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     

Binding of Avibirnavirus VP3 to the PIK3C3-PDPK1 complex inhibits autophagy by activating the AKT-MTOR pathway

ABSTRACT

Macroautophagy/autophagy is a host natural defense response. Viruses have developed various strategies to subvert autophagy during their life cycle. Recently, we revealed that autophagy was activated by binding of Avibirnavirus to cells. In the present study, we report the inhibition of autophagy initiated by PIK3C3/VPS34 via the PDPK1-dependent AKT-MTOR pathway. Autophagy detection revealed that viral protein VP3 triggered inhibition of autophagy at the early stage of Avibirnavirus replication. Subsequent interaction analysis showed that the CC1 domain of VP3 disassociated PIK3C3-BECN1 complex by direct interaction with BECN1 and blocked autophagosome formation, while the CC3 domain of VP3 disrupted PIK3C3-PDPK1 complex via directly binding to PIK3C3 and inhibited both formation and maturation of autophagosome. Furthermore, we found that PDPK1 activated AKT-MTOR pathway for suppressing autophagy via binding to AKT. Finally, we proved that CC3 domain was critical for role of VP3 in regulating replication of Avibirnavirus through autophagy. Taken together, our study identified that Avibirnavirus VP3 links PIK3C3-PDPK1 complex to AKT-MTOR pathway and inhibits autophagy, a critical step for controlling virus replication.     

Selective autophagy inhibition through disruption of the PIK3C3-containing complex I

ABSTRACT

The PIK3C3/VPS34-containing phosphatidylinositol 3-kinase (PtdIns3K) initiation complex (complex I) is necessary for macroautophagy/autophagy initiation and is comprised of PIK3R4/VPS15-PIK3C3/VPS34-BECN1-ATG14, while the endosomal trafficking complex (complex II) is necessary for vesicle trafficking and is comprised of PIK3R4/VPS15-PIK3C3/VPS34-BECN1-UVRAG. This composition difference was exploited to identify novel and specific autophagy inhibitors that disrupted the BECN1-ATG14 protein-protein interaction, without affecting vesicle trafficking. A cellular NanoBRET assay was implemented to identify these inhibitors, and one compound was able to successfully disrupt the BECN1-ATG14 interaction and inhibit autophagy, with limited impact on vesicle trafficking. These results reveal the first protein-protein interaction inhibitor targeting the autophagy initiation machinery and demonstrate the viability of targeting protein-protein interactions for the discovery of autophagy-specific modulators.     

Regulation of PIK3C3/VPS34 complexes by MTOR in nutrient stress-induced autophagy

Autophagy is a cellular defense response to stress conditions, such as nutrient starvation. The type III phosphatidylinositol (PtdIns) 3-kinase, whose catalytic subunit is PIK3C3/VPS34, plays a critical role in intracellular membrane trafficking and autophagy induction. PIK3C3 forms multiple complexes and the ATG14-containing PIK3C3 is specifically involved in autophagy induction. Mechanistic target of rapamycin (MTOR) complex 1, MTORC1, is a key cellular nutrient sensor and integrator to stimulate anabolism and inhibit catabolism. Inactivation of TORC1 by nutrient starvation plays a critical role in autophagy induction. In this report we demonstrated that MTORC1 inactivation is critical for the activation of the autophagy-specific (ATG14-containing) PIK3C3 kinase, whereas it has no effect on ATG14-free PIK3C3 complexes. MTORC1 inhibits the PtdIns 3-kinase activity of ATG14-containing PIK3C3 by phosphorylating ATG14, which is required for PIK3C3 inhibition by MTORC1 both in vitro and in vivo. Our data suggest a mechanistic link between amino acid starvation and autophagy induction via the direct activation of the autophagy-specific PIK3C3 kinase.     

Mammalian PIK3C3/VPS34

PIK3C3/Vps34 is the class III PtdIns3K that is evolutionarily conserved from yeast to mammals. Its central role in mammalian autophagy has been suggested through the use of pharmacological inhibitors and the study of its binding partners. However, the precise role of PIK3C3 in mammals is not clear. Using mouse strains that allow tissue-specific deletion of PIK3C3, we have described an essential role of PIK3C3 in regulating autophagy, and liver and heart function.     

