Flexible Atg1/ULK complex composition activates selective autophagy for phosphate starvation.

The molecular basis for bulk autophagy activation due to a deficiency in essential nutrients such as carbohydrates, amino acids, and nitrogen is well understood. Given autophagy functions to reduce surplus to compensate for scarcity, it theoretically possesses the capability to selectively degrade specific substrates to meet distinct metabolic demands. However, direct evidence is still lacking that substantiates the idea that autophagy selectively targets specific substrates (known as selective autophagy) to address particular nutritional needs. Recently, Gross et al. found that during phosphate starvation (P-S), rather than nitrogen starvation (N-S), yeasts selectively eliminate peroxisomes by dynamically altering the composition of the Atg1/ULK kinase complex (AKC) to adapt to P-S. This study elucidates how the metabolite sensor Pho81 flexibly interacts with AKC and guides selective autophagic clearance of peroxisomes during P-S, providing novel insights into the metabolic contribution of autophagy to special nutritional needs.

FIP200 Phosphorylation Regulates Late Steps in Mitophagy.

Mitophagy is a specific type of autophagy responsible for the selective elimination of dysfunctional or superfluous mitochondria, ensuring the maintenance of mitochondrial quality control. The initiation of mitophagy is coordinated by the ULK1 kinase complex, which engages mitophagy receptors via its FIP200 subunit. Whether FIP200 performs additional functions in the subsequent later phases of mitophagy beyond this initial step and how its regulation occurs, remains unclear. Our findings reveal that multiple phosphorylation events on FIP200 differentially control the early and late stages of mitophagy. Furthermore, these phosphorylation events influence FIP200's interaction with ATG16L1. In summary, our results highlight the necessity for precise and dynamic regulation of FIP200, underscoring its importance in the progression of mitophagy.

The HSP90 chaperone code regulates the crosstalk between proteostasis and autophagy.

Proteostasis, the maintenance of proper protein folding, stability, and degradation within cells, is fundamental for cellular function. Two key players in this intricate cellular process are macroautophagy/autophagy and chaperoning of nascent proteins. Here, we explore the crosstalk between autophagy and the HSP90 chaperone in maintaining proteostasis, highlighting their interplay and significance in cellular homeostasis.Abbreviation: HSP90: heat shock protein 90; PTMs: post-translational modifications.

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.

A new regulator of autophagy initiation in glia.

Macroautophagy/autophagy is the major degradation pathway in neurons for eliminating damaged proteins and organelles in Parkinson disease (PD). Like neurons, glial cells are important contributors to PD, yet how autophagy is executed in glia and whether it is using similar interplay as in neurons or other tissues, remain largely elusive. Recently, we reported that the PD risk factor, GAK/aux (cyclin-G-associated kinase/auxilin), regulates the onset of glial autophagy. In the absence of GAK/aux, the number and size of the autophagosomes and autophagosomal precursors increase in adult fly glia and mouse microglia. The protein levels of components in the initiation and class III phosphatidylinositol 3-kinase (PtdIns3K) complexes are generally upregulated. GAK/aux interacts with the master initiation regulator ULK1/Atg1 (unc-51 like autophagy activating kinase 1) via its uncoating domain, hinders autophagy activation by competing with ATG13 (autophagy related 13) for binding to the ULK1 C terminus, and regulates ULK1 trafficking to phagophores. Nonetheless, lack of GAK/aux impairs the autophagic flux and blocks substrate degradation, suggesting that GAK/aux might play additional roles. Overall, our findings reveal a new regulator of autophagy initiation in glia, advancing our understanding on how glia contribute to PD in terms of eliminating pathological protein aggregates.Abbreviations: ATG13: autophagy related 13; GAK/aux: cyclin G associated kinase/auxilin; PtdIns3K: phosphatidylinositol 3-kinase; PD: Parkinson disease; ULK1/Atg1: unc-51 like autophagy activating kinase 1.

Mec1-Rad53 Signaling Regulates DNA Damage-Induced Autophagy and Pathogenicity in Candida albicans.

DNA damage activates the DNA damage response and autophagy in C. albicans; however, the relationship between the DNA damage response and DNA damage-induced autophagy in C. albicans remains unclear. Mec1-Rad53 signaling is a critical pathway in the DNA damage response, but its role in DNA damage-induced autophagy and pathogenicity in C. albicans remains to be further explored. In this study, we compared the function of autophagy-related (Atg) proteins in DNA damage-induced autophagy and traditional macroautophagy and explored the role of Mec1-Rad53 signaling in regulating DNA damage-induced autophagy and pathogenicity. We found that core Atg proteins are required for these two types of autophagy, while the function of Atg17 is slightly different. Our results showed that Mec1-Rad53 signaling specifically regulates DNA damage-induced autophagy but has no effect on macroautophagy. The recruitment of Atg1 and Atg13 to phagophore assembly sites (PAS) was significantly inhibited in the mec1Δ/Δ and rad53Δ/Δ strains. The formation of autophagic bodies was obviously affected in the mec1Δ/Δ and rad53Δ/Δ strains. We found that DNA damage does not induce mitophagy and ER autophagy. We also identified two regulators of DNA damage-induced autophagy, Psp2 and Dcp2, which regulate DNA damage-induced autophagy by affecting the protein levels of Atg1, Atg13, Mec1, and Rad53. The deletion of Mec1 or Rad53 significantly reduces the ability of C. albicans to systematically infect mice and colonize the kidneys, and it makes C. albicans more susceptible to being killed by macrophages.

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.

Neuronal atg1 Coordinates Autophagy Induction and Physiological Adaptations to Balance mTORC1 Signalling.

The mTORC1 nutrient-sensing pathway integrates metabolic and endocrine signals into the brain to evoke physiological responses to food deprivation, such as autophagy. Nevertheless, the impact of neuronal mTORC1 activity on neuronal circuits and organismal metabolism remains obscure. Here, we show that mTORC1 inhibition acutely perturbs serotonergic neurotransmission via proteostatic alterations evoked by the autophagy inducer atg1. Neuronal ATG1 alters the intracellular localization of the serotonin transporter, which increases the extracellular serotonin and stimulates the 5HTR7 postsynaptic receptor. 5HTR7 enhances food-searching behaviour and ecdysone-induced catabolism in Drosophila. Along similar lines, the pharmacological inhibition of mTORC1 in zebrafish also stimulates food-searching behaviour via serotonergic activity. These effects occur in parallel with neuronal autophagy induction, irrespective of the autophagic activity and the protein synthesis reduction. In addition, ectopic neuronal atg1 expression enhances catabolism via insulin pathway downregulation, impedes peptidergic secretion, and activates non-cell autonomous cAMP/PKA. The above exert diverse systemic effects on organismal metabolism, development, melanisation, and longevity. We conclude that neuronal atg1 aligns neuronal autophagy induction with distinct physiological modulations, to orchestrate a coordinated physiological response against reduced mTORC1 activity.

Autophagy impairment and lifespan reduction caused by Atg1 RNAi or Atg18 RNAi expression in adult fruit flies (Drosophila melanogaster).

Autophagy, an autophagosome and lysosome-based eukaryotic cellular degradation system, has previously been implicated in lifespan regulation in different animal models. In this report, we show that expression of the RNAi transgenes targeting the transcripts of the key autophagy genes Atg1 or Atg18 in adult fly muscle or glia does not affect the overall levels of autophagosomes in those tissues and does not change the lifespan of the tested flies but the lifespan reduction phenotype has become apparent when Atg1 RNAi or Atg18 RNAi is expressed ubiquitously in adult flies or after autophagy is eradicated through the knockdown of Atg1 or Atg18 in adult fly adipocytes. Lifespan reduction was also observed when Atg1 or Atg18 was knocked down in adult fly enteroblasts and midgut stem cells. Overexpression of wild-type Atg1 in adult fly muscle or adipocytes reduces the lifespan and causes accumulation of high levels of ubiquitinated protein aggregates in muscles. Our research data have highlighted the important functions of the key autophagy genes in adult fly adipocytes, enteroblasts, and midgut stem cells and their undetermined roles in adult fly muscle and glia for lifespan regulation.

Activation of autophagy depends on Atg1/Ulk1-mediated phosphorylation and inhibition of the Hsp90 chaperone machinery.

Cellular homeostasis relies on both the chaperoning of proteins and the intracellular degradation system that delivers cytoplasmic constituents to the lysosome, a process known as autophagy. The crosstalk between these processes and their underlying regulatory mechanisms is poorly understood. Here, we show that the molecular chaperone heat shock protein 90 (Hsp90) forms a complex with the autophagy-initiating kinase Atg1 (yeast)/Ulk1 (mammalian), which suppresses its kinase activity. Conversely, environmental cues lead to Atg1/Ulk1-mediated phosphorylation of a conserved serine in the amino domain of Hsp90, inhibiting its ATPase activity and altering the chaperone dynamics. These events impact a conformotypic peptide adjacent to the activation and catalytic loop of Atg1/Ulk1. Finally, Atg1/Ulk1-mediated phosphorylation of Hsp90 leads to dissociation of the Hsp90:Atg1/Ulk1 complex and activation of Atg1/Ulk1, which is essential for initiation of autophagy. Our work indicates a reciprocal regulatory mechanism between the chaperone Hsp90 and the autophagy kinase Atg1/Ulk1 and consequent maintenance of cellular proteostasