Golgi-Resident Gαo Promotes Protrusive Membrane Dynamics.

To form protrusions like neurites, cells must coordinate their induction and growth. The first requires cytoskeletal rearrangements at the plasma membrane (PM), the second requires directed material delivery from cell's insides. We find that the Gαo-subunit of heterotrimeric G proteins localizes dually to PM and Golgi across phyla and cell types. The PM pool of Gαo induces, and the Golgi pool feeds, the growing protrusions by stimulated trafficking. Golgi-residing KDELR binds and activates monomeric Gαo, atypically for G protein-coupled receptors that normally act on heterotrimeric G proteins. Through multidimensional screenings identifying > 250 Gαo interactors, we pinpoint several basic cellular activities, including vesicular trafficking, as being regulated by Gαo. We further find small Golgi-residing GTPases Rab1 and Rab3 as direct effectors of Gαo. This KDELR → Gαo → Rab1/3 signaling axis is conserved from insects to mammals and controls material delivery from Golgi to PM in various cells and tissues.

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.

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.

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.

Conformational control of small GTPases by AMPylation.

Posttranslational modifications (PTMs) are important physiological means to regulate the activities and structures of central regulatory proteins in health and disease. Small GTPases have been recognized as important molecules that are targeted by PTMs during infections of mammalian cells by bacterial pathogens. The enzymes DrrA/SidM and AnkX from Legionella pneumophila AMPylate and phosphocholinate Rab1b during infection, respectively. Cdc42 is AMPylated by IbpA from Histophilus somni at tyrosine 32 or by VopS from Vibrio parahaemolyticus at threonine 35. These modifications take place in the important regulatory switch I or switch II regions of the GTPases. Since Rab1b and Cdc42 are central regulators of intracellular vesicular trafficking and of the actin cytoskeleton, their modifications by bacterial pathogens have a profound impact on the course of infection. Here, we addressed the biochemical and structural consequences of GTPase AMPylation and phosphocholination. By combining biochemical experiments and NMR analysis, we demonstrate that AMPylation can overrule the activity state of Rab1b that is commonly dictated by binding to guanosine diphosphate or guanosine triphosphate. Thus, PTMs may exert conformational control over small GTPases and may add another previously unrecognized layer of activity control to this important regulatory protein family.

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.

Rab29 activation of the Parkinson's disease-associated LRRK2 kinase.

Parkinson's disease predisposing LRRK2 kinase phosphorylates a group of Rab GTPase proteins including Rab29, within the effector-binding switch II motif. Previous work indicated that Rab29, located within the PARK16 locus mutated in Parkinson's patients, operates in a common pathway with LRRK2. Here, we show that Rab29 recruits LRRK2 to the trans-Golgi network and greatly stimulates its kinase activity. Pathogenic LRRK2 R1441G/C and Y1699C mutants that promote GTP binding are more readily recruited to the Golgi and activated by Rab29 than wild-type LRRK2. We identify conserved residues within the LRRK2 ankyrin domain that are required for Rab29-mediated Golgi recruitment and kinase activation. Consistent with these findings, knockout of Rab29 in A549 cells reduces endogenous LRRK2-mediated phosphorylation of Rab10. We show that mutations that prevent LRRK2 from interacting with either Rab29 or GTP strikingly inhibit phosphorylation of a cluster of highly studied biomarker phosphorylation sites (Ser910, Ser935, Ser955 and Ser973). Our data reveal that Rab29 is a master regulator of LRRK2, controlling its activation, localization, and potentially biomarker phosphorylation.

TRAPP complexes in membrane traffic: convergence through a common Rab.

Transport protein particle (TRAPP; also known as trafficking protein particle), a multimeric guanine nucleotide-exchange factor for the yeast GTPase Ypt1 and its mammalian homologue, RAB1, regulates multiple membrane trafficking pathways. TRAPP complexes exist in three forms, each of which activates Ypt1 or RAB1 through a common core of subunits and regulates complex localization through distinct subunits. Whereas TRAPPI and TRAPPII tether coated vesicles during endoplasmic reticulum to Golgi and intra-Golgi traffic, respectively, TRAPPIII has recently been shown to be required for autophagy. These advances illustrate how the TRAPP complexes link Ypt1 and RAB1 activation to distinct membrane-tethering events.

Screening Legionella effectors for antiviral effects reveals Rab1 GTPase as a proviral factor coopted for tombusvirus replication.

Bacterial virulence factors or effectors are proteins targeted into host cells to coopt or interfere with cellular proteins and pathways. Viruses often coopt the same cellular proteins and pathways to support their replication in infected cells. Therefore, we screened the Legionella pneumophila effectors to probe virus-host interactions and identify factors that modulate tomato bushy stunt virus (TBSV) replication in yeast surrogate host. Among 302 Legionella effectors tested, 28 effectors affected TBSV replication. To unravel a coopted cellular pathway in TBSV replication, the identified DrrA effector from Legionella was further exploited. We find that expression of DrrA in yeast or plants blocks TBSV replication through inhibiting the recruitment of Rab1 small GTPase and endoplasmic reticulum-derived COPII vesicles into the viral replication compartment. TBSV hijacks Rab1 and COPII vesicles to create enlarged membrane surfaces and optimal lipid composition within the viral replication compartment. To further validate our Legionella effector screen, we used the Legionella effector LepB lipid kinase to confirm the critical proviral function of PI(3)P phosphoinositide and the early endosomal compartment in TBSV replication. We demonstrate the direct inhibitory activity of LegC8 effector on TBSV replication using a cell-free replicase reconstitution assay. LegC8 inhibits the function of eEF1A, a coopted proviral host factor. Altogether, the identified bacterial effectors with anti-TBSV activity could be powerful reagents in cell biology and virus-host interaction studies. This study provides important proof of concept that bacterial effector proteins can be a useful toolbox to identify host factors and cellular pathways coopted by (+)RNA viruses.

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.

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.

COPI-TRAPPII activates Rab18 and regulates its lipid droplet association.

The transport protein particle (TRAPP) was initially identified as a vesicle tethering factor in yeast and as a guanine nucleotide exchange factor (GEF) for Ypt1/Rab1. In mammals, structures and functions of various TRAPP complexes are beginning to be understood. We found that mammalian TRAPPII was a GEF for both Rab18 and Rab1. Inactivation of TRAPPII-specific subunits by various methods including siRNA depletion and CRISPR-Cas9-mediated deletion reduced lipolysis and resulted in aberrantly large lipid droplets. Recruitment of Rab18 onto lipid droplet (LD) surface was defective in TRAPPII-deleted cells, but the localization of Rab1 on Golgi was not affected. COPI regulates LD homeostasis. We found that the previously documented interaction between TRAPPII and COPI was also required for the recruitment of Rab18 to the LD We hypothesize that the interaction between COPI and TRAPPII helps bring TRAPPII onto LD surface, and TRAPPII, in turn, activates Rab18 and recruits it on the LD surface to facilitate its functions in LD homeostasis.

The GTPase Rab1 Is Required for NLRP3 Inflammasome Activation and Inflammatory Lung Injury.

