Coordinated regulation of autophagy by p38α MAPK through mAtg9 and p38IP

Autophagy, a lysosomal degradation pathway, is essential for homeostasis, development, neurological diseases, and cancer. Regulation of autophagy in human disease is not well understood. Atg9 is a transmembrane protein required for autophagy, and it has been proposed that trafficking of Atg9 may regulate autophagy. Mammalian Atg9 traffics between the TGN and endosomes in basal conditions, and newly formed autophagosomes in response to signals inducing autophagy. We identified p38IP as a new mAtg9 interactor and showed that this interaction is regulated by p38α MAPK. p38IP is required for starvation‐induced mAtg9 trafficking and autophagosome formation. Manipulation of p38IP and p38α alters mAtg9 localization, suggesting p38α regulates, through p38IP, the starvation‐induced mAtg9 trafficking to forming autophagosomes. Furthermore, we show that p38α is a negative regulator of both basal autophagy and starvation‐induced autophagy, and suggest that this regulation may be through a direct competition with mAtg9 for binding to p38IP. Our results provide evidence for a link between the MAPK pathway and the control of autophagy through mAtg9 and p38IP.     

TBC1D14 regulates autophagosome formation via Rab11- and ULK1-positive recycling endosomes

Autophagy is a bulk degradation process characterized by the formation of double membrane vesicles called autophagosomes. The exact molecular mechanism of autophagosome formation and the origin of the autophagosomal membrane remain unclear. We screened 38 human Tre-2/Bub2/Cdc16 domain-containing Rab guanosine triphosphatase-activating proteins (GAPs) and identified 11 negative regulators of starvation-induced autophagy. One of these putative RabGAPs, TBC1D14, colocalizes and interacts with the autophagy kinase ULK1. Overexpressed TBC1D14 tubulates ULK1-positive recycling endosomes (REs), impairing their function and inhibiting autophagosome formation. TBC1D14 binds activated Rab11 but is not a GAP for Rab11, and loss of Rab11 prevents TBC1D14-induced tubulation of REs. Furthermore, Rab11 is required for autophagosome formation. ULK1 and Atg9 are found on Rab11- and transferrin (Tfn) receptor (TfnR)-positive recycling endosomes. Amino acid starvation causes TBC1D14 to relocalize from REs to the Golgi complex, whereas TfnR and Tfn localize to forming autophagosomes, which are ULK1 and LC3 positive. Thus, TBC1D14- and Rab11-dependent vesicular transport from REs contributes to and regulates starvation-induced autophagy.     

