RAB3GAP1 and RAB3GAP2 modulate basal and rapamycin-induced autophagy

Macroautophagy is a degradative pathway that sequesters and transports cytosolic cargo in autophagosomes to lysosomes, and its deterioration affects intracellular proteostasis. Membrane dynamics accompanying autophagy are mostly elusive and depend on trafficking processes. RAB GTPase activating proteins (RABGAPs) are important factors for the coordination of cellular vesicle transport systems, and several TBC (TRE2-BUB2-CDC16) domain-containing RABGAPs are associated with autophagy. Employing C. elegans and human primary fibroblasts, we show that RAB3GAP1 and RAB3GAP2, which are components of the TBC domain-free RAB3GAP complex, influence protein aggregation and affect autophagy at basal and rapamycin-induced conditions. Correlating the activity of RAB3GAP1/2 with ATG3 and ATG16L1 and analyzing ATG5 punctate structures, we illustrate that the RAB3GAPs modulate autophagosomal biogenesis. Significant levels of RAB3GAP1/2 colocalize with members of the Atg8 family at lipid droplets, and their autophagy modulatory activity depends on the GTPase-activating activity of RAB3GAP1 but is independent of the RAB GTPase RAB3. Moreover, we analyzed RAB3GAP1/2 in relation to the previously reported suppressive autophagy modulators FEZ1 and FEZ2 and demonstrate that both reciprocally regulate autophagy. In conclusion, we identify RAB3GAP1/2 as novel conserved factors of the autophagy and proteostasis network.     

The integration of autophagy and cellular trafficking pathways via RAB GAPs

Macroautophagy is a conserved degradative pathway in which a double-membrane compartment sequesters cytoplasmic cargo and delivers the contents to lysosomes for degradation. Efficient formation and maturation of autophagic vesicles, so-called phagophores that are precursors to autophagosomes, and their subsequent trafficking to lysosomes relies on the activity of small RAB GTPases, which are essential factors of cellular vesicle transport systems. The activity of RAB GTPases is coordinated by upstream factors, which include guanine nucleotide exchange factors (RAB GEFs) and RAB GTPase activating proteins (RAB GAPs). A role in macroautophagy regulation for different TRE2-BUB2-CDC16 (TBC) domain-containing RAB GAPs has been established. Recently, however, a positive modulation of macroautophagy has also been demonstrated for the TBC domain-free RAB3GAP1/2, adding to the family of RAB GAPs that coordinate macroautophagy and additional cellular trafficking pathways.     

Warburg Micro syndrome is caused by RAB18 deficiency or dysregulation

RAB18, RAB3GAP1, RAB3GAP2 and TBC1D20 are each mutated in Warburg Micro syndrome, a rare autosomal recessive multisystem disorder. RAB3GAP1 and RAB3GAP2 form a binary ‘RAB3GAP’ complex that functions as a guanine-nucleotide exchange factor (GEF) for RAB18, whereas TBC1D20 shows modest RAB18 GTPase-activating (GAP) activity in vitro. Here, we show that in the absence of functional RAB3GAP or TBC1D20, the level, localization and dynamics of cellular RAB18 is altered. In cell lines where TBC1D20 is absent from the endoplasmic reticulum (ER), RAB18 becomes more stably ER-associated and less cytosolic than in control cells. These data suggest that RAB18 is a physiological substrate of TBC1D20 and contribute to a model in which a Rab-GAP can be essential for the activity of a target Rab. Together with previous reports, this indicates that Warburg Micro syndrome can be caused directly by loss of RAB18, or indirectly through loss of RAB18 regulators RAB3GAP or TBC1D20.     

Control of RAB7 activity and localization through the retromer‐TBC1D5 complex enables RAB7‐dependent mitophagy

Retromer is an endosomal multi‐protein complex that organizes the endocytic recycling of a vast range of integral membrane proteins. Here, we establish an additional retromer function in controlling the activity and localization of the late endosomal small GTPase RAB7. Surprisingly, we found that RAB7 not only decorates late endosomes or lysosomes, but is also present on the endoplasmic reticulum, trans‐Golgi network, and mitochondrial membranes, a localization that is maintained by retromer and the retromer‐associated RAB7‐specific GAP TBC1D5. In the absence of either TBC1D5 or retromer, RAB7 activity state and localization are no longer controlled and hyperactivated RAB7 expands over the entire lysosomal domain. This lysosomal accumulation of hyperactivated RAB7 results in a striking loss of RAB7 mobility and overall depletion of the inactive RAB7 pool on endomembranes. Functionally, we establish that this control of RAB7 activity is not required for the recycling of retromer‐dependent cargoes, but instead enables the correct sorting of the autophagy related transmembrane protein ATG9a and autophagosome formation around damaged mitochondria during Parkin‐mediated mitophagy.     

Group A Streptococcus modulates RAB1- and PIK3C3 complex-dependent autophagy

ABSTRACT

Autophagy selectively targets invading bacteria to defend cells, whereas bacterial pathogens counteract autophagy to survive in cells. The initiation of canonical autophagy involves the PIK3C3 complex, but autophagy targeting Group A Streptococcus (GAS) is PIK3C3-independent. We report that GAS infection elicits both PIK3C3-dependent and -independent autophagy, and that the GAS effector NAD-glycohydrolase (Nga) selectively modulates PIK3C3-dependent autophagy. GAS regulates starvation-induced (canonical) PIK3C3-dependent autophagy by secreting streptolysin O and Nga, and Nga also suppresses PIK3C3-dependent GAS-targeting-autophagosome formation during early infection and facilitates intracellular proliferation. This Nga-sensitive autophagosome formation involves the ATG14-containing PIK3C3 complex and RAB1 GTPase, which are both dispensable for Nga-insensitive RAB9A/RAB17-positive autophagosome formation. Furthermore, although MTOR inhibition and subsequent activation of ULK1, BECN1, and ATG14 occur during GAS infection, ATG14 recruitment to GAS is impaired, suggesting that Nga inhibits the recruitment of ATG14-containing PIK3C3 complexes to autophagosome-formation sites. Our findings reveal not only a previously unrecognized GAS-host interaction that modulates canonical autophagy, but also the existence of multiple autophagy pathways, using distinct regulators, targeting bacterial infection.

Abbreviations: ATG5: autophagy related 5; ATG14: autophagy related 14; ATG16L1: autophagy related 16 like 1; BECN1: beclin 1; CALCOCO2: calcium binding and coiled-coil domain 2; GAS: group A streptococcus; GcAV: GAS-containing autophagosome-like vacuole; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; Nga: NAD-glycohydrolase; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns4P: phosphatidylinositol-4-phosphate; RAB: RAB, member RAS oncogene GTPases; RAB1A: RAB1A, member RAS oncogene family; RAB11A: RAB11A, member RAS oncogene family; RAB17: RAB17, member RAS oncogene family; RAB24: RAB24, member RAS oncogene family; RPS6KB1: ribosomal protein S6 kinase B1; SLO: streptolysin O; SQSTM1: sequestosome 1; ULK1: unc-51 like autophagy activating kinase 1; WIPI2: WD repeat domain, phosphoinositide interacting 2     

The small GTPase RAB37 functions as an organizer for autophagosome biogenesis

Macroautophagy/autophagy is a catabolic process that is essential for cellular homeostasis. How autophagosomal vesicle forms in a spatio-temporally regulated manner remains elusive. Our recent study revealed that small GTPase, RAB37 (RAB37, member RAS oncogene family), functions as a key organizer of autophagosomal membrane biogenesis. RAB37 interacts with ATG5 (autophagy related 5) and promotes autophagosome formation by modulating ATG12–ATG5-ATG16L1 complex assembly. These findings provide new insights into autophagy regulation.     