Uncontrolled inflammatory response during sepsis predominantly contributes to the development of multiorgan failure and lethality. However, the cellular and molecular mechanisms for excessive production and release of proinflammatory cytokines are not clearly defined. In this study, we show the crucial role of the GTPase Ras-related protein in brain (Rab)1a in regulating the nucleotide binding domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation and lung inflammatory injury. Expression of dominant negative Rab1 N124I plasmid in bone marrow-derived macrophages prevented the release of IL-1ß and IL-18, NLRP3 inflammasome activation, production of pro-IL-1ß and pro-IL-18, and attenuated TLR4 surface expression and NF-кB activation induced by bacterial LPS and ATP compared with control cells. In alveolar macrophage-depleted mice challenged with cecal ligation and puncture, pulmonary transplantation of Rab1a-inactivated macrophages by expression of Rab1 N124I plasmid dramatically reduced the release of IL-1ß and IL-18, neutrophil count in bronchoalveolar lavage fluid, and inflammatory lung injury. Rab1a activity was elevated in alveolar macrophages from septic patients and positively associated with severity of sepsis and respiratory dysfunction. Thus, inhibition of Rab1a activity in macrophages resulting in the suppression of NLRP3 inflammasome activation may be a promising target for the treatment of patients with sepsis.

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.

Human myotubularin-related protein 9 regulates ER-to-Golgi trafficking and modulates WNT3A secretion.

Regulation of phosphatidylinositol phosphates plays a crucial role in signal transduction, membrane trafficking or autophagy. Members of the myotubularin family of lipid phosphatases contribute to phosphoinositide metabolism by counteracting the activity of phosphoinositide kinases. The mechanisms determining their subcellular localization and targeting to specific membrane compartments are still poorly understood. We show here that the inactive phosphatase MTMR9 localizes to the intermediate compartment and to the Golgi apparatus and is able to recruit its active phosphatase partners MTMR6 and MTMR8 to these locations. Furthermore, MTMR8 and MTMR9 co-localize with the small GTPase RAB1A and regulate its localization. Loss of MTMR9 expression compromises the integrity of the Golgi apparatus and results in altered distribution of RAB1A and actin nucleation-promoting factor WHAMM. Loss or overexpression of MTMR9 leads to decreased rate of protein secretion. We demonstrate that secretion of physiologically relevant cargo exemplified by the WNT3A protein is affected after perturbation of MTMR9 levels.

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.

The small GTPase RAB1B promotes antiviral innate immunity by interacting with TNF receptor-associated factor 3 (TRAF3).

Innate immune detection of viral nucleic acids during viral infection activates a signaling cascade that induces type I and type III IFNs as well as other cytokines, to generate an antiviral response. This signaling is initiated by pattern recognition receptors, such as the RNA helicase retinoic acid-inducible gene I (RIG-I), that sense viral RNA. These sensors then interact with the adaptor protein mitochondrial antiviral signaling protein (MAVS), which recruits additional signaling proteins, including TNF receptor-associated factor 3 (TRAF3) and TANK-binding kinase 1 (TBK1), to form a signaling complex that activates IFN regulatory factor 3 (IRF3) for transcriptional induction of type I IFNs. Here, using several immunological and biochemical approaches in multiple human cell types, we show that the GTPase-trafficking protein RAB1B up-regulates RIG-I pathway signaling and thereby promotes IFN-ß induction and the antiviral response. We observed that RAB1B overexpression increases RIG-I-mediated signaling to IFN-ß and that RAB1B deletion reduces signaling of this pathway. Additionally, loss of RAB1B dampened the antiviral response, indicated by enhanced Zika virus infection of cells depleted of RAB1B. Importantly, we identified the mechanism of RAB1B action in the antiviral response, finding that it forms a protein complex with TRAF3 to facilitate the interaction of TRAF3 with mitochondrial antiviral signaling protein. We conclude that RAB1B regulates TRAF3 and promotes the formation of innate immune signaling complexes in response to nucleic acid sensing during RNA virus infection.

Autophagy in fibroblasts induced by cigarette smoke extract promotes invasion in lung cancer cells.

