A Selective Autophagy Pathway for Phase-Separated Endocytic Protein Deposits.

Autophagy eliminates cytoplasmic content selected by autophagy receptors, which link cargo to the membrane-bound autophagosomal ubiquitin-like protein Atg8/LC3. Here, we report a selective autophagy pathway for protein condensates formed by endocytic proteins in yeast. In this pathway, the endocytic protein Ede1 functions as a selective autophagy receptor. Distinct domains within Ede1 bind Atg8 and mediate phase separation into condensates. Both properties are necessary for an Ede1-dependent autophagy pathway for endocytic proteins, which differs from regular endocytosis and does not involve other known selective autophagy receptors but requires the core autophagy machinery. Cryo-electron tomography of Ede1-containing condensates, at the plasma membrane and in autophagic bodies, shows a phase-separated compartment at the beginning and end of the Ede1-mediated selective autophagy route. Our data suggest a model for autophagic degradation of macromolecular protein complexes by the action of intrinsic autophagy receptors.

Decoding the function of Atg13 phosphorylation reveals a role of Atg11 in bulk autophagy initiation.

Autophagy is initiated by the assembly of multiple autophagy-related proteins that form the phagophore assembly site where autophagosomes are formed. Atg13 is essential early in this process, and a hub of extensive phosphorylation. How these multiple phosphorylations contribute to autophagy initiation, however, is not well understood. Here we comprehensively analyze the role of phosphorylation events on Atg13 during nutrient-rich conditions and nitrogen starvation. We identify and functionally characterize 48 in vivo phosphorylation sites on Atg13. By generating reciprocal mutants, which mimic the dephosphorylated active and phosphorylated inactive state of Atg13, we observe that disrupting the dynamic regulation of Atg13 leads to insufficient or excessive autophagy, which are both detrimental to cell survival. We furthermore demonstrate an involvement of Atg11 in bulk autophagy even during nitrogen starvation, where it contributes together with Atg1 to the multivalency that drives phase separation of the phagophore assembly site. These findings reveal the importance of post-translational regulation on Atg13 early during autophagy initiation, which provides additional layers of regulation to control bulk autophagy activity and integrate cellular signals.

Two Independent Pathways within Selective Autophagy Converge to Activate Atg1 Kinase at the Vacuole.

Autophagy is a potent cellular degradation pathway, and its activation needs to be tightly controlled. Cargo receptors mediate selectivity during autophagy by bringing cargo to the scaffold protein Atg11 and, in turn, to the autophagic machinery, including the central autophagy kinase Atg1. Here we show how selective autophagy is tightly regulated in space and time to prevent aberrant Atg1 kinase activation and autophagy induction. We established an induced bypass approach (iPass) that combines genetic deletion with chemically induced dimerization to evaluate the roles of Atg13 and cargo receptors in Atg1 kinase activation and selective autophagy progression. We show that Atg1 activation does not require cargo receptors, cargo-bound Atg11, or Atg13 per se. Rather, these proteins function in two independent pathways that converge to activate Atg1 at the vacuole. This pathway architecture underlies the spatiotemporal control of Atg1 kinase activity, thereby preventing inappropriate autophagosome formation.

The ESCRT-III complex contributes to macromitophagy in yeast.

Mitochondria play important roles in energy generation and homeostasis maintenance in eukaryotic cells. The damaged or superfluous mitochondria can be nonselectively or selectively removed through the autophagy/lysosome pathway, which was referred as mitophagy. According to the molecular machinery for degrading mitochondria, the selectively removed mitochondria can occur through macromitophagy or micromitophagy. In this study, we show that the endosomal sorting complex required for transport III (ESCRT-III) in budding yeast regulates macromitophagy induced by nitrogen starvation, but not by the post-logarithmic phase growth in lactate medium by monitoring a mitochondrial marker, Om45. Firstly, loss of ESCRT-III subunit Snf7 or Vps4-Vta1 complex subunit Vps4, two representative subunits of the ESCRT complex, suppresses the delivery and degradation of Om45-GFP to vacuoles. Secondly, we show that the mitochondrial marker Om45 and mitophagy receptor Atg32 accumulate on autophagosomes marked with Atg8 (mitophagosomes, MPs) in ESCRT mutants. Moreover, the protease-protection assay indicates that Snf7 and Vps4 are involved in MP closure. Finally, Snf7 interacts with Atg11, which was detected by two ways, glutathione-S-transferase (GST) pulldown and bimolecular fluorescence complementation (BiFC) assay, and this BiFC interaction happens on mitochondrial reticulum. Therefore, we proposed that the ESCRT-III machinery mediates nitrogen starvation-induced macromitophagy by the interaction between Snf7 and Atg11 so that Snf7 is recruited to Atg32-marked MPs by the known Atg11-Atg32 interaction to seal them. These results reveal that the ESCRT-III complex plays a new role in yeast on macromitophagy.