Transiently expressed ATG16L1 inhibits autophagosome biogenesis and aberrantly targets RAB11-positive recycling endosomes

The membrane source for autophagosome biogenesis is an unsolved mystery in the study of autophagy. ATG16L1 forms a complex with ATG12–ATG5 (the ATG16L1 complex). The ATG16L1 complex is recruited to autophagic membranes to convert MAP1LC3B-I to MAP1LC3B-II. The ATG16L1 complex dissociates from the phagophore before autophagosome membrane closure. Thus, ATG16L1 can be used as an early event marker for the study of autophagosome biogenesis. We found that among 3 proteins in the ATG16L1 complex, only ATG16L1 formed puncta-like structures when transiently overexpressed. ATG16L1⁺ puncta formed by transient expression could represent autophagic membrane structures. We thoroughly characterized the transiently expressed ATG16L1 in several mammalian cell lines. We found that transient expression of ATG16L1 not only inhibited autophagosome biogenesis, but also aberrantly targeted RAB11-positive recycling endosomes, resulting in recycling endosome aggregates. We conclude that transient expression of ATG16L1 is not a physiological model for the study of autophagy. Caution is warranted when reviewing findings derived from a transient expression model of ATG16L1.     

RAB26-dependent autophagy protects adherens junctional integrity in acute lung injury

Microvascular barrier dysfunction is the central pathophysiological feature of acute lung injury (ALI). RAB26 is a newly identified small GTPase involved in the regulation of endothelial cell (EC) permeability. However, the mechanism behind this protection has not been clearly elucidated. Here we found that RAB26 promoted the integrity of adherens junctions (AJs) in a macroautophagy/autophagy-dependent manner in ALI. RAB26 is frequently downregulated in mouse lungs after LPS treatment. Mice lacking Rab26 exhibited phosphorylated SRC expression and increased CDH5/VE-cadherin phosphorylation, leading to AJ destruction. rab26-null mice showed further aggravation of the effects of endotoxin insult on lung vascular permeability and water content. Depletion of RAB26 resulted in upregulation of phosphorylated SRC, enhancement of CDH5 phosphorylation, and aggravation of CDH5 internalization, thereby weakening AJ integrity and endothelial barrier function in human pulmonary microvascular endothelial cells (HPMECs). RAB26 overexpression caused active interaction between SRC and the autophagy marker LC3-II and promoted degradation of phosphorylated SRC. Furthermore, RAB26 was involved in a direct and activation-dependent manner in autophagy induction through interaction with ATG16L1 in its GTP-bound form. These findings demonstrate that RAB26 exerts a protective effect on endothelial cell (EC) permeability, which is in part dependent on autophagic targeting of active SRC, and the resultant CDH5 dephosphorylation maintains AJ stabilization. Thus, RAB26-mediated autophagic targeting of phosphorylated SRC can maintain barrier integrity when flux through the RAB26-SRC pathway is protected. These findings suggest that activation of RAB26-SRC signaling provides a new therapeutic opportunity to prevent vascular leakage in ALI.

Abbreviations: AJs: adherens junctions; ALI: acute lung injury; ARDS: acute respiratory distress syndrome; ATG5: autophagy related 5; ATG12: autophagy related 12; ATG 16L1: autophagy related 16 like; 1 BALF: bronchoalveolar lavage fluidCQ: chloroquine; Ctrl: control; EC: endothelial cell; GFP: green fluorescent protein; HA-tagged; RAB26^WT: HA-tagged wild-type; RAB26 HA-tagged; RAB26^QL: HA-tagged; RAB26^Q123LHA-tagged; RAB26^NI: HA-tagged; RAB26^N177IHPMECs: human pulmonary microvascular endothelial cells; H&E: hematoxylin & eosin; IgG: immunoglobulin; GIF: immunofluorescence; IP: immunoprecipitationi;. p.: intraperitoneal; LPS: lipopolysaccharide; PBS: phosphate-buffered salinesi; RNA: small interfering;RNASQSTM1/p62, sequestosome; 1TBS: Tris-buffered saline; VEGF: vascular endothelial growth factor; WB: western blot; WT: wild-type     

Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase

Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes a potentially fatal emerging zoonosis, human monocytic ehrlichiosis. E. chaffeensis has a limited capacity for biosynthesis and metabolism and thus depends mostly on host-synthesized nutrients for growth. Although the host cell cytoplasm is rich with these nutrients, as E. chaffeensis is confined within the early endosome-like membrane-bound compartment, only host nutrients that enter the compartment can be used by this bacterium. How this occurs is unknown. We found that ehrlichial replication depended on autophagy induction involving class III phosphatidylinositol 3-kinase (PtdIns3K) activity, BECN1 (Beclin 1), and ATG5 (autophagy-related 5). Ehrlichia acquired host cell preincorporated amino acids in a class III PtdIns3K-dependent manner and ehrlichial growth was enhanced by treatment with rapamycin, an autophagy inducer. Moreover, ATG5 and RAB5A/B/C were routed to ehrlichial inclusions. RAB5A/B/C siRNA knockdown, or overexpression of a RAB5-specific GTPase-activating protein or dominant-negative RAB5A inhibited ehrlichial infection, indicating the critical role of GTP-bound RAB5 during infection. Both native and ectopically expressed ehrlichial type IV secretion effector protein, Etf-1, bound RAB5 and the autophagy-initiating class III PtdIns3K complex, PIK3C3/VPS34, and BECN1, and homed to ehrlichial inclusions. Ectopically expressed Etf-1 activated class III PtdIns3K as in E. chaffeensis infection and induced autophagosome formation, cleared an aggregation-prone mutant huntingtin protein in a class III PtdIns3K-dependent manner, and enhanced ehrlichial proliferation. These data support the notion that E. chaffeensis secretes Etf-1 to induce autophagy to repurpose the host cytoplasm and capture nutrients for its growth through RAB5 and class III PtdIns3K, while avoiding autolysosomal killing.     

Crystal structure of TBC1D15 GTPase‐activating protein (GAP) domain and its activity on Rab GTPases

TBC1D15 belongs to the TBC (Tre‐2/Bub2/Cdc16) domain family and functions as a GTPase‐activating protein (GAP) for Rab GTPases. So far, the structure of TBC1D15 or the TBC1D15·Rab complex has not been determined, thus, its catalytic mechanism on Rab GTPases is still unclear. In this study, we solved the crystal structures of the Shark and Sus TBC1D15 GAP domains, to 2.8 Å and 2.5 Å resolution, respectively. Shark‐TBC1D15 and Sus‐TBC1D15 belong to the same subfamily of TBC domain‐containing proteins, and their GAP‐domain structures are highly similar. This demonstrates the evolutionary conservation of the TBC1D15 protein family. Meanwhile, the newly determined crystal structures display new variations compared to the structures of yeast Gyp1p Rab GAP domain and TBC1D1. GAP assays show that Shark and Sus GAPs both have higher catalytic activity on Rab11a·GTP than Rab7a·GTP, which differs from the previous study. We also demonstrated the importance of arginine and glutamine on the catalytic sites of Shark GAP and Sus GAP. When arginine and glutamine are changed to alanine or lysine, the activities of Shark GAP and Sus GAP are lost.     

Global effects of RAB3GAP1 dysexpression on the proteome of mouse cortical neurons

Mounting studies have demonstrated that RAB3GAP1 expression is modified in brain diseases with multiple neurobiological functions and processes and acts as a potentially significant target. However, the cellular and molecular events arising from RAB3GAP1 dysexpression are still incompletely understood. In this work, underexpression and overexpression of RAB3GAP1 were first induced into cultured mouse cortical neurons by transfection with lentivirus plasmids. Then we globally explored the effects of RAB3GAP1 dysexpression on the proteome of the neurons through the use of isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomics with bioinformatics. A total of 364 proteins in the RAB3GAP1-underexpression group and 314 proteins in the RAB3GAP1-overexpression group were identified to be differentially expressed. Subsequent bioinformatics analysis indicated that the proteome functional expression profiles induced by RAB3GAP1 underexpression and overexpression were different, suggesting the potential differences in biological processes and cellular effects. Subsequent intergroup cross-comparison revealed some candidate target proteins regulated directly by RAB3GAP1. Further parallel reaction monitoring (PRM) analysis illustrated that Sub1, Ssrp1, and Top1 proteins might serve as new potentially important linkers in the RAB3GAP1-mediated autophagy pathway in the cortical neurons. Collectively, the current proteomics data furnished new valuable insights to better understand the regulatory molecular mechanism of neuronal RAB3GAP1.

