SNX18 regulates ATG9A trafficking from recycling endosomes by recruiting Dynamin‐2

Trafficking of mammalian ATG9A between the Golgi apparatus, endosomes and peripheral ATG9A compartments is important for autophagosome biogenesis. Here, we show that the membrane remodelling protein SNX18, previously identified as a positive regulator of autophagy, regulates ATG9A trafficking from recycling endosomes. ATG9A is recruited to SNX18‐induced tubules generated from recycling endosomes and accumulates in juxtanuclear recycling endosomes in cells lacking SNX18. Binding of SNX18 to Dynamin‐2 is important for ATG9A trafficking from recycling endosomes and for formation of ATG16L1‐ and WIPI2‐positive autophagosome precursor membranes. We propose a model where upon autophagy induction, SNX18 recruits Dynamin‐2 to induce budding of ATG9A and ATG16L1 containing membranes from recycling endosomes that traffic to sites of autophagosome formation.     

WIPI β-propellers function as scaffolds for STK11/LKB1-AMPK and AMPK-related kinase signaling in autophagy

The article discusses new findings on the role of the 4 human WIPI proteins at the onset of macroautophagy/autophagy. New insights revealing a circuit scaffold function of WIPI β-propellers that interconnect autophagy signaling control with appropriate autophagosome formation are summarized.     

TMEM41B is a novel regulator of autophagy and lipid mobilization

Autophagy maintains cellular homeostasis by targeting damaged organelles, pathogens, or misfolded protein aggregates for lysosomal degradation. The autophagic process is initiated by the formation of autophagosomes, which can selectively enclose cargo via autophagy cargo receptors. A machinery of well‐characterized autophagy‐related proteins orchestrates the biogenesis of autophagosomes; however, the origin of the required membranes is incompletely understood. Here, we have applied sensitized pooled CRISPR screens and identify the uncharacterized transmembrane protein TMEM41B as a novel regulator of autophagy. In the absence of TMEM41B, autophagosome biogenesis is stalled, LC3 accumulates at WIPI2‐ and DFCP1‐positive isolation membranes, and lysosomal flux of autophagy cargo receptors and intracellular bacteria is impaired. In addition to defective autophagy, TMEM41B knockout cells display significantly enlarged lipid droplets and reduced mobilization and β‐oxidation of fatty acids. Immunostaining and interaction proteomics data suggest that TMEM41B localizes to the endoplasmic reticulum (ER). Taken together, we propose that TMEM41B is a novel ER‐localized regulator of autophagosome biogenesis and lipid mobilization.     

Modulation of intracellular calcium homeostasis blocks autophagosome formation

Cellular stress responses often involve elevation of cytosolic calcium levels, and this has been suggested to stimulate autophagy. Here, however, we demonstrated that agents that alter intracellular calcium ion homeostasis and induce ER stress—the calcium ionophore A23187 and the sarco/endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin (TG)—potently inhibit autophagy. This anti-autophagic effect occurred under both nutrient-rich and amino acid starvation conditions, and was reflected by a strong reduction in autophagic degradation of long-lived proteins. Furthermore, we found that the calcium-modulating agents inhibited autophagosome biogenesis at a step after the acquisition of WIPI1, but prior to the closure of the autophagosome. The latter was evident from the virtually complete inability of A23187- or TG-treated cells to sequester cytosolic lactate dehydrogenase. Moreover, we observed a decrease in both the number and size of starvation-induced EGFP-LC3 puncta as well as reduced numbers of mRFP-LC3 puncta in a tandem fluorescent mRFP-EGFP-LC3 cell line. The anti-autophagic effect of A23187 and TG was independent of ER stress, as chemical or siRNA-mediated inhibition of the unfolded protein response did not alter the ability of the calcium modulators to block autophagy. Finally, and remarkably, we found that the anti-autophagic activity of the calcium modulators did not require sustained or bulk changes in cytosolic calcium levels. In conclusion, we propose that local perturbations in intracellular calcium levels can exert inhibitory effects on autophagy at the stage of autophagosome expansion and closure.     

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.     

Modulation of Local PtdIns3P Levels by the PI Phosphatase MTMR3 Regulates Constitutive Autophagy

Autophagy is a catabolic process that delivers cytoplasmic material to the lysosome for degradation. The mechanisms regulating autophagosome formation and size remain unclear. Here, we show that autophagosome formation was triggered by the overexpression of a dominant‐negative inactive mutant of Myotubularin‐related phosphatase 3 (MTMR3). Mutant MTMR3 partially localized to autophagosomes, and PtdIns3P and two autophagy‐related PtdIns3P‐binding proteins, GFP‐DFCP1 and GFP‐WIPI‐1α (WIPI49/Atg18), accumulated at sites of autophagosome formation. Knock‐down of MTMR3 increased autophagosome formation, and overexpression of wild‐type MTMR3 led to significantly smaller nascent autophagosomes and a net reduction in autophagic activity. These results indicate that autophagy initiation depends on the balance between PI 3‐kinase and PI 3‐phosphatase activity. Local levels of PtdIns3P at the site of autophagosome formation determine autophagy initiation and the size of the autophagosome membrane structure.     

Recombinant protein rVP1 upregulates BECN1-independent autophagy,MAPK1/3 phosphorylation and MMP9 activity via WIPI1/WIPI2 to promote macrophage migration

The monocyte/macrophage is critical for regulating immune and antitumor responses. Recombinant capsid protein VP1 (rVP1) of foot-and-mouth disease virus induces apoptosis and inhibits migration/metastasis of cancer cells. Here, we explored the effects of rVP1 on macrophages. Our results showed that rVP1 increased LC3-related autophagosome formation via WIPI1 and WIPI2 in a BECN1-independent manner. rVP1 treatment increased macrophage migration that was attenuated by knockdown of ATG5, ATG7, WIPI1 or WIPI2 and was abolished when both WIPI1 and WIPI2 were depleted. Treatment of macrophages with rVP1 increased matrix metalloproteinase-9 (MMP9) activity and phosphorylated mitogen-activated protein kinase 1/3 (MAPK1/3), two major mediators of cell migration. Knockdown of WIPI1, WIPI2, ATG5 and ATG7 but not BECN1 attenuated the rVP1-mediated increase in MAPK1/3 phosphorylation and MMP9 activity. These results indicated that rVP1 upregulated autophagy, MAPK1/3 phosphorylation and MMP9 activity to promote macrophage migration, which was dependent on WIPI1, WIPI2, ATG5 and ATG7 but not BECN1.     

Acidocalcisome is required for autophagy in Trypanosoma brucei

Lysosomes play important roles in autophagy, not only in autophagosome degradation, but also in autophagy initiation. In Trypanosoma brucei, an early divergent protozoan parasite, we discovered a previously unappreciated function of the acidocalcisome, a lysosome-related organelle characterized by acidic pH and large content of Ca²⁺ and polyphosphates, in autophagy regulation. Starvation- and chemical-induced autophagy is accompanied with acidocalcisome acidification, and blocking the acidification completely inhibits autophagosome formation. Blocking acidocalcisome biogenesis by depleting the adaptor protein-3 complex, which does not affect lysosome biogenesis or function, also inhibits autophagy. Overall, our results support the role of the acidocalcisome, a conserved organelle from bacteria to human, as a relevant regulator in autophagy.     

Commentary: The ER-Golgi intermediate compartment feeds the phagophore membrane

A long-standing quest in the autophagy field is to define the membrane origin of the autophagosome. We have established a cell-free assay based on LC3 lipidation that recapitulates multiple regulatory hallmarks of early autophagosome biogenesis. Using a systematic membrane fractionation approach, we have identified the ER-Golgi intermediate compartment (ERGIC) as the most efficient membrane substrate for LC3 lipidation. Further studies indicate that the ERGIC plays an essential role to trigger LC3 lipidation and autophagosome biogenesis by recruiting the key early autophagic factor ATG14.     

Evidence for the involvement of lipid rafts localized at the ER-mitochondria associated membranes in autophagosome formation

Mitochondria-associated membranes (MAMs) are subdomains of the endoplasmic reticulum (ER) that interact with mitochondria. This membrane scrambling between ER and mitochondria appears to play a critical role in the earliest steps of autophagy. Recently, lipid microdomains, i.e. lipid rafts, have been identified as further actors of the autophagic process. In the present work, a series of biochemical and molecular analyses has been carried out in human fibroblasts with the specific aim of characterizing lipid rafts in MAMs and to decipher their possible implication in the autophagosome formation. In fact, the presence of lipid microdomains in MAMs has been detected and, in these structures, a molecular interaction of the ganglioside GD3, a paradigmatic “brick” of lipid rafts, with core-initiator proteins of autophagy, such as AMBRA1 and WIPI1, was revealed. This association seems thus to take place in the early phases of autophagic process in which MAMs have been hypothesized to play a key role. The functional activity of GD3 was suggested by the experiments carried out by knocking down ST8SIA1 gene expression, i.e., the synthase that leads to the ganglioside formation. This experimental condition results in fact in the impairment of the ER-mitochondria crosstalk and the subsequent hindering of autophagosome nucleation. We thus hypothesize that MAM raft-like microdomains could be pivotal in the initial organelle scrambling activity that finally leads to the formation of autophagosome.     

The ER-Golgi intermediate compartment feeds the phagophore membrane

A long-standing quest in the autophagy field is to define the membrane origin of the autophagosome. We have established a cell-free assay based on LC3 lipidation that recapitulates multiple regulatory hallmarks of early autophagosome biogenesis. Using a systematic membrane fractionation approach, we have identified the ER-Golgi intermediate compartment (ERGIC) as the most efficient membrane substrate for LC3 lipidation. Further studies indicate that the ERGIC plays an essential role to trigger LC3 lipidation and autophagosome biogenesis by recruiting the key early autophagic factor ATG14.     

Mammalian Atg18 (WIPI2) localizes to omegasome-anchored phagophores and positively regulates LC3 lipidation

Autophagosome formation is a complex process that begins with the nucleation of a pre-autophagosomal structure (PAS) that expands into a phagophore or isolation membrane, the precursor of the autophagosome. A key event in the formation of the phagophore is the production of PtdIns3P by the phosphatidylinsitol kinase Vps34. In yeast the two closely related proteins, Atg18 and Atg21, are the only known effectors of PtdIns3P that act in the autophagy pathway. The recruitment of Atg18 or Atg21 to the PAS is an essential step in the formation of the phagophore. Our bioinformatic analysis of the Atg18 and Atg21 orthologues in all eukaryotes shows that WIPI1 and WIPI2 are both mammalian orthologues of Atg18. We show that WIPI2 is a mammalian effector of PtdIns3P and is ubiquitously expressed in a variety of cell lines. WIPI2 is recruited to early autophagosomal structures along with Atg16L and ULK1 and is required for the formation of LC3-positive autophagosomes. Furthermore, when WIPI2 is depleted, we observe a remarkable accumulation of omegasomes, ER-localized PtdIns3P-containing structures labeled by DFCP1 (double FYVE domain-containing protein 1), which are thought to act as platforms for autophagosome formation. In view of our data we propose a role for WIPI2 in the progression of omegasomes into autophagosomes.     

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.     

Acidocalcisome is required for autophagy in Trypanosoma brucei

Lysosomes play important roles in autophagy, not only in autophagosome degradation, but also in autophagy initiation. In Trypanosoma brucei, an early divergent protozoan parasite, we discovered a previously unappreciated function of the acidocalcisome, a lysosome-related organelle characterized by acidic pH and large content of Ca²⁺ and polyphosphates, in autophagy regulation. Starvation- and chemical-induced autophagy is accompanied with acidocalcisome acidification, and blocking the acidification completely inhibits autophagosome formation. Blocking acidocalcisome biogenesis by depleting the adaptor protein-3 complex, which does not affect lysosome biogenesis or function, also inhibits autophagy. Overall, our results support the role of the acidocalcisome, a conserved organelle from bacteria to human, as a relevant regulator in autophagy.     

Architecture of the ATG2B-WDR45 complex and an aromatic Y/HF motif crucial for complex formation

PtdIns3P signaling is critical for dynamic membrane remodeling during autophagosome formation. Proteins in the Atg18/WIPI family are PtdIns3P-binding effectors which can form complexes with proteins in the Atg2 family, and both families are essential for macroautophagy/autophagy. However, little is known about the biophysical properties and biological functions of the Atg2-Atg18/WIPI complex as a whole. Here, we demonstrate that an ortholog of yeast Atg18, mammalian WDR45/WIPI4 has a stronger binding capacity for mammalian ATG2A or ATG2B than the other 3 WIPIs. We purified the full-length Rattus norvegicus ATG2B and found that it could bind to liposomes independently of PtdIns3P or WDR45. We also purified the ATG2B-WDR45 complex and then performed 3-dimensional reconstruction of the complex by single-particle electron microscopy, which revealed a club-shaped heterodimer with an approximate length of 22 nm. Furthermore, we performed cross-linking mass spectrometry and identified a set of highly cross-linked intermolecular and intramolecular lysine pairs. Finally, based on the cross-linking data followed by bioinformatics and mutagenesis analysis, we determined the conserved aromatic H/YF motif in the C terminus of ATG2A and ATG2B that is crucial for complex formation.     

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.