Function of Atg11 in non-selective autophagy and selective autophagy of Candida albicans.

Candida albicans is an important opportunistic pathogenic fungus in the human body. It is a common microbe inhabiting on the mucosa surfaces of healthy individuals, but may cause infections when the host immune system is weak. Autophagy is a "self-eating" process in eukaryotes, which can recover and utilize damaged organelles and misfolded proteins. Here we investigated the role of the autophagy-related protein Atg11 in C. albicans. Deletion of ATG11 led to the defect in growth under the nitrogen starvation condition. Western blotting and GFP localization further revealed that the transport and degradation of Atg8 was blocked in the atg11Δ/Δ mutant under both the nitrogen starvation and hypha-inducing conditions. Moreover, degradation of both Lap41 (the indicator of the cytoplasm-to-vacuole pathway) and Csp37 (the indicator of mitophagy) was also thoroughly suppressed in this mutant under nitrogen starvation. These results indicated that Atg11 plays an essential role in both non-selective and selective autophagy in C. albicans.

Hrr25 kinase promotes selective autophagy by phosphorylating the cargo receptor Atg19.

Autophagy is the major pathway for the delivery of cytoplasmic material to the vacuole or lysosome. Selective autophagy is mediated by cargo receptors, which link the cargo to the scaffold protein Atg11 and to Atg8 family proteins on the forming autophagosomal membrane. We show that the essential kinase Hrr25 activates the cargo receptor Atg19 by phosphorylation, which is required to link cargo to the Atg11 scaffold, allowing selective autophagy to proceed. We also find that the Atg34 cargo receptor is regulated in a similar manner, suggesting a conserved mechanism.

Pho81 is a novel regulator of pexophagy induced by phosphate starvation.

In the budding yeast Saccharomyces cerevisiae, macroautophagy/autophagy can be induced by various types of starvation. It is thought that potential autophagic substrates vary to meet specific nutritional demands under different starvation conditions. In a recent study, Gross et al. found that autophagy induced by phosphate starvation includes many selective aspects. For example, this work identified Pho81 as a regulator of pexophagy under conditions of phosphate starvation. Pho81 senses phosphate metabolites and directly interacts with Atg11 to promote Atg1-mediated Atg11 phosphorylation. This finding provides an example of how modulation of the Atg1/ULK kinase complex can convey specific metabolic information to regulate autophagic substrates.Abbreviation: AKC: Atg1/ULK kinase complex.

Ca2+ as an essential signaling molecule controlling Snf1-mediated Atg1 activation.

Macroautophagy/autophagy is essential for maintaining glucose homeostasis, but the mechanisms by which cells sense glucose starvation and initiate autophagy are not yet fully understood. Recently, we reported that the assembly of a Ca2+-triggered Snf1-Bmh1/Bmh2-Atg11 complex initiates autophagy in response to glucose starvation. Our research reveals that during glucose starvation, the efflux of vacuolar Ca2+ increases cytoplasmic Ca2+ levels, which activates the protein kinase Rck2. Rck2-mediated phosphorylation of Atg11 enhances its interaction with Bmh1 and Bmh2. This interaction recruits the Snf1-Sip1-Snf4 complex, which is located on the vacuolar membrane, to the phagophore assembly site (PAS), leading to the activation of Atg1 and the initiation of autophagy. In summary, we have identified a previously unrecognized signaling pathway involved in glucose starvation-induced autophagy, where Ca2+ acts as a fundamental signaling molecule that links energy stress to the formation of the autophagy initiation complex.Abbreviation: AMPK: AMP-activated protein kinase; ATG: autophagy related; co-IP: co-immunoprecipitation; MAPK: mitogen-activated protein kinase; PAS: phagophore assembly site; ULK1: unc-51 like autophagy activating kinase 1.