     

TBC1D5 and the AP2 complex regulate ATG9 trafficking and initiation of autophagy

The RabGAP protein TBC1D5 controls cellular endomembrane trafficking processes and binds the retromer subunit VPS29 and the ubiquitin‐like protein ATG8 (LC3). Here, we describe that TBC1D5 also associates with ATG9 and the active ULK1 complex during autophagy. Moreover, ATG9 and TBC1D5 interact with clathrin and the AP2 complex. Depletion of TBC1D5 leads to missorting of ATG9 to late endosomes upon activation of autophagy, whereas inhibition of clathrin‐mediated endocytosis or AP2 depletion alters ATG9 trafficking and its association with TBC1D5. Taken together, our data show that TBC1D5 and the AP2 complex are important novel regulators of the rerouting of ATG9‐containing vesicular carriers toward sites of autophagosome formation.     

Vacuolin-1 potently and reversibly inhibits autophagosome-lysosome fusion by activating RAB5A

Autophagy is a catabolic lysosomal degradation process essential for cellular homeostasis and cell survival. Dysfunctional autophagy has been associated with a wide range of human diseases, e.g., cancer and neurodegenerative diseases. A large number of small molecules that modulate autophagy have been widely used to dissect this process and some of them, e.g., chloroquine (CQ), might be ultimately applied to treat a variety of autophagy-associated human diseases. Here we found that vacuolin-1 potently and reversibly inhibited the fusion between autophagosomes and lysosomes in mammalian cells, thereby inducing the accumulation of autophagosomes. Interestingly, vacuolin-1 was less toxic but at least 10-fold more potent in inhibiting autophagy compared with CQ. Vacuolin-1 treatment also blocked the fusion between endosomes and lysosomes, resulting in a defect in general endosomal-lysosomal degradation. Treatment of cells with vacuolin-1 alkalinized lysosomal pH and decreased lysosomal Ca²⁺ content. Besides marginally inhibiting vacuolar ATPase activity, vacuolin-1 treatment markedly activated RAB5A GTPase activity. Expression of a dominant negative mutant of RAB5A or RAB5A knockdown significantly inhibited vacuolin-1-induced autophagosome-lysosome fusion blockage, whereas expression of a constitutive active form of RAB5A suppressed autophagosome-lysosome fusion. These data suggest that vacuolin-1 activates RAB5A to block autophagosome-lysosome fusion. Vacuolin-1 and its analogs present a novel class of drug that can potently and reversibly modulate autophagy.     

Vacuolin-1 potently and reversibly inhibits autophagosome-lysosome fusion by activating RAB5A

Autophagy is a catabolic lysosomal degradation process essential for cellular homeostasis and cell survival. Dysfunctional autophagy has been associated with a wide range of human diseases, e.g., cancer and neurodegenerative diseases. A large number of small molecules that modulate autophagy have been widely used to dissect this process and some of them, e.g., chloroquine (CQ), might be ultimately applied to treat a variety of autophagy-associated human diseases. Here we found that vacuolin-1 potently and reversibly inhibited the fusion between autophagosomes and lysosomes in mammalian cells, thereby inducing the accumulation of autophagosomes. Interestingly, vacuolin-1 was less toxic but at least 10-fold more potent in inhibiting autophagy compared with CQ. Vacuolin-1 treatment also blocked the fusion between endosomes and lysosomes, resulting in a defect in general endosomal-lysosomal degradation. Treatment of cells with vacuolin-1 alkalinized lysosomal pH and decreased lysosomal Ca²⁺ content. Besides marginally inhibiting vacuolar ATPase activity, vacuolin-1 treatment markedly activated RAB5A GTPase activity. Expression of a dominant negative mutant of RAB5A or RAB5A knockdown significantly inhibited vacuolin-1-induced autophagosome-lysosome fusion blockage, whereas expression of a constitutive active form of RAB5A suppressed autophagosome-lysosome fusion. These data suggest that vacuolin-1 activates RAB5A to block autophagosome-lysosome fusion. Vacuolin-1 and its analogs present a novel class of drug that can potently and reversibly modulate autophagy.     