We investigated the effects of cigarette smoke extract (CSE) on lung fibroblasts and found that the invasiveness of lung cancer cells was facilitated by the conditioned medium from CSE-treated fibroblasts. CSE induced autophagy in fibroblasts and increased the expression of autophagy-related proteins, including optineurin and Ras-related protein Rab1B. Afterward, the fibroblasts produced high levels of interleukin-8 (IL-8), which promoted cancer cell invasion. The inhibition of either optineurin or Rab1B abrogated a rise in microtubule-associated protein 1 light chain 3 ß and a decrease in p62 protein, as well as the production of IL-8, in CSE-treated fibroblasts. A three-dimensional invasion assay using cancer cell spheroids revealed that the invasion of cancer cells alone and the fibroblast-led cancer cell invasion were both enhanced by the conditioned media from CSE-treated fibroblasts. These results suggest that cigarette smoke may induce autophagy and IL-8 secretion in lung fibroblasts and modify the microenvironment to favor invasion of lung cancer cells.

LRRK2 phosphorylates membrane-bound Rabs and is activated by GTP-bound Rab7L1 to promote recruitment to the trans-Golgi network.

Human genetic studies implicate LRRK2 and RAB7L1 in susceptibility to Parkinson disease (PD). These two genes function in the same pathway, as knockout of Rab7L1 results in phenotypes similar to LRRK2 knockout, and studies in cells and model organisms demonstrate LRRK2 and Rab7L1 interact in the endolysosomal system. Recently, a subset of Rab proteins have been identified as LRRK2 kinase substrates. Herein, we find that Rab8, Rab10, and Rab7L1 must be membrane and GTP-bound for LRRK2 phosphorylation. LRRK2 mutations that cause PD including R1441C, Y1699C, and G2019S all increase LRRK2 phosphorylation of Rab7L1 four-fold over wild-type LRRK2 in cells, resulting in the phosphorylation of nearly one-third the available Rab7L1 protein in cells. In contrast, the most common pathogenic LRRK2 mutation, G2019S, does not upregulate LRRK2-mediated phosphorylation of Rab8 or Rab10. LRRK2 interaction with membrane and GTP-bound Rab7L1, but not Rab8 or Rab10, results in the activation of LRRK2 autophosphorylation at the serine 1292 position, required for LRRK2 toxicity. Further, Rab7L1 controls the proportion of LRRK2 that is membrane-associated, and LRRK2 mutations enhance Rab7L1-mediated recruitment of LRRK2 to the trans-Golgi network. Interaction studies with the Rab8 and Rab10 GTPase-activating protein TBC1D4/AS160 demonstrate that LRRK2 phosphorylation may block membrane and GTP-bound Rab protein interaction with effectors. These results suggest reciprocal regulation between LRRK2 and Rab protein substrates, where Rab7L1-mediated upregulation of LRRK2 kinase activity results in the stabilization of membrane and GTP-bound Rab proteins that may be unable to interact with Rab effector proteins.

TBC1D14 regulates autophagy via the TRAPP complex and ATG9 traffic.

Macroautophagy requires membrane trafficking and remodelling to form the autophagosome and deliver its contents to lysosomes for degradation. We have previously identified the TBC domain-containing protein, TBC1D14, as a negative regulator of autophagy that controls delivery of membranes from RAB11-positive recycling endosomes to forming autophagosomes. In this study, we identify the TRAPP complex, a multi-subunit tethering complex and GEF for RAB1, as an interactor of TBC1D14. TBC1D14 binds to the TRAPP complex via an N-terminal 103 amino acid region, and overexpression of this region inhibits both autophagy and secretory traffic. TRAPPC8, the mammalian orthologue of a yeast autophagy-specific TRAPP subunit, forms part of a mammalian TRAPPIII-like complex and both this complex and TBC1D14 are needed for RAB1 activation. TRAPPC8 modulates autophagy and secretory trafficking and is required for TBC1D14 to bind TRAPPIII. Importantly, TBC1D14 and TRAPPIII regulate ATG9 trafficking independently of ULK1. We propose a model whereby TBC1D14 and TRAPPIII regulate a constitutive trafficking step from peripheral recycling endosomes to the early Golgi, maintaining the cycling pool of ATG9 required for initiation of autophagy.