Plasma membrane helps autophagosomes grow

The membrane origin of autophagosomes has long been a mystery and it may involve multiple sources. In this punctum, we discuss our recent finding that the plasma membrane contributes to the formation of pre-autophagic structures via clathrin-mediated endocytosis. Our study suggests that Atg16L1 interacts with clathrin heavy-chain/AP2 and is also localized on vesicles (positive for clathrin or cholera toxin B) close to the plasma membrane. Live-cell imaging studies revealed that the plasma membrane contributes to Atg16L1-positive structures and that this process and autophagosome formation are impaired by knockdowns of genes regulating clathrin-mediated endocytosis.Key words: autophagy, plasma membrane, endocytosis, phagophore, originWhere do autophagosomes get their membrane from? Although the field of autophagy has grown tremendously since its discovery a few decades ago, the origin(s) of the membranes that contribute to autophagosome biogenesis has been a mystery among autophagy researchers until recently. Mammalian autophagosomes are formed randomly throughout the cytoplasm via a process that involves elongation and fusion of phagophores to form double-membraned autophagosomes. This process involves two ubiquitin-like conjugation systems: conjugation of Atg12 to Atg5 that later forms a macromolecular complex with Atg16L1, and conjugation of phosphatidylethanolamine (PE) with Atg8/LC3-I. The Atg12-Atg5-Atg16L1 complex is targeted to the preautophagic structures, which then acquire Atg8. Atg12-Atg5-Atg16L1 dissociates from completed autophagosomes, while LC3-PE (LC3-II) is associated both with pre-autophagic structures and completed autophagosomes.Some recent studies have explored the contribution of membranes from different organelles supporting the general idea that autophagosomes derive membranes from pre-existing organelles. It is quite possible that there may be multiple membrane sources involved. A few groups have revisited the hypothesis that the endoplasmic reticulum (ER) may be one of the membrane donors. High-resolution 2D electron microscopy (EM) and 3D EM-tomography studies have revealed connections between the ER and the growing autophagosomes. Whether the ER contributes to general autophagy or a specific form of autophagy, reticulophagy, remains to be determined. In addition, it has not been shown if ER membrane is required for autophagosome formation. Recently another study has reported that autophagosomes receive lipids from the outer mitochondrial membrane, but only under starvation conditions, again fueling the multiple-membrane source hypothesis.We have now found evidence for plasma membrane contribution to pre-autophagic structures via endocytosis. Unlike the previous studies, which have focused on LC3- positive structures, we looked specifically at the Atg5-, Atg12- and Atg16-positive pre-autophagic structures, an idea that stemmed from our finding that clathrin heavy-chain immunoprecipitates with Atg16L1. We think that this interaction is partly mediated by the adaptor protein AP2, since knockdown of AP2 decreases the clathrin heavy-chain-Atg16L1 interaction. Immunogold EM also shows clathrin localization on Atg16L1-labeled vesicles close to the plasma membrane.These findings led us to test whether knockdown of proteins involved in clathrin-mediated endocytosis affected Atg16L1-positive pre-autophagic structures. Indeed, knockdown of key proteins in the clathrin-mediated endocytic pathway results in a decrease in the formation of Atg16L1-positive structures both under basal or autophagy-induced conditions (starvation or trehalose treatment). This correlates with a decrease in the number of LC3-labeled autophagosomes. When we directly analyzed vesicle fusion by livecell microscopy, we observed that vesicles endocytosed from the plasma membrane fuse to the Atg16L1-positive vesicles close to the plasma membrane. This was confirmed by immuno-EM when we found cholera toxin B-labeling (used to label plasma membrane that is subsequently internalized by endocytosis) on Atg16L1-vesicles. We noticed that overexpression of an Atg16L1 mutant that does not bind clathrin heavy-chain does not form Atg16L1-vesicular structures in the way we see with wild-type Atg16L1, suggesting that the binding of Atg16L1 to AP2/clathrin is required for the subsequent formation of the Atg16L1 vesicles.When we blocked endocytic vesicle scission (using both genetic and chemical inhibitors) we found that Atg16L1 strongly immunoprecipitates with clathrin-heavy chain probably due to the accumulation of clathrin-Atg16L1 structures at the plasma membrane that failed to pinch off. This was strongly supported by our fluorescence microscopy and immuno-EM studies that showed what we predicted—accumulation of Atg16L1 at the plasma membrane. This suggests that Atg16L1 in a complex with AP2/clathrin is targeted to the plasma membrane and subsequently internalized as Atg16L1-positive structures. Thus, our data strongly suggest that plasma membrane contributes to early autophagic precursors that subsequently mature to form phagophores (Fig. 1).Open in a separate windowFigure 1Plasma membrane contributes to the formation of early autophagic precursors. Previous studies show that delivery of fully formed autophagosomes to lysosomes requires fusion of such autophagosomes with early or late endosomes to form amphisomes, which are Atg16L1-negative, LC3-positive and are also positive for endosomal markers. We show that blocking clathrin-mediated endocytosis inhibits formation of Atg16L1-positive structures that mature to form phagophores and later autophagosomes. These Atg16L1-vesicles are positive for other early autophagosomal markers like Atg5 and Atg12, but are negative for early endosomal markers like EEA1, suggesting that they are high up in the autophagosome biogenesis cascade. Inhibition of dynamin with Dynsasore or the use of a dominant negative K44A mutant blocks scission and results in Atg16L1 accumulation on the plasma membrane, suggesting that endosomal scission is critical for this process.Although previous studies suggest that completely formed autophagosomes need to fuse with early or late endosomes in order for subsequent autophagosomelysosome fusion to occur, they did not look at the formation of pre-autophagic structures. Our study shows that active endocytosis is required both for the formation of autophagosomes, when very early endocytic intermediates immediately pinching off the plasma membrane (not early endosomes) fuse with Atg16L1-positive structures to form phagophores, and also for maturation of autophagosomes when early or late endosomes fuse with Atg16L1-negative but LC3-positive autophagosomes to form amphisomes. Since blocking clathrin-mediated endocytosis does not completely abrogate autophagosome formation, we believe that other endocytic pathways may have a similar role. Depending on the cell type or the physiological conditions, the contributions from the different endocytic pathways may vary accordingly. It will be interesting to know if the endocytic pathway continuously delivers membrane for early steps in autophagy as the preautophagic structures grow and mature to form autophagosomes, deriving membrane from other sources.     

Autophagosome precursor maturation requires homotypic fusion

Autophagy is a catabolic process in which lysosomes degrade intracytoplasmic contents transported in double-membraned autophagosomes. Autophagosomes are formed by the elongation and fusion of phagophores, which can be derived from preautophagosomal structures coming from the plasma membrane and other sites like the endoplasmic reticulum and mitochondria. The mechanisms by which preautophagosomal structures elongate their membranes and mature toward fully formed autophagosomes still remain unknown. Here, we show that the maturation of the early Atg16L1 precursors requires homotypic fusion, which is essential for subsequent autophagosome formation. Atg16L1 precursor homotypic fusion depends on the SNARE protein VAMP7 together with partner SNAREs. Atg16L1 precursor homotypic fusion is a critical event in the early phases of autophagy that couples membrane acquisition and autophagosome biogenesis, as this step regulates the size of the vesicles, which in turn appears to influence their subsequent maturation into LC3-positive autophagosomes.     

Atg9 trafficking in Mammalian cells

The molecular mechanisms of autophagy have been best characterized in the yeast Saccharomyces cerevisiae, where a number of proteins have been identified to be essential for this degradative pathway. ATG (autophagy-related) proteins(1) localize to a unique compartment, the pre-autophagosomal structure (PAS). Isolation membranes are suggested to originate from the PAS, enwrapping cytoplasmic components to form a double membrane autophagosome, which then fuses with the vacuole. Although many Atg proteins have been identified, the source of the PAS membrane in yeast is unknown. Identification of the source of the PAS in yeast has been hindered due to the transient association of Atg proteins with forming autophagosomes.(2) Likewise, in mammalian cells, it is not known if a PAS equivalent exists or if the formation of autophagosomes occurs from numerous membrane sources. The identification of stably associated markers would allow us to address this question further. Thus, characterization of the only transmembrane autophagy protein so far identified, Atg9, may aid in the search for the source of the PAS. Recent data from our lab suggests that mammalian Atg9 (mAtg9) traffics between the Golgi and endosomes, and suggests an involvement of the Golgi complex in the autophagic pathway.(3) Here we address the implications of our model with regard to membrane trafficking events in mammalian cells after starvation.     

Phagophores evolve from recycling endosomes

The membrane origins of autophagosomes have been a key unresolved question in the field. The earliest morphologically recognizable structure in the macroautophagy/autophagy itinerary is the double-membraned cup-shaped phagophore. Newly formed phosphatidylinositol 3-phosphate (PtdIns3P) on the membranes destined to become phagophores recruits WIPI2, which, in turn, binds ATG16L1 to define the sites of autophagosome formation. Here we review our recent study showing that membrane recruitment of WIPI2 requires coincident detection of PtdIns3P and RAB11A, a protein that marks recycling endosomes. We found that multiple core autophagy proteins are more tightly associated with the recycling endosome compartment than with endoplasmic reticulum (ER)-mitochondrial contact sites. Furthermore, biochemical isolation of the recycling endosomes confirmed that they recruit autophagy proteins. Finally, fixed and live-cell imaging data revealed that recycling endosomes engulf autophagic substrates. Indeed, the sequestration of mitochondria after mitophagy stimulation depends on early autophagy regulators. These data suggest that autophagosomes evolve from the RAB11A compartment.     

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.     

Microtubules facilitate autophagosome formation and fusion of autophagosomes with endosomes

Nutrient deprivation of eukaryotic cells provokes a variety of stress responses, including autophagy. Autophagy is carried out by autophagosomes which sequester cytosolic components and organelles for degradation after fusion with protease-containing endosomes. To determine the role of microtubules in autophagy, we used nocodazole and vinblastine to disrupt microtubules and independently measured formation and fusion of autophagsosomes in primary rat hepatocytes. By measuring the translocation of GFP-LC3, an autophagosomal marker, to autophagosomes and the lipidation of GFP-LC3, we quantified the rate and magnitude of autophagosome formation. Starvation increased both the rate of autophagosome formation over the basal level and the total number of autophagosomes per cell. Maximal autophagosome formation required an intact microtubule network. Fusion of autophagosomes with endosomes, assayed by acquisition of protease-inhibitor sensitivity as well as overlap with LysoTracker Red-positive endosomes, required intact microtubules. Live-cell imaging demonstrated that autophagosomes were motile structures, and their movement also required microtubules. Interestingly, vinblastine stimulated autophagosome formation more than twofold before any discernable change in the microtubule network was observed. Stimulation of autophagosome formation by vinblastine was independent of nutrients and mTOR activity but was inhibited by depletion of the Autophagy proteins Atg5 and Atg6, known to be required for autophagy.     

Bif-1 regulates Atg9 trafficking by mediating the fission of Golgi membranes during autophagy

Atg9 is a transmembrane protein essential for autophagy which cycles between the Golgi network, late endosomes and LC3-positive autophagosomes in mammalian cells during starvation through a mechanism that is dependent on ULK1 and requires the activity of the class III phosphatidylinositol-3-kinase (PI3KC3). In this study, we demonstrate that the N-BAR-containing protein, Bif-1, is required for Atg9 trafficking and the fission of Golgi membranes during the induction of autophagy. Upon starvation, Atg9-positive membranes undergo continuous tubulation and fragmentation to produce cytoplasmic punctate structures that are positive for Rab5, Atg16L and LC3. Loss of Bif-1 or inhibition of the PI3KC3 complex II suppresses starvation-induced fission of Golgi membranes and peripheral cytoplasmic redistribution of Atg9. Moreover, Bif-1 mutants, which lack the functional regions of the N-BAR domain that are responsible for membrane binding and/or bending activity, fail to restore the fission of Golgi membranes as well as the formation of Atg9 foci and autophagosomes in Bif-1-deficient cells starved of nutrients. Taken together, these findings suggest that Bif-1 acts as a critical regulator of Atg9 puncta formation presumably by mediating Golgi fission for autophagosome biogenesis during starvation.     

Unraveling the role of myosin in forming autophagosomes

Macroautophagy (hereafter autophagy) is a membrane-mediated catabolic process that occurs in response to a variety of intra- and extra-cellular stresses. It is characterized by the formation of specialized double-membrane vesicles, autophagosomes, which engulf organelles and long-lived proteins, and in turn fuse with lysosomes for degradation and recycling. How autophagosomes emerge is still unclear. The Atg1 kinase plays a crucial role in the induction of autophagosome formation. While several Atg (autophagy-related) proteins have been associated with, and have been found to regulate, Atg1 kinase activity, the downstream targets of Atg1 that trigger autophagy remain unknown. Our recent studies have identified a myosin light chain kinase (MLCK)-like kinase as the Atg1 kinase effector that induces the activation of myosin II, and have found it to be required for autophagosome formation during nutrient deprivation. We further demonstrated that Atg1-mediated myosin II activation is crucial for the movement of the Atg9 transmembrane protein between the Golgi and the forming autophagosome, which provides a membrane source for the formation of autophagosomes during starvation.     

Unraveling the role of myosin in forming autophagosomes

Macroautophagy (hereafter autophagy) is a membrane-mediated catabolic process that occurs in response to a variety of intra- and extra-cellular stresses. It is characterized by the formation of specialized double-membrane vesicles, autophagosomes, which engulf organelles and long-lived proteins, and in turn fuse with lysosomes for degradation and recycling. How autophagosomes emerge is still unclear. The Atg1 kinase plays a crucial role in the induction of autophagosome formation. While several Atg (autophagy-related) proteins have been associated with, and have been found to regulate, Atg1 kinase activity, the downstream targets of Atg1 that trigger autophagy remain unknown. Our recent studies have identified a myosin light chain kinase (MLCK)-like kinase as the Atg1 kinase effector that induces the activation of myosin II, and have found it to be required for autophagosome formation during nutrient deprivation. We further demonstrated that Atg1-mediated myosin II activation is crucial for the movement of the Atg9 transmembrane protein between the Golgi and the forming autophagosome, which provides a membrane source for the formation of autophagosomes during starvation.     

Atg9 vesicles are an important membrane source during early steps of autophagosome formation

During the process of autophagy, cytoplasmic materials are sequestered by double-membrane structures, the autophagosomes, and then transported to a lytic compartment to be degraded. One of the most fundamental questions about autophagy involves the origin of the autophagosomal membranes. In this study, we focus on the intracellular dynamics of Atg9, a multispanning membrane protein essential for autophagosome formation in yeast. We found that the vast majority of Atg9 existed on cytoplasmic mobile vesicles (designated Atg9 vesicles) that were derived from the Golgi apparatus in a process involving Atg23 and Atg27. We also found that only a few Atg9 vesicles were required for a single round of autophagosome formation. During starvation, several Atg9 vesicles assembled individually into the preautophagosomal structure, and eventually, they are incorporated into the autophagosomal outer membrane. Our findings provide conclusive linkage between the cytoplasmic Atg9 vesicles and autophagosomal membranes and offer new insight into the requirement for Atg9 vesicles at the early step of autophagosome formation.     

Atg9 Trafficking in Mammalian Cells

The molecular mechanisms of autophagy have been best characterized in the yeast Saccharomyces cerevisiae, where a number of proteins have been identified to be essential for this degradative pathway. ATG (autophagy-related) proteins localize to a unique compartment, the pre-autophagosomal structure (PAS). Isolation membranes are suggested to originate from the PAS, enwrapping cytoplasmic components to form a double membrane autophagosome, which then fuses with the vacuole. Although many Atg proteins have been identified, the source of the PAS membrane in yeast is unknown. Identification of the source of the PAS in yeast has been hindered due to the transient association of Atg proteins with forming autophagosomes. Likewise, in mammalian cells, it is not known if a PAS equivalent exists or if the formation of autophagosomes occurs from numerous membrane sources. The identification of stably associated markers would allow us to address this question further. Thus, characterization of the only transmembrane autophagy protein so far identified, Atg9, may aid in the search for the source of the PAS. Recent data from our lab suggests that mammalian Atg9 (mAtg9) traffics between the Golgi and endosomes, and suggests an involvement of the Golgi complex in the autophagic pathway. Here we address the implications of our model with regard to membrane trafficking events in mammalian cells after starvation.

Addendum to:

Starvation and ULK1-Dependent Cycling of Mammalian Atg9 Between the TGN and Andosomes

A.R.J. Young, E.Y.W. Chan, X.W. Hu, R. Köchl, S.G. Crawshaw, S. High, D.W. Hailey, J. Lippincott-Schwartz and S.A. Tooze

J Cell Sci 2006; 119:3888-900     

Atg27 is required for autophagy-dependent cycling of Atg9

Autophagy is a catabolic pathway for the degradation of cytosolic proteins or organelles and is conserved among all eukaryotic cells. The hallmark of autophagy is the formation of double-membrane cytosolic vesicles, termed autophagosomes, which sequester cytoplasm; however, the mechanism of vesicle formation and the membrane source remain unclear. In the yeast Saccharomyces cerevisiae, selective autophagy mediates the delivery of specific cargos to the vacuole, the analog of the mammalian lysosome. The transmembrane protein Atg9 cycles between the mitochondria and the pre-autophagosomal structure, which is the site of autophagosome biogenesis. Atg9 is thought to mediate the delivery of membrane to the forming autophagosome. Here, we characterize a second transmembrane protein Atg27 that is required for specific autophagy in yeast. Atg27 is required for Atg9 cycling and shuttles between the pre-autophagosomal structure, mitochondria, and the Golgi complex. These data support a hypothesis that multiple membrane sources supply the lipids needed for autophagosome formation.     

HS1BP3 inhibits autophagy by regulation of PLD1

Macroautophagy/autophagy is a membrane trafficking and intracellular degradation process involving the formation of double-membrane autophagosomes and their ultimate fusion with lysosomes. Much is yet to be learned about the regulation of this process, especially at the level of the membranes and lipids involved. We have recently found that the PX domain protein HS1BP3 (HCLS1 binding protein 3) is a negative regulator of autophagosome formation. HS1BP3 depletion increases the formation of LC3-positive autophagosomes both in human cells and zebrafish. HS1BP3 localizes to ATG16L1- and ATG9-positive autophagosome precursors deriving from recycling endosomes, which appear to fuse with LC3-positive phagophores. The HS1BP3 PX domain interacts with phosphatidic acid (PA) and 3’-phosphorylated phosphoinositides. When HS1BP3 is depleted, the total cellular PA content is upregulated stemming from increased activity of the PA-producing enzyme PLD (phospholipase D) and increased localization of PLD1 to ATG16L1-positive membranes. We propose that HS1BP3 negatively regulates autophagy by decreasing the PA content of the ATG16L1-positive autophagosome precursor membranes through inhibition of PLD1 activity and localization.     

Atg5 regulates late endosome and lysosome biogenesis

Autophagy is an evolutionarily conserved lysosome-based degradation process.Atg5 plays a very important role in autophagosome formation.Here we show that Atg5 is required for biogenesis of late endosomes and lysosomes in an autophagy-independent manner.In Atg5 cells,but not in other essential autophagy genes defecting cells,recycling and retrieval of late endosomal components from hybrid organelles are impaired,causing persistent hybrid organelles and defective formation of late endosomes and lysosomes.Defective retrieval of late endosomal components from hybrid organelles resulting from impaired recruitment of a component of V1-ATPase to acidic organelles blocks the pH-dependent retrieval of late endosomal components from hybrid organelles.Lowering the intracellular pH restores late endosome/lysosome biogenesis in Atg5 cells.Our data demonstrate an unexpected role of Atg5 and shed new light on late endosome and lysosome biogenesis.     

The role of membrane proteins in mammalian autophagy

Autophagy is an evolutionarily conserved degradative process that is initiated by autophagosomes, double-membrane structures that sequester cytoplasmic material and fuse with endosomes and lysosomes to become autolysosomes. Recent progress in the identification of proteins required for autophagy has led to a substantial understanding of the process involved in making an autophagosome. Mammalian Atg9, a multi-spanning transmembrane protein, is one of the possible keys to understanding how autophagosomes are formed. Current and future advances in understanding the function of mammalian Atg9 will provide a basis for further progress. In addition, the identification of so far uncharacterized transmembrane proteins which are involved in autophagy will also help to address the important questions of where, how, and why autophagosomes form.     

Membrane Delivery to the Yeast Autophagosome from the Golgi�CEndosomal System

While many of the proteins required for autophagy have been identified, the source of the membrane of the autophagosome is still unresolved with the endoplasmic reticulum (ER), endosomes, and mitochondria all having been evoked. The integral membrane protein Atg9 is delivered to the autophagosome during starvation and in the related cytoplasm-to-vacuole (Cvt) pathway that occurs constitutively in yeast. We have examined the requirements for delivery of Atg9-containing membrane to the yeast autophagosome. Atg9 does not appear to originate from mitochondria, and Atg9 cannot reach the forming autophagosome directly from the ER or early Golgi. Components of traffic between Golgi and endosomes are known to be required for the Cvt pathway but do not appear required for autophagy in starved cells. However, we find that pairwise combinations of mutations in Golgi-endosomal traffic components apparently only required for the Cvt pathway can cause profound defects in Atg9 delivery and autophagy in starved cells. Thus it appears that membrane that contains Atg9 is delivered to the autophagosome from the Golgi-endosomal system rather than from the ER or mitochondria. This is underestimated by examination of single mutants, providing a possible explanation for discrepancies between yeast and mammalian studies on Atg9 localization and autophagosome formation.     

New insights into the function of Atg9

Autophagy is a lysosomal degradation pathway that is essential for cellular homeostasis. Identification of more than 30 autophagy related proteins including a multi-spanning membrane protein, Atg9, has increased our understanding of the molecular mechanisms involved in autophagy. Atg9 is required for autophagy in several eukaryotic organisms although its function is unknown. Recently, we identified a novel interacting partner of mAtg9, p38 MAPK interacting protein, p38IP. We summarise recent data on the role of Atg9 trafficking in yeast and mammalian autophagy and discuss the role of p38IP and p38 MAPK in regulation of mAtg9 trafficking and autophagy.     

Atg16L2, a novel isoform of mammalian Atg16L that is not essential for canonical autophagy despite forming an Atg12–5-16L2 complex

A large protein complex consisting of Atg5, Atg12 and Atg16L1 has recently been shown to be essential for the elongation of isolation membranes (also called phagophores) during mammalian autophagy. However, the precise function and regulation of the Atg12–5-16L1 complex has largely remained unknown. In this study we identified a novel isoform of mammalian Atg16L, termed Atg16L2, that consists of the same domain structures as Atg16L1. Biochemical analysis revealed that Atg16L2 interacts with Atg5 and self-oligomerizes to form an ~800-kDa complex, the same as Atg16L1 does. A subcellular distribution analysis indicated that, despite forming the Atg12–5-16L2 complex, Atg16L2 is not recruited to phagophores and is mostly present in the cytosol. The results also showed that Atg16L2 is unable to compensate for the function of Atg16L1 in autophagosome formation, and knockdown of endogenous Atg16L2 did not affect autophagosome formation, indicating that Atg16L2 does not possess the ability to mediate canonical autophagy. Moreover, a chimeric analysis between Atg16L1 and Atg16L2 revealed that their difference in function in regard to autophagy is entirely attributable to the difference between their middle regions that contain a coiled-coil domain. Based on the above findings, we propose that formation of the Atg12–5-16L complex is necessary but insufficient to mediate mammalian autophagy and that an additional function of the middle region (especially around amino acid residues 229–242) of Atg16L1 (e.g., interaction with an unidentified binding partner on phagophores) is required for autophagosome formation.