AMPK connects energy stress to PIK3C3/VPS34 regulation

The class III phosphatidylinositol (PtdIns)-3 kinase, PIK3C3/VPS34, forms multiple complexes and regulates a variety of cellular functions, especially in intracellular vesicle trafficking and autophagy. Even though PtdIns3P, the product of PIK3C3, is thought to be a critical membrane marker for the autophagosome, it is unclear how PIK3C3 is regulated in response to autophagy-inducing stimuli. A complexity of PIK3C3 biology is due in part to the existence of multiple complexes, of which the ATG14- or UVRAG-containing complexes play important roles in autophagy. We recently discovered differential regulation of distinct PIK3C3 complexes in response to energy starvation and showed a mechanism by which AMPK directly phosphorylates PIK3C3 and BECN1 to regulate non- and pro-autophagic PIK3C3 complexes, respectively.     

Effects of neuronal PIK3C3/Vps34 deletion on autophagy and beyond

PIK3C3/Vps34 plays important roles in the endocytic and autophagic pathways, both of which are essential for maintaining neuronal integrity. However, it is unclear how inactivating PIK3C3 may affect neuronal endosomal versus autophagic processes in vivo. We generated a conditional null allele of the Pik3c3 gene in mouse, and specifically deleted it in postmitotic sensory neurons. Subsequent analyses reveal several interesting and surprising findings.Key words: PIK3C3/Vps34, ATG7, sensory neurons, neurodegeneration, autophagy, abnormal endosomePIK3C3 (commonly known as Vps34) is the class III phosphatidylinositol 3-kinase (PtdIns3K) that specifically catalyzes the formation of phosphatidylinositol-3-phosphate (PtdIns3P). It is the only PtdIns3K that is conserved from lower eukaryotes to mammals, and represents the most ancient form of PtdIns3Ks. Studies in invertebrate organisms as well as mammalian cell lines show that PIK3C3/Vps34 regulates multiple aspects of both the endocytic and the autophagic pathways. On one hand, PIK3C3 is important for the progression of early endosome to late endosome, and the biogenesis of multivesicular bodies. On the other hand, PIK3C3 is critical for the initiation of autophagosome formation. A chemical inhibitor of PIK3C3, 3-MA, has been commonly used as a specific inhibitor for autophagy. The distinct functions of PIK3C3 are thought to be carried out by at least two different PIK3C3 complexes. In yeast, complex I (Vps34, Vps15, Atg6 and Atg14) is involved in autophagy, whereas complex II (Vps34, Vps15, Atg6 and Vps38) functions in the vacuolar protein sorting process. In mammals, the homologue of complex I (PIK3C3, p150, Beclin 1 and Atg14L) activates autophagy, whereas the homologue of complex II (PIK3C3, p150, Beclin 1 and UVRAG/Vps38) regulates endocytic trafficking.To characterize the in vivo function of PIK3C3 in mammals, we generated a conditional allele of the Pik3c3 gene in mouse and specifically deleted it in postmitotic sensory neurons (Pik3c3-cKO mouse). We focused our analyses on sensory neurons because Pik3c3 is most abundantly expressed in these neurons. Detailed analyses of the sensory ganglia in the knockout mice reveal rapid but differential neurodegenerations of different types of sensory neurons within a few days after birth. Large-diameter myelinated mechanosensory and proprioceptive neurons undergo fast degeneration, whereas mutant small-diameter unmyelinated nociceptive neurons degenerate slower and survive longer.Interestingly, the large-diameter Pik3c3-deleted neurons rapidly accumulate ubiquitin-positive aggregates as well as numerous enlarged vesicles, which are likely abnormal endosomes. The accumulation of enlarged vesicles not only sequesters the cellular membrane source, but also could create trafficking jams that block the transport of prosurvival signals and/or material and organelles, and thus may underlie the rapid demise of large neurons. By contrast, the small-diameter Pik3c3-deleted neurons contain a limited number of vacuoles but gradually build up lysosome- like organelles. The marked increase of lysosomes seems to be more tolerable by neurons, but the mechanism underlying this phenotype is unclear. It could represent a protective and homeostatic response of neurons challenged with stress and insults to their endomembrane system. Alternatively, since sorting of many lysosomal proteins requires PtdIns3P, this phenotype may also result from a build-up of nonfunctional lysosomes as was the case in cathepsin B and L knockout mice. It is also unclear why two types of sensory neurons respond differently to a universal insult. One speculative explanation is that the large-diameter neurons are constantly activated under normal physiological conditions by touch and body movement and thus they contain more active endocytic and membrane trafficking processes; whereas small-diameter pain-sensing neurons are normally not activated and have less endocytic events. These differences might allow the two types of neurons to respond differently to PIK3C3 deletion.We further show that the fast and differential degeneration phenotypes in the Pik3c3-cKO mice are caused primarily by a disruption in the endosomal but not the autophagic pathway. This is validated by comparing the neuronal phenotypes of Pik3c3-cKO mice with those of Atg7-cKO mice, in which the autophagy-specific gene Atg7 is deleted using the same sensory neuron-specific cre driver. Disrupting autophagy leads to a slow degeneration of all types of sensory neurons over a period of several months, and formation of very large intracellular inclusion bodies in all sensory neurons. No increase of lysosomes or accumulation of enlarged vesicles is observed. The completely distinct phenotypes observed in Atg7-cKO versus Pik3c3-cKO mice suggest that inactivation of PIK3C3 primarily disrupts the endosomal pathway rather than inhibiting autophagy (at least in neurons). It calls into attention that care needs to be taken to interpret the results of using PIK3C3 inhibitors such as 3-MA as autophagy-specific inhibitors.The most surprising finding is the existence and activation of a noncanonical, PIK3C3-independent macroautophagy pathway in small-diameter Pik3c3-mutant neurons. Although PIK3C3 is traditionally viewed as indispensable for autophagy initiation, several recent studies suggest a possible PIK3C3-independent autophagy pathway in various cell lines and in Drosophila. We show that this noncanonical autophagy pathway can occur in sensory neurons in vivo using three different assays: crossing Pik3c3-cKO mice to the GFP-LC3 reporter line, western blot analyses of LC3 isoforms, and performing autophagy flux experiments. Interestingly, analyses of Pik3c3/Atg7 double-mutant neurons indicate that this alternative autophagosome initiation pathway still requires ATG7 and hence the conventional conjugation systems. Therefore, this non-canonical autophagy is distinct from the newly reported ATG5/ATG7-independent but PIK3C3-dependent autophagy. We speculate that activation of this PIK3C3-independent autophagy in small-diameter mutant neurons is part of the reason for their longer survival period.The molecular mechanism underlying the PIK3C3-independent autophagosome formation is unknown. It is possible that PtdIns3P can be generated at a low level on the membrane of pre-autophagosomes/phagophores by salvage pathways using other lipid kinases or phosphatases. Alternatively, other mechanisms may direct the formation of the crescent-shaped double membrane structures. For instance, asymmetric insertion into the membrane of proteins with amphipathic helices can induce membrane curvature; BAR domain-containing proteins can also detect and facilitate the formation of curved membrane structures. Thus, these types of proteins might potentially be recruited to nucleate the formation of pre-autophagosomes in the absence of PIK3C3. Finally, the role of this PIK3C3-independent autophagy under normal physiological conditions in vivo needs to be explored.     

Mammalian PIK3C3/VPS34: the key to autophagic processing in liver and heart

PIK3C3/Vps34 is the class III PtdIns3K that is evolutionarily conserved from yeast to mammals. Its central role in mammalian autophagy has been suggested through the use of pharmacological inhibitors and the study of its binding partners. However, the precise role of PIK3C3 in mammals is not clear. Using mouse strains that allow tissue-specific deletion of PIK3C3, we have described an essential role of PIK3C3 in regulating autophagy, and liver and heart function.     

MTORC1-mediated NRBF2 phosphorylation functions as a switch for the class III PtdIns3K and autophagy

NRBF2/Atg38 has been identified as the fifth subunit of the macroautophagic/autophagic class III phosphatidylinositol 3-kinase (PtdIns3K) complex, along with ATG14/Barkor, BECN1/Vps30, PIK3R4/p150/Vps15 and PIK3C3/Vps34. However, its functional mechanism and regulation are not fully understood. Here, we report that NRBF2 is a fine tuning regulator of PtdIns3K controlled by phosphorylation. Human NRBF2 is phosphorylated by MTORC1 at S113 and S120. Upon nutrient starvation or MTORC1 inhibition, NRBF2 phosphorylation is diminished. Phosphorylated NRBF2 preferentially interacts with PIK3C3/PIK3R4. Suppression of NRBF2 phosphorylation by MTORC1 inhibition alters its binding preference from PIK3C3/PIK3R4 to ATG14/BECN1, leading to increased autophagic PtdIns3K complex assembly, as well as enhancement of ULK1 protein complex association. Consequently, NRBF2 in its unphosphorylated form promotes PtdIns3K lipid kinase activity and autophagy flux, whereas its phosphorylated form blocks them. This study reveals NRBF2 as a critical molecular switch of PtdIns3K and autophagy activation, and its on/off state is precisely controlled by MTORC1 through phosphorylation.     

PIK3C3/VPS34 control by acetylation

PIK3C3/VPS34 (phosphatidylinositol 3-kinase catalytic subunit type 3) converts phosphatidylinositol (PtdIns) to phosphatidylinositol-3-phosphate (PtdIns3P), sustaining macroautophagy/autophagy and endosomal transport. So far, facilitating the assembly of the PIK3C3/VPS34-BECN1-PIK3R4/VPS15/p150 core complex at distinct membranes is the only known way to activate PIK3C3/VPS34 in cells. We have recently revealed a novel mechanism that regulates PIK3C3/VPS34 activation; cellular PIK3C3/VPS34 is repressed under nutrient-rich conditions by EP300/p300-mediated acetylation. Following nutrient-deprivation that drops EP300 activity, PIK3C3/VPS34 is liberated by deacetylation. Intriguingly, while deacetylation of the N-terminal K29 residue accounts for core complex formation, deacetylation at the C-terminal K771 site determines the binding of PIK3C3/VPS34 to its substrate PtdIns. In vitro and in cell evidence shows that EP300-dependent acetylation and deacetylation is a switch for turning off/on PIK3C3/VPS34 in which deacetylation of K771 is required for its full activation. This PIK3C3/VPS34 activation mechanism is utilized not only by starvation-induced autophagy but also by autophagy without the involvement of AMPK, MTORC1 or ULK1. These findings suggest an alternative circuit in cells for PIK3C3/VPS34 activation, which is involved in membrane transformations in response to metabolic and nonmetabolic cues.     

The NEDD4-USP13 axis facilitates autophagy via deubiquitinating PIK3C3

ABSTRACT

Macroautophagy/autophagy, an evolutionarily conserved eukaryotic bioprocess, plays an important role in the bulk degradation of intracellular macromolecules, organelles, and invading pathogens. PIK3C3/VPS34 (phosphatidylinositol 3-kinase catalytic subunit type 3) functions as a key protein in autophagy initiation and progression. The activity of PIK3C3 is tightly regulated by multiple post-translational modifications, including ubiquitination, however, the regulatory mechanisms underpinning the reversible deubiquitination of PIK3C3 remain poorly understood. Recently, we identified the E3 ubiquitin ligase NEDD4/NEDD4-1 as a positive regulator of autophagy through decreasing the K48-linked ubiquitination of PIK3C3 by recruiting USP13.     