A novel ER-localized transmembrane protein,EMC6, interacts with RAB5A and regulates cell autophagy

Autophagy is mediated by a unique organelle, the autophagosome, which encloses a portion of the cytoplasm for delivery to the lysosome. Phosphatidylinositol 3-phosphate (PtdIns3P) produced by the class III phosphatidylinositol 3-kinase (PtdIns3K) complex is essential for canonical autophagosome formation. RAB5A, a small GTPase localized to early endosomes, has been shown to associate with the class III PtdIns3K complex, regulate its activity and promote autophagosome formation. However, little is known about how endosome-localized RAB5A functions with the class III PtdIns3K complex. Here we identified a novel endoplasmic reticulum (ER)-localized transmembrane protein, ER membrane protein complex subunit 6 (EMC6), which interacted with both RAB5A and BECN1/Beclin 1 and colocalized with the omegasome marker ZFYVE1/DFCP1. It was shown to regulate autophagosome formation, and its deficiency caused the accumulation of autophagosomal precursor structures and impaired autophagy. Our study showed for the first time that EMC6 is a novel regulator involved in autophagy.     

Mutation in Rab3 GTPase-activating protein (RAB3GAP) noncatalytic subunit in a kindred with Martsolf syndrome

We identified a homozygous missense mutation in the noncatalytic subunit (RAB3GAP2) of RAB3GAP that results in abnormal splicing in a family with congenital cataracts, hypogonadism, and mild mental retardation (Martsolf syndrome). Recently, mutations in the catalytic subunit of RAB3GAP (RAB3GAP1), a key regulator of calcium-mediated hormone and neurotransmitter exocytosis, were reported in Warburg micro syndrome, a severe neurodevelopmental condition with overlapping clinical features. RAB3GAP is a heterodimeric protein that consists of a catalytic subunit and a noncatalytic subunit encoded by RAB3GAP1 and RAB3GAP2, respectively. We performed messenger RNA-expression studies of RAB3GAP1 and RAB3GAP2 orthologues in Danio rerio embryos and demonstrated that, whereas developmental expression of rab3gap1 was generalized (similar to that reported elsewhere in mice), rab3gap2 expression was restricted to the central nervous system. These findings are consistent with RAB3GAP2 having a key role in neurodevelopment and may indicate that Warburg micro and Martsolf syndromes represent a spectrum of disorders. However, we did not detect RAB3GAP2 mutations in patients with Warburg micro syndrome. These findings suggest that RAB3GAP dysregulation may result in a spectrum of phenotypes that range from Warburg micro syndrome to Martsolf syndrome.     

SEACing the GAP that nEGOCiates TORC1 activation

The target of rapamycin complex 1 (TORC1) regulates eukaryotic cell growth in response to a variety of input signals. In S. cerevisiae, amino acids activate TORC1 through the Rag guanosine triphosphatase (GTPase) heterodimer composed of Gtr1 and Gtr2 found together with Ego1 and Ego3 in the EGO complex (EGOC). The GTPase activity of Gtr1 is regulated by the SEA complex (SEAC). Specifically, SEACIT, a SEAC subcomplex containing Iml1, Npr2, and Npr3 functions as a GTPase activator (GAP) for Gtr1 to decrease the activity of TORC1 and, consequently, growth, after amino acid deprivation. Here, we present genetic epistasis data, which show that SEACAT, the other SEAC subcomplex, containing Seh1, Sea2–4, and Sec13, antagonizes the GAP function of SEACIT. Orthologs of EGOC (Ragulator), SEACIT (GATOR1), and SEACAT (GATOR2) are present in higher eukaryotes, highlighting the remarkable conservation, from yeast to man, of Rag GTPase and TORC1 regulation.