Atg1-mediated Atg11 phosphorylation is required for selective autophagy by regulating its association with receptor proteins.

Atg11 is an adaptor protein required for the induction of selective autophagy via receptor binding. However, our understanding of the molecular mechanisms by which it regulates selective autophagy remains incomplete. Here, we show that Atg11 is phosphorylated by Atg1. Rapamycin treatment or starvation conditions induced slower electrophoretic mobility of Atg11 in an Atg1 kinase activity-dependent manner. Through in vitro kinase assays combined with mutagenesis, we determined that Atg1 phosphorylates S949, S1057, and S1064 residues in CC4 domain of Atg11. Replacing the three residues with alanine suppressed the cleavage of selective autophagy substrates for the cytoplasm-to-vacuole targeting (Cvt) pathway, mitophagy, reticulophagy, and pexophagy. The Atg11 mutant was defective in binding to related selective autophagy receptors. These results demonstrate a previously unknown function of Atg1 in regulation of selective autophagy via Atg11 phosphorylation.Abbreviations: AMPK: AMP-activated protein kinase; ATG: autophagy-related; Cvt: cytoplasm-to-vacuole targeting; FUNDC1: FUN14 domain-containing protein 1; GFP: green fluorescent protein; MTOR: mechanistic target of rapamycin kinase; PAS: phagophore assembly site; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PRKAC/PKA: protein kinase cAMP-activated; SD-G: glucose starvation; SD-N: nitrogen starvation; ULK1: unc-51 like autophagy activating kinase 1; λ-PPase: lambda protein phosphatase.

Autophagy-mediated surveillance of Rim4-mRNA interaction safeguards programmed meiotic translation.

In yeast meiosis, autophagy is active and essential. Here, we investigate the fate of Rim4, a meiosis-specific RNA-binding protein (RBP), and its associated transcripts during meiotic autophagy. We demonstrate that Rim4 employs a nuclear localization signal (NLS) to enter the nucleus, where it loads its mRNA substrates before nuclear export. Upon reaching the cytoplasm, active autophagy selectively spares the Rim4-mRNA complex. During meiotic divisions, autophagy preferentially degrades Rim4 in an Atg11-dependent manner, coinciding with the release of Rim4-bound mRNAs for translation. Intriguingly, these released mRNAs also become vulnerable to autophagy. In vitro, purified Rim4 and its RRM-motif-containing variants activate Atg1 kinase in meiotic cell lysates and in immunoprecipitated (IP) Atg1 complexes. This suggests that the conserved RNA recognition motifs (RRMs) of Rim4 are involved in stimulating Atg1 and thereby facilitating selective autophagy. Taken together, our findings indicate that autophagy surveils Rim4-mRNA interaction to ensure stage-specific translation during meiosis.

An overview of the molecular mechanisms of mitophagy in yeast.

Autophagy-dependent selective degradation of excess or damaged mitochondria, termed mitophagy, is a tightly regulated process necessary for mitochondrial quality and quantity control. Mitochondria are highly dynamic and major sites for vital cellular processes such as ATP and iron­sulfur cluster biogenesis. Due to their pivotal roles for immunity, apoptosis, and aging, the maintenance of mitochondrial function is of utmost importance for cellular homeostasis. In yeast, mitophagy is mediated by the receptor protein Atg32 that is localized to the outer mitochondrial membrane. Upon mitophagy induction, Atg32 expression is transcriptionally upregulated, which leads to its accumulation on the mitochondrial surface and to recruitment of the autophagic machinery via its direct interaction with Atg11 and Atg8. Importantly, post-translational modifications such as phosphorylation further fine-tune the mitophagic response. This review summarizes the current knowledge about mitophagy in yeast and its connection with mitochondrial dynamics and the ubiquitin-proteasome system.

The emerging roles of ATG1/ATG13 kinase complex in plants.

Autophagy is a conserved system from yeast to mammals that mediates the degradation and renovation of cellular components. This process is mainly driven by numerous autophagy-related (ATG) proteins. Among these components, the ATG1/ATG13 complex plays an essential role in initiating autophagy, sensing nutritional status signals, recruiting downstream ATG proteins to the autophagosome formation site, and governing autophagosome formation. In this review, we will focus on the ATG1/ATG13 kinase complex, summarizing and discussing the current views on the composition, structure, function, and regulation of this complex in plants.

Autophagy ENDing unproductive phase-separated endocytic protein deposits.

Selective disposal of a wide range of cellular entities by macroautophagy/autophagy is achieved through a special class of proteins called autophagy receptors, which link corresponding cargo to the membrane-bound autophagosomal protein Atg8/LC3. In pursuit of novel autophagy receptors and their cargo, we uncovered a previously undescribed autophagy pathway for removal of aberrant clathrin-mediated endocytosis (CME) protein condensates in S. cerevisiae. Of these CME proteins, Ede1 functions as an autophagy receptor, harboring distinct Atg8-binding domains and driving phase separation into condensates. The aberrant CME condensates at the plasma membrane (PM) exhibit a drop-like structure surrounded by a fenestrated ER, which are engulfed in pieces in an Ede1-dependent manner by autophagy. Thus, our work suggests that aberrant CME is a target for autophagic degradation, with the scaffold protein Ede1 serving as a built-in autophagy receptor that monitors the assembly status of the CME machinery.

Atg11-mediated activation of Atg1 kinase in fission yeast.

The protein kinase Atg1 is a key player in macroautophagy/autophagy, but how its activity is regulated in various organisms is inadequately understood. Our recent study showed that in the fission yeast Schizosaccharomyces pombe, Atg1 kinase activity depends on Atg11, but not Atg13, Atg17, or Atg101. Notably, a 62 amino acid region of S. pombe Atg11 is sufficient for activating Atg1. This region is composed of two parts: an Atg1-binding domain and a homodimerization domain. Atg11 uses this region to dimerize Atg1. Dimerized Atg1 is activated through cis-autophosphorylation.

Mapping Critical Residues in ATG11's Coiled-Coil 2 Domain that Block Multiple Interactions and Disrupt Selective Autophagy.

Selective autophagy is a conserved subcellular process that maintains the health of eukaryotic cells by targeting damaged or toxic cytoplasmic components to the vacuole/lysosome for degradation. A key player in the initiation of selective autophagy in S. Cerevisiae (baker's yeast) is a large adapter protein called Atg11. Atg11 has multiple predicted coiled-coil domains and intrinsically disordered regions, is known to dimerize, and binds and organizes other essential components of the autophagosome formation machinery, including Atg1 and Atg9. We performed systematic directed mutagenesis on the coiled-coil 2 domain of Atg11 in order to map which residues were required for its structure and function. Using yeast-2-hybrid and coimmunoprecipitation, we found only three residues to be critical: I562, Y565, and I569. Mutation of any of these, but especially Y565, could interfere with Atg11 dimerization and block its interaction with Atg1 and Atg9, thereby inactivating selective autophagy.

The Third Coiled Coil Domain of Atg11 Is Required for Shaping Mitophagy Initiation Sites.

Selective autophagy is the capture of specific cytosolic contents in double-membrane vesicles that subsequently fuse with the vacuole or lysosome, thereby delivering cargo for degradation. Selective autophagy receptors (SARs) mark the cargo for degradation and, in yeast, recruit Atg11, the scaffolding protein for selective autophagy initiation. The mitochondrial protein Atg32 is the yeast SAR that mediates mitophagy, the selective autophagic capture of mitochondria. Atg11-Atg32 interactions concentrate Atg32 into puncta that are thought to represent sites of mitophagy initiation. However, it is unclear how Atg11 concentrates Atg32 to generate mitophagy initiation sites. We show here that the coiled coil 3 (CC3) domain of Atg11 is required for concentrating Atg32 into puncta. We determined the structure of the majority of the CC3, demonstrating that the CC3 forms a parallel homodimer whose dimer interface is formed by a small number of hydrophobic residues. We further show that the CC3 interface is not required for Atg11 dimerization but is required for shaping Atg32 into functional mitophagy initiation sites and for delivery of mitochondria to the vacuole. Our findings suggest that Atg11 self-interactions help concentrate SARs as a necessary precondition for cargo capture.

Scaffold proteins in bulk and selective autophagy.

Autophagy is a crucial cellular degradation and recycling pathway. During autophagy double-membrane vesicles, called autophagosomes, encapsulate cellular components and deliver their cargo to the lytic compartment for degradation. Formation of autophagosomes is regulated by the Atg1 kinase complex in yeast and the homologous ULK1 kinase complex in mammals. While research on Atg1 and ULK1 has advanced our understanding of how these protein kinases function in autophagy, the other Atg1/ULK1 kinase complex members have received much less attention. Here, we focus on the functions of the Atg1 kinase complex members Atg11 and Atg17 as well as the ULK1 kinase complex member FIP200 in autophagy. These three proteins act as scaffolds in their respective complexes. Recent studies have made it evident that they have similar but also distinct functions. In this article, we review our current understanding of how these scaffold proteins function from autophagosome formation to fusion and also discuss their possible roles in diseases.

Mechanistic Insights into the Role of Atg11 in Selective Autophagy.

Macroautophagy (referred to hereafter as autophagy) is an intracellular degradation pathway in which the formation of a double-membrane vesicle called the autophagosome is a key event in the transport of multiple cytoplasmic cargo (e.g., proteins, protein aggregates, lipid droplets or organelles) to the vacuole (lysosome in mammals) for degradation and recycling. During this process, autophagosomes are formed de novo by membrane fusion events leading to phagophore formation initiated at the phagophore assembly site. In yeast, Atg11 and Atg17 function as protein scaffolds, essential for selective and non-selective types of autophagy, respectively. While Atg17 functions in non-selective autophagy are well-defined in the literature, less attention is concentrated on recent findings regarding the roles of Atg11 in selective autophagy. Here, we summarize current knowledge about the Atg11 scaffold protein and review recent findings in the context of its role in selective autophagy initiation and autophagosome formation.

Atg1 kinase in fission yeast is activated by Atg11-mediated dimerization and cis-autophosphorylation.

Autophagy is a proteolytic pathway that is conserved from yeasts to mammals. Atg1 kinase is essential for autophagy, but how its activity is controlled remains insufficiently understood. Here, we show that, in the fission yeast Schizosaccharomyces pombe, Atg1 kinase activity requires Atg11, the ortholog of mammalian FIP200/RB1CC1, but does not require Atg13, Atg17, or Atg101. Remarkably, a 62 amino acid region of Atg11 is sufficient for the autophagy function of Atg11 and for supporting the Atg1 kinase activity. This region harbors an Atg1-binding domain and a homodimerization domain. Dimerizing Atg1 is the main role of Atg11, as it can be bypassed by artificially dimerizing Atg1. In an Atg1 dimer, only one Atg1 molecule needs to be catalytically active, suggesting that Atg1 activation can be achieved through cis-autophosphorylation. We propose that mediating Atg1 oligomerization and activation may be a conserved function of Atg11/FIP200 family proteins and cis-autophosphorylation may be a general mechanism of Atg1 activation.

Atg11 is required for initiation of glucose starvation-induced autophagy.

How energy deprivation induces macroautophagy/autophagy is not fully understood. Here, we show that Atg11, a receptor protein for cargo recognition in selective autophagy, is required for the initiation of glucose starvation-induced autophagy. Upon glucose starvation, Atg11 facilitates the interaction between Snf1 and Atg1, thus is required for Snf1-dependent Atg1 activation. Phagophore assembly site (PAS) formation requires Atg11 via its control of the association of Atg17 with Atg29-Atg31. The binding of Atg11 with Atg9 is crucial for recruiting Atg9 vesicles to the PAS and, thus, glucose starvation-induced autophagy. We propose Atg11 as a key initiation factor controlling multiple key steps in energy deprivation-induced autophagy. Abbreviations: AMPK: AMP-activated protein kinase; Ams1: α-mannosidase; Ape1: aminopeptidase I; Cvt: cytoplasm-to-vacuole targeting; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; GFP: green fluorescent protein; MBP: myelin basic protein; MMS: methanesulfonate; PAS: phagophore assembly site; PNBM: p-nitrobenzyl mesylate; SD-G: glucose starvation medium; SD-N: nitrogen starvation medium; ULK1, unc-51 like autophagy activating kinase 1; WT: wild type.