Two-layer regulation of PAQR3 on ATG14-linked class III PtdIns3K activation upon glucose starvation

As a central node of the macroautophagy/autophagy process, the BECN1/Beclin1-PIK3C3/VPS34 complex participates in different steps of autophagy by interacting with multiple molecules. The ATG14-associated PIK3C3 complex is involved in autophagy initiation, whereas the UVRAG-associated complex mainly modulates autophagosome maturation and endosome fusion. However, the molecular mechanism that coordinates the sequential execution of the autophagy program remains unknown. We have recently discovered that a Golgi-resident protein, PAQR3, regulates autophagy initiation as it preferentially facilitates the formation of the ATG14-linked PIK3C3 complex instead of the UVRAG-associated complex. Upon glucose starvation, AMPK directly phosphorylates T32 of PAQR3, which is crucial for the activation of the ATG14-associated class III PtdIns3K. Furthermore, Paqr3-deleted mice have a deficiency in exercise-induced autophagy as well as behavioral disorders. Thus, this work not only uncovers the regulatory mechanism of PAQR3 on autophagy initiation, but also provides a potential candidate therapeutic target for neurodegenerative diseases.     

Local detection of PtdIns3P at autophagosome biogenesis membrane platforms

Phosphatidylinositol 3-phosphate (PtdIns3P) is a key player of membrane trafficking regulation, mostly synthesized by the PIK3C3 lipid kinase. The presence of PtdIns3P on endosomes has been demonstrated; however, the role and dynamics of the pool of PtdIns3P dedicated to macroautophagy/autophagy remains elusive. Here we addressed this question by studying the mobilization of PtdIns3P in time and space during autophagosome biogenesis. We compared different dyes known to specifically detect PtdIns3P by fluorescence microscopy analysis, based on PtdIns3P-binding FYVE and PX domains, and show that these transfected dyes induce defects in endosomal dynamics as well as artificial and sustained autophagosome formation. In contrast, indirect use of recombinant FYVE enabled us to track and discriminate endosomal and autophagosomal pools of PtdIns3P. We used this method to analyze localization and dynamics of PtdIns3P subdomains on the endoplasmic reticulum, at sites of pre-autophagosome associated protein recruitment such as the PtdIns3P-binding ZFYVE1/DFCP1 and WIPI2 autophagy regulators. This approach thus revealed the presence of a specific pool of PtdIns3P at the site where autophagosome assembly is initiated.     

BECN1/Beclin 1 is recruited into lipid rafts by prion to activate autophagy in response to amyloid β 42

Prion protein (PRNP) has been implicated in various types of neurodegenerative diseases. Although much is known about prion diseases, the function of cellular PRNP remains cryptic. Here, we show that PRNP mediates amyloid β_1–42 (Aβ₄₂)-induced autophagy activation through its interaction with BECN1. Treatment with Aβ₄₂ enhanced autophagy flux in neuronal cells. Aβ₄₂-induced autophagy activation, however, was impaired in prnp-knockout primary cortical neurons and Prnp-knockdown or prnp-knockout neuronal cells. Immunoprecipitation assays revealed that PRNP interacted with BECN1 via the BCL2-binding domain of BECN1. This interaction promoted the subcellular localization of BECN1 into lipid rafts of the plasma membrane and enhanced activity of PtdIns3K (whose catalytic subunit is termed PIK3C3, mammalian ortholog of yeast VPS34) in lipid rafts by generating PtdIns3P in response to Aβ₄₂. Further, the levels of lipid rafts that colocalized with BECN1, decreased in the brains of aged C57BL/6 mice, as did PRNP. These results suggested that PRNP interacts with BECN1 to recruit the PIK3C3 complex into lipid rafts and thus activates autophagy in response to Aβ₄₂, defining a novel role of PRNP in the regulation of autophagy.     

A fine-tuning mechanism underlying self-control for autophagy: deSUMOylation of BECN1 by SENP3

ABSTRACT

The roles of SUMOylation and the related enzymes in autophagic regulation are unclear. Based on our previous studies that identified the SUMO2/3-specific peptidase SENP3 as an oxidative stress-responsive molecule, we investigated the correlation between SUMOylation and macroautophagy/autophagy. We found that Senp3^± mice showed increased autophagy in the liver under basal and fasting conditions, compared to Senp3^+/+ mice. We constructed a liver-specific senp3 knockout mouse; these Senp3-deficient liver tissues showed increased autophagy as well. Autophagic flux was accelerated in hepatic and other cell lines following knockdown of SENP3, both before and after the cells underwent starvation in the form of the serum and amino acid deprivation. We demonstrated that BECN1/beclin 1, the core molecule of the BECN1-PIK3C3 complex, could be SUMO3-conjugated by PIAS3 predominantly at K380 and deSUMOylated by SENP3. The basal SUMOylation of BECN1 was increased upon cellular starvation, which enhanced autophagosome formation by facilitating BECN1 interaction with other complex components UVRAG, PIK3C3 and ATG14, thus promoting PIK3C3 activity. In contrast, SENP3 deSUMOylated BECN1, which impaired BECN1-PIK3C3 complex formation or stability to suppress the PIK3C3 activity. DeSUMOylation of BECN1 restrained autophagy induction under basal conditions and especially upon starvation when SENP3 had accumulated in response to the increased generation of reactive oxygen species. Thus, while reversible SUMOylation regulated the degree of autophagy, SENP3 provided an intrinsic overflow valve for fine-tuning autophagy induction.     

PI3P phosphatase activity is required for autophagosome maturation and autolysosome formation

Autophagosome formation is promoted by the PI3 kinase complex and negatively regulated by myotubularin phosphatases, indicating that regulation of local phosphatidylinositol 3‐phosphate (PtdIns3P) levels is important for this early phase of autophagy. Here, we show that the Caenorhabditis elegans myotubularin phosphatase MTM‐3 catalyzes PtdIns3P turnover late in autophagy. MTM‐3 acts downstream of the ATG‐2/EPG‐6 complex and upstream of EPG‐5 to promote autophagosome maturation into autolysosomes. MTM‐3 is recruited to autophagosomes by PtdIns3P, and loss of MTM‐3 causes increased autophagic association of ATG‐18 in a PtdIns3P‐dependent manner. Our data reveal critical roles of PtdIns3P turnover in autophagosome maturation and/or autolysosome formation.     

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.     

Primary cilium and autophagy: The avengers of cell-size regulation

The maintenance of cellular homeostasis in response to extracellular stresses by autophagy is vital for the health of various tissues. Extracellular stimuli may include nutrient starvation, endoplasmic reticulum stress, hypoxia, cytotoxic agents, or mechanical stress. The primary cilium (PC) is a microtubule-based sensory organelle that regulates the integration of various extracellular stimuli. The interconnection between macroautophagy/autophagy and the PC is beginning to be illuminated. In this punctum, we discuss our recent study of PC-dependent autophagy in response to fluid flow in kidney epithelial cells. Urinary flow in kidney tubules creates a shear stress that regulates epithelial cell volume. PC-mediated autophagy is necessary for the regulation of cell size. The signal from the PC is transduced by the activation of STK11/LKB1 and by MTOR inhibition. Our results clarify the physiological role of PC-dependent autophagy in the kidney and suggest that autophagy manipulation may provide a route to the treatment of ciliopathies.     

WIPI2B links PtdIns3P to LC3 lipidation through binding ATG16L1

WIPI proteins, phosphatidylinositol 3-phosphate (PtdIns3P) binding proteins with β-propeller folds, are recruited to the omegasome following PtdIns3P production. The functions of the WIPI proteins in autophagosome formation are poorly understood. In a recent study, we reported that WIPI2B directly binds ATG16L1 and functions by recruiting the ATG12–ATG5-ATG16L1 complex to forming autophagosomes during starvation- or pathogen-induced autophagy. Our model of WIPI2 function provides an explanation for the PtdIns3P-dependent recruitment of the ATG12–ATG5-ATG16L1 complex during initiation of autophagy.