Atg1 kinase organizes autophagosome formation by phosphorylating Atg9

The conserved Ser/Thr kinase Atg1/ULK1 plays a crucial role in the regulation of autophagy. However, only very few Atg1 targets have been identified, impeding elucidation of the mechanisms by which Atg1 regulates autophagy. In our study, we determined the Saccharomyces cerevisiae Atg1 consensus phosphorylation sequence using a peptide array-based approach. Among proteins containing this sequence we identified Atg9, another essential component of the autophagic machinery. We showed that phosphorylation of Atg9 by Atg1 is required for phagophore elongation, shedding light on the mechanism by which Atg1 regulates early steps of autophagy.     

Atg23 and Atg27 Act at the Early Stages of Atg9 Trafficking in S. cerevisiae

Atg9 is a conserved multipass transmembrane protein with an essential role in autophagy. In Saccharomyces cerevisiae, it travels through the secretory pathway to a unique compartment, the Atg9 peripheral structures. These structures are then targeted to the phagophore assembly site (PAS), where they are proposed to help deliver membrane to the forming autophagosome. We used ‘in vivo reconstitution’ of this process in a multiple‐knockout strain to define four proteins, Atg11, Atg19, Atg23 and Atg27, as the core minimal machinery necessary and sufficient for the trafficking of Atg9 to the PAS. Atg23 and Atg27 function in the formation of the Atg9 peripheral structures. Overexpression of Atg9 can bypass the need for Atg23, suggesting that the amount of Atg9 in each peripheral structure is a critical factor in their targeting to the PAS. In contrast, overexpression of Atg23 or Atg27 interferes with Atg9 trafficking, suggesting that these proteins must be present in the appropriate stoichiometry in order to function properly. These data allow us to resolve existing controversies regarding the role of Atg23 and Atg27, and propose a model that ties together previous observations regarding the role of Atg9 in autophagosome formation.      

Lap3 is a selective target of autophagy in yeast, Saccharomyces cerevisiae

Autophagy is a primarily non-selective degradation system of cytoplasmic constituents in lysosomes/vacuoles during starvation. In yeast, autophagy is also involved in the selective transport of Ape1, a vacuolar hydrolase, as a biosynthetic route. Ald6, a soluble cytoplasmic enzyme, is preferentially eliminated from cytoplasm via autophagy. However, little is known about the mechanisms of Ald6 targeting to autophagosomes. Here, we show that Lap3, a soluble cytosolic cysteine protease, is spatially associated with Ape1 and selectively transported to the vacuole during nitrogen starvation. The rate of Lap3 transport is much higher than that of Ald6 and is similar to that of Ape1. Moreover, ATG11 and ATG19, essential factors for Ape1 transport, are important for Lap3 transport. Most Lap3 is degraded within a couple of hours in the vacuole in contrast to Ape1; therefore, we conclude that the machinery required for Ape1 biosynthesis is used for selective degradation of Lap3.     

Liquidity Is a Critical Determinant for Selective Autophagy of Protein Condensates

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Multiple roles of the cytoskeleton in autophagy

Autophagy is involved in a wide range of physiological processes including cellular remodeling during development, immuno‐protection against heterologous invaders and elimination of aberrant or obsolete cellular structures. This conserved degradation pathway also plays a key role in maintaining intracellular nutritional homeostasis and during starvation, for example, it is involved in the recycling of unnecessary cellular components to compensate for the limitation of nutrients. Autophagy is characterized by specific membrane rearrangements that culminate with the formation of large cytosolic double‐membrane vesicles called autophagosomes. Autophagosomes sequester cytoplasmic material that is destined for degradation. Once completed, these vesicles dock and fuse with endosomes and/or lysosomes to deliver their contents into the hydrolytically active lumen of the latter organelle where, together with their cargoes, they are broken down into their basic components. Specific structures destined for degradation via autophagy are in many cases selectively targeted and sequestered into autophagosomes. A number of factors required for autophagy have been identified, but numerous questions about the molecular mechanism of this pathway remain unanswered. For instance, it is unclear how membranes are recruited and assembled into autophagosomes. In addition, once completed, these vesicles are transported to cellular locations where endosomes and lysosomes are concentrated. The mechanism employed for this directed movement is not well understood. The cellular cytoskeleton is a large, highly dynamic cellular scaffold that has a crucial role in multiple processes, several of which involve membrane rearrangements and vesicle‐mediated events. Relatively little is known about the roles of the cytoskeleton network in autophagy. Nevertheless, some recent studies have revealed the importance of cytoskeletal elements such as actin microfilaments and microtubules in specific aspects of autophagy. In this review, we will highlight the results of this work and discuss their implications, providing possible working models. In particular, we will first describe the findings obtained with the yeast Saccharomyces cerevisiae, for long the leading organism for the study of autophagy, and, successively, those attained in mammalian cells, to emphasize possible differences between eukaryotic organisms.     

The sorting nexin FgAtg20 is involved in the Cvt pathway,non‐selective macroautophagy,pexophagy and pathogenesis in Fusarium graminearum

The sorting nexin Atg20/Snx42 plays an important role in autophagy. The wheat head blight pathogen Fusarium graminearum contains an FgAtg20 protein orthologous to Saccharomyces cerevisiae Atg20/Snx42, but its function remains largely unknown. Here, we report a role for FgAtg20 in regulating morphogenesis and fungal pathogenicity. Cytological observation and Western blot analysis revealed that ΔFgAtg20 mutants are defective in vacuolar transport and proteolysis of GFP‐FgAtg8, indicating that FgAtg20 is required for non‐selective macroautophagy. Furthermore, we found that FgATG20 is necessary for the maturation of FgApe1, an indicator of the cytoplasm‐to‐vacuole targeting (Cvt) pathway. Immunoblot analysis displayed lower level of FgPex14, a peroxisomal integral membrane protein in ΔFgAtg20 mutants, suggesting that pexophagy is impaired. Furthermore, we demonstrate that FgAtg20 forms a complex with FgAtg1, FgAtg11, FgAtg17 and FgAtg24. When considered together, we conclude that FgAtg20 plays a critical role in vegetative growth, conidiation and pathogenicity of the head blight pathogen, and is involved in the Cvt pathway, non‐selective macroautophagy and pexophagy.     

Molecular machinery of autophagosome formation in yeast, Saccharomyces cerevisiae

Autophagy is a degradation process accompanied by dynamic membrane organization. In the yeast, Saccharomyces cerevisiae, about 30 ATG (autophagy-related) genes have been identified as important genes for autophagy. Among them, 17 are indispensable for formation of the autophagosome, an organelle enclosed by a double lipid bilayer during starvation-induced autophagy. Recently, a central structure for autophagosome generation, termed the pre-autophagosomal structure, was identified. Despite intensive study, many questions regarding the mechanisms underlying autophagosome formation remain unanswered. In this review, we will give an overview of recent studies on the mechanisms of autophagosome formation and discuss these unresolved questions.     

The Arl3 and Arl1 GTPases co‐operate with Cog8 to regulate selective autophagy via Atg9 trafficking

The Arl3‐Arl1 GTPase cascade plays important roles in vesicle trafficking at the late Golgi and endosomes. Subunits of the conserved oligomeric Golgi (COG) complex, a tethering factor, are important for endosome‐to‐Golgi transport and contribute to the efficient functioning of the cytoplasm‐to‐vacuole targeting (Cvt) pathway, a well‐known selective autophagy pathway. According to our findings, the Arl3‐Arl1 GTPase cascade co‐operates with Cog8 to regulate the Cvt pathway via Atg9 trafficking. arl3cog8Δ and arl1cog8Δ exhibit profound defects in aminopeptidase I maturation in rich medium. In addition, the Arl3‐Arl1 cascade acts on the Cvt pathway via dynamic nucleotide binding. Furthermore, Atg9 accumulates at the late Golgi in arl3cog8Δ and arl1cog8Δ cells under normal growth conditions but not under starvation conditions. Thus, our results offer insight into the requirement for multiple components in the Golgi‐endosome system to determine Atg9 trafficking at the Golgi, thereby regulating selective autophagy.      

Cis1/Atg31 is required for autophagosome formation in Saccharomyces cerevisiae

Autophagy is the bulk degradation of cytosolic materials in lysosomes/vacuoles of eukaryotic cells. In the yeast Saccharomyces cerevisiae, 17 Atg proteins are known to be involved in autophagosome formation. Genome wide analyses have shown that Atg17 interacts with numerous proteins. Further studies on these interacting proteins may provide further insights into membrane dynamics during autophagy. Here, we identify Cis1/Atg31 as a protein that exhibits similar phenotypes to Atg17. ATG31 null cells were defective in autophagy and lost viability under starvation conditions. Localization of Atg31 to pre-autophagosomal structures (PAS) was dependent on Atg17. Coimmunoprecipitation experiments indicated that Atg31 interacts with Atg17. Together, Atg31 is a novel protein that, in concert with Atg17, is required for proper autophagosome formation.     

Biomolecular condensates in autophagy regulation

Autophagy is an intracellular degradation system that contributes to cellular homeostasis. Autophagosome formation is a landmark event in autophagy, which sequesters and delivers cytoplasmic components to the lysosome for degradation. Based on selectivity, autophagy can be classified into bulk and selective autophagy, which are mechanistically distinct from each other, especially in the requirement of cargos for autophagosome formation. Recent studies revealed that liquid-like biomolecular condensates, which are formed through liquid–liquid phase separation, regulate the autophagosome formation of both bulk and selective autophagy. Here, we focus on recent findings on the involvement of biomolecular condensates in autophagy regulation and discuss their significance.