Evolutionarily conserved role and physiological relevance of a STX17/Syx17 (syntaxin 17)-containing SNARE complex in autophagosome fusion with endosomes and lysosomes

Phagophores engulf cytoplasmic material and give rise to autophagosomes, double-membrane vesicles mediating cargo transport to lysosomes for degradation. The regulation of autophagosome fusion with endosomes and lysosomes during autophagy has remained poorly characterized. Two recent papers conclude that STX17/syntaxin 17 (Syx17 in Drosophila) has an evolutionarily conserved role in autophagosome fusion with endosomes and lysosomes, acting in one SNARE complex with SNAP29 (ubisnap in Drosophila) and the endosomal/lysosomal VAMP8 (CG1599/Vamp7 in Drosophila). Surprisingly, a third report suggests that STX17 might also contribute to proper phagophore assembly. Although several experiments presented in the two human cell culture studies yielded controversial results, the essential role of STX17 in autophagic flux is now firmly established, both in cultured cells and in an animal model. Based on these data, we propose that genetic inhibition of STX17/Syx17 may be a more specific tool in autophagic flux experiments than currently used drug treatments, which impair all lysosomal degradation routes and also inactivate MTOR (mechanistic target of rapamycin), a major negative regulator of autophagy. Finally, the neuronal dysfunction and locomotion defects observed in Syx17 mutant animals point to the possible contribution of defective autophagosome clearance to various human diseases.     

Interaction of the HOPS complex with Syntaxin 17 mediates autophagosome clearance in Drosophila

Homotypic fusion and vacuole protein sorting (HOPS) is a tethering complex required for trafficking to the vacuole/lysosome in yeast. Specific interaction of HOPS with certain SNARE (soluble NSF attachment protein receptor) proteins ensures the fusion of appropriate vesicles. HOPS function is less well characterized in metazoans. We show that all six HOPS subunits (Vps11 [vacuolar protein sorting 11]/CG32350, Vps18/Dor, Vps16A, Vps33A/Car, Vps39/CG7146, and Vps41/Lt) are required for fusion of autophagosomes with lysosomes in Drosophila. Loss of these genes results in large-scale accumulation of autophagosomes and blocks autophagic degradation under basal, starvation-induced, and developmental conditions. We find that HOPS colocalizes and interacts with Syntaxin 17 (Syx17), the recently identified autophagosomal SNARE required for fusion in Drosophila and mammals, suggesting their association is critical during tethering and fusion of autophagosomes with lysosomes. HOPS, but not Syx17, is also required for endocytic down-regulation of Notch and Boss in developing eyes and for proper trafficking to lysosomes and eye pigment granules. We also show that the formation of autophagosomes and their fusion with lysosomes is largely unaffected in null mutants of Vps38/UVRAG (UV radiation resistance associated), a suggested binding partner of HOPS in mammals, while endocytic breakdown and lysosome biogenesis is perturbed. Our results establish the role of HOPS and its likely mechanism of action during autophagy in metazoans.     

Atg2: A novel phospholipid transfer protein that mediates de novo autophagosome biogenesis

The degradation of cytoplasmic components via autophagy is crucial for intracellular homeostasis. In the process of autophagy, a newly synthesized isolation membrane (IM) is developed to sequester degradation targets and eventually the IM seals, forming an autophagosome. One of the most poorly understood autophagy‐related proteins is Atg2, which is known to localize to a contact site between the edge of the expanding IM and the exit site of the endoplasmic reticulum (ERES). Recent advances in structural and biochemical analyses have been applied to Atg2 and have revealed it to be a novel multifunctional protein that tethers membranes and transfers phospholipids between them. Considering that Atg2 is essential for the expansion of the IM that requires phospholipids as building blocks, it is suggested that Atg2 transfers phospholipids from the ERES to the IM during the process of autophagosome formation, suggesting that lipid transfer proteins can mediate de novo organelle biogenesis.     

The C. elegans ATG101 homolog EPG-9 directly interacts with EPG-1/Atg13 and is essential for autophagy

Autophagy is an evolutionarily conserved catabolic process that involves the engulfment of cytoplasmic contents in a closed double-membrane structure, called the autophagosome, and their subsequent delivery to the vacuole/lysosomes for degradation. Genetic screens in Saccharomyces cerevisiae have identified more than 30 autophagy-related (Atg) genes that are essential for autophagosome formation. Here we isolated a novel autophagy gene, epg-9, whose loss of function causes defective autophagic degradation of a variety of protein aggregates during C. elegans embryogenesis. Mutations in epg-9 also reduce survival of animals under food depletion conditions. epg-9 mutants exhibit autophagy phenotypes characteristic of those associated with loss of function of unc-51/Atg1 and epg-1/Atg13. epg-9 encodes a protein with significant homology to mammalian ATG101. EPG-9 directly interacts with EPG-1/Atg13. Our study indicates that EPG-9 forms a complex with EPG-1 in the aggrephagy pathway in C. elegans.     

ER-mitochondria signaling regulates autophagy

The endoplasmic reticulum (ER) and mitochondria form tight functional contacts that regulate several key cellular processes. The formation of these contacts involves “tethering proteins” that function to recruit regions of ER to mitochondria. The integral ER protein VAPB (VAMP associated protein B and C) binds to the outer mitochondrial membrane protein, RMDN3/PTPIP51 (regulator of microtubule dynamics 3) to form one such set of tethers. Recently, we showed that the VAPB-RMDN3 tethers regulate macroautophagy/autophagy. Small interfering RNA (siRNA) knockdown of VAPB or RMDN3 to loosen ER-mitochondria contacts stimulates autophagosome formation, whereas overexpression of VAPB or RMDN3 to tighten contacts inhibit their formation. Artificial tethering of ER and mitochondria via expression of a synthetic linker protein also reduces autophagy and this artificial tether rescues the effects of VAPB- or RMDN3-targeted siRNA loss on autophagosome formation. Finally, our studies revealed that the modulatory effects of ER-mitochondria contacts on autophagy involve their role in mediating ITPR (inositol 1,4,5-trisphosphate receptor) delivery of Ca²⁺ from ER stores to mitochondria.     

Mechanism and functions of membrane binding by the Atg5–Atg12/Atg16 complex during autophagosome formation

Autophagy is a conserved process for the bulk degradation of cytoplasmic material. Triggering of autophagy results in the formation of double membrane‐bound vesicles termed autophagosomes. The conserved Atg5–Atg12/Atg16 complex is essential for autophagosome formation. Here, we show that the yeast Atg5–Atg12/Atg16 complex directly binds membranes. Membrane binding is mediated by Atg5, inhibited by Atg12 and activated by Atg16. In a fully reconstituted system using giant unilamellar vesicles and recombinant proteins, we reveal that all components of the complex are required for efficient promotion of Atg8 conjugation to phosphatidylethanolamine and are able to assign precise functions to all of its components during this process. In addition, we report that in vitro the Atg5–Atg12/Atg16 complex is able to tether membranes independently of Atg8. Furthermore, we show that membrane binding by Atg5 is downstream of its recruitment to the pre‐autophagosomal structure but is essential for autophagy and cytoplasm‐to‐vacuole transport at a stage preceding Atg8 conjugation and vesicle closure. Our findings provide important insights into the mechanism of action of the Atg5–Atg12/Atg16 complex during autophagosome formation.     

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.     

Sphingolipid synthesis is involved in autophagy in Saccharomyces cerevisiae

In eukaryotes, autophagy is a conserved protein degradation system that degrades cytoplasmic components by encompassing them with double-membrane structures, called autophagosomes, and delivering them to the lytic compartments of vacuoles/lysosomes. Certain Atg proteins are known to be involved in autophagy, yet the identity and function of lipid molecules involved remain largely unknown. We investigated the involvement of sphingolipids in autophagy using Saccharomyces cerevisiae. Inhibiting synthesis of the simplest complex sphingolipid, inositol phosphorylceramide (IPC), resulted in reduced autophagic activities. Similar results were obtained using myriocin, an inhibitor of the first step in sphingolipid synthesis. Our results indicate that sphingolipids, especially IPC, are required for autophagy. Inhibition of sphingolipid synthesis had no effect on formation of Atg12-Atg5 or Atg8-phosphatidylethanolamine conjugates, on maturation of vacuolar proteases, or on formation of the pre-autophagosomal structure (PAS). These results suggest that sphingolipids are not involved in the cellular signaling that leads to formation of the PAS, but may be involved in the process of autophagosome formation.     

Autophagosome Formation Depends on the Small GTPase Rab1 and Functional ER Exit Sites

Autophagy is an important cellular degradation pathway present in all eukaryotic cells. Via this pathway, portions of the cytoplasm and/or organelles are sequestered in double‐membrane structures called autophagosomes. In spite of the significant advance achieved in autophagy, the long‐standing question about the source of the autophagic membrane remains unsolved. We have investigated the role of the secretory pathway in autophagosome biogenesis. Sar1 and Rab1b are monomeric GTPases that control traffic from the endoplasmic reticulum (ER) to the Golgi. We present evidence indicating that the activity of both proteins is required for autophagosome formation. Overexpression of dominant‐negative mutants and the use of siRNAs impaired autophagosome generation as determined by LC3 puncta formation and light chain 3 (LC3)‐II processing. In addition, our results indicate that the autophagic and secretory pathways intersect at a level preceding the brefeldin A blockage, suggesting that the transport from the cis/medial Golgi is not necessary for autophagosome biogenesis. Our present results highlight the role of transport from the ER in the initial events of the autophagic vacuole development.     

β‐Guanidinopropionic acid extends the lifespan of Drosophila melanogaster via an AMP‐activated protein kinase‐dependent increase in autophagy

Previous studies have demonstrated that AMP‐activated protein kinase (AMPK) controls autophagy through the mammalian target of rapamycin (mTOR) and Unc‐51 like kinase 1 (ULK1/Atg1) signaling, which augments the quality of cellular housekeeping, and that β‐guanidinopropionic acid (β‐GPA), a creatine analog, leads to a chronic activation of AMPK. However, the relationship between β‐GPA and aging remains elusive. In this study, we hypothesized that feeding β‐GPA to adult Drosophila produces the lifespan extension via activation of AMPK‐dependent autophagy. It was found that dietary administration of β‐GPA at a concentration higher than 900 mm induced a significant extension of the lifespan of Drosophila melanogaster in repeated experiments. Furthermore, we found that Atg8 protein, the homolog of microtubule‐associated protein 1A/1B‐light chain 3 (LC3) and a biomarker of autophagy in Drosophila, was significantly upregulated by β‐GPA treatment, indicating that autophagic activity plays a role in the effect of β‐GPA. On the other hand, when the expression of Atg5 protein, an essential protein for autophagy, was reduced by RNA interference (RNAi), the effect of β‐GPA on lifespan extension was abolished. Moreover, we found that AMPK was also involved in this process. β‐GPA treatment significantly elevated the expression of phospho‐T172‐AMPK levels, while inhibition of AMPK by either AMPK‐RNAi or compound C significantly attenuated the expression of autophagy‐related proteins and lifespan extension in Drosophila. Taken together, our results suggest that β‐GPA can induce an extension of the lifespan of Drosophila via AMPK‐Atg1‐autophagy signaling pathway.     

Getting ready for building: signaling and autophagosome biogenesis

Autophagy is the main cellular catabolic process responsible for degrading organelles and large protein aggregates. It is initiated by the formation of a unique membrane structure, the phagophore, which engulfs part of the cytoplasm and forms a double‐membrane vesicle termed the autophagosome. Fusion of the outer autophagosomal membrane with the lysosome and degradation of the inner membrane contents complete the process. The extent of autophagy must be tightly regulated to avoid destruction of proteins and organelles essential for cell survival. Autophagic activity is thus regulated by external and internal cues, which initiate the formation of well‐defined autophagy‐related protein complexes that mediate autophagosome formation and selective cargo recruitment into these organelles. Autophagosome formation and the signaling pathways that regulate it have recently attracted substantial attention. In this review, we analyze the different signaling pathways that regulate autophagy and discuss recent progress in our understanding of autophagosome biogenesis.     

Membrane remodeling by the PX-BAR protein SNX18 promotes autophagosome formation

The membrane remodeling events required for autophagosome biogenesis are still poorly understood. Because PX domain proteins mediate membrane remodeling and trafficking, we conducted an imaging-based siRNA screen for autophagosome formation targeting human PX proteins. The PX-BAR protein SNX18 was identified as a positive regulator of autophagosome formation, and its Drosophila melanogaster homologue SH3PX1 was found to be required for efficient autophagosome formation in the larval fat body. We show that SNX18 is required for recruitment of Atg16L1-positive recycling endosomes to a perinuclear area and for delivery of Atg16L1- and LC3-positive membranes to autophagosome precursors. We identify a direct interaction of SNX18 with LC3 and show that the pro-autophagic activity of SNX18 depends on its membrane binding and tubulation capacity. We also show that the function of SNX18 in membrane tubulation and autophagy is negatively regulated by phosphorylation of S233. We conclude that SNX18 promotes autophagosome formation by virtue of its ability to remodel membranes and provide membrane to forming autophagosomes.     

STK38 at the crossroad between autophagy and apoptosis

We describe the STK38 protein kinase as a conserved regulator of autophagy. We discovered STK38 as a novel binding partner of Beclin1, a key regulator of autophagy. By combining molecular, cell biological and genetic approaches, we show that STK38 promotes autophagosome formation in human cells and in Drosophila. Furthermore, we also provide evidence demonstrating that STK38 with the small GTPase RalB, assist the co-ordination between autophagic and apoptotic events upon autophagy induction, hence proposing a role for STK38 in determining cellular fate in response to autophagic conditions.     

Autophagy favors <Emphasis Type="Italic">Brucella melitensis</Emphasis> survival in infected macrophages

This study investigated the role of autophagy in the survival of the invasive bacterium Brucella melitensis strain 16M in murine macrophages. Here, Brucella melitensis 16M was found to trigger autophagosome formation, enhance autophagy flux and increase the expression level of the autophagy marker protein LC3-II. When autophagy was pharmacologically inhibited by 3-methyladenine (3-MA), Brucella replication efficiency was significantly decreased (p < 0.05). These results suggest that autophagy favors Brucella melitensis 16M survival in murine macrophages.     

UNC51‐like kinase 1, autophagic regulator and cancer therapeutic target

Autophagy, the cell process of self‐digestion, plays a pivotal role in maintaining energy homoeostasis and protein synthesis. When required, it causes degradation of long‐lived proteins and damaged organelles, indicating that it may play a dual role in cancer, by both protecting against and promoting cell death. The autophagy‐related gene (Atg) family, with more than 35 members, regulates multiple stages of the process. Serine/threonine protein kinase Atg1 in yeast, for example, can interact with other ATG gene products, functioning in autophagosome formation. One mammalian homologue of Atg1, UNC‐51‐like kinase 1 (ULK1) and its related complex ULK1–mAtg13–FIP200 can mediate autophagy under nutrient‐deprived conditions, by protein–protein interactions and post‐translational modifications. Although specific mechanisms of how ULK1 and its complex transduces upstream signals to the downstream central autophagy pathways is not fully understood, past studies have indicated that ULK1 can both suppress and promote tumour growth under different conditions. Here, we summarize some properties of ULK1 which can regulate autophagy in cancer, which may shed new light on future cancer therapy strategies, utilizing ULK1 as a potential new target.     

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.     

Disruption of Autophagy Results in Constitutive Oxidative Stress in Arabidopsis

Plant cells frequently encounter oxidative stress, leading to oxidative damage and inactivation of proteins. We have recently demonstrated that oxidative stress induces autophagy in Arabidopsis seedlings in an AtATG18a-dependent manner and that RNAi-AtATG18a transgenic lines, which are defective in autophagosome formation, are hypersensitive to reactive oxygen species. Analysis of protein oxidation indicated that oxidized proteins are degraded in the vacuole after uptake by autophagy, and this degradation is impaired in RNAi-AtATG18a lines. Our results also suggest that in the absence of a functional autophagy pathway, plants are under increased oxidative stress, even under normal growth conditions.

Addendum to:

Degradation of Oxidized Proteins by Autophagy during Oxidative Stress in Arabidopsis

Y. Xiong, A.L. Contento, N.Q. Phan and D.C. Bassham

Plant Physiol 2007; 143:291-9     

Hidden Behind Autophagy: The Unconventional Roles of ATG Proteins

Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved intracellular catabolic transport route that generally allows the lysosomal degradation of cytoplasmic components, including bulk cytosol, protein aggregates, damaged or superfluous organelles and invading microbes. Target structures are sequestered by double‐membrane vesicles called autophagosomes, which are formed through the concerted action of the autophagy (ATG)‐related proteins. Until recently it was assumed that ATG proteins were exclusively involved in autophagy. A growing number of studies, however, have attributed functions to some of them that are distinct from their classical role in autophagosome biogenesis. Autophagy‐independent roles of the ATG proteins include the maintenance of cellular homeostasis and resistance to pathogens. For example, they assist and enhance the turnover of dead cells and microbes upon their phagocytic engulfment, and inhibit murine norovirus replication. Moreover, bone resorption by osteoclasts, innate immune regulation triggered by cytoplasmic DNA and the ER‐associated degradation regulation all have in common the requirement of a subset of ATG proteins. Microorganisms such as coronaviruses, Chlamydia trachomatis or Brucella abortus have even evolved ways to manipulate autophagy‐independent functions of ATG proteins in order to ensure the completion of their intracellular life cycle. Taken together these novel mechanisms add to the repertoire of functions and extend the number of cellular processes involving the ATG proteins.