rab1A is a small GTPase known for its role in vesicular trafficking. Recent evidence indicates that Rab1A is essential for amino acids (aas) sensing and signaling to regulate mTORC1 in normal and cancer cells. However, Rab1A's in vivo function in mammals is not known. Here, we report the generation of tamoxifen (TAM)-induced whole body Rab1A knockout (Rab1A-/-) in adult mice. Rab1A-/- mice are viable but become hyperglycemic and glucose intolerant due to impaired insulin transcription and ß-cell proliferation and maintenance. Mechanistically, Rab1A mediates AA-mTORC1 signaling, particularly branched chain amino acids (BCAA), to regulate the stability and localization of the insulin transcription factor Pdx1. Collectively, these results reveal a physiological role of aa-Rab1A-mTORC1 signaling in the control of whole-body glucose homeostasis in mammals. Intriguingly, Rab1A expression is reduced in ß-cells of type 2 diabetes (T2D) patients, which is correlated with loss of insulin expression, suggesting that Rab1A downregulation contributes to T2D progression.

Rab1-AMPylation by Legionella DrrA is allosterically activated by Rab1.

Legionella pneumophila infects eukaryotic cells by forming a replicative organelle - the Legionella containing vacuole. During this process, the bacterial protein DrrA/SidM is secreted and manipulates the activity and post-translational modification (PTM) states of the vesicular trafficking regulator Rab1. As a result, Rab1 is modified with an adenosine monophosphate (AMP), and this process is referred to as AMPylation. Here, we use a chemical approach to stabilise low-affinity Rab:DrrA complexes in a site-specific manner to gain insight into the molecular basis of the interaction between the Rab protein and the AMPylation domain of DrrA. The crystal structure of the Rab:DrrA complex reveals a previously unknown non-conventional Rab-binding site (NC-RBS). Biochemical characterisation demonstrates allosteric stimulation of the AMPylation activity of DrrA via Rab binding to the NC-RBS. We speculate that allosteric control of DrrA could in principle prevent random and potentially cytotoxic AMPylation in the host, thereby perhaps ensuring efficient infection by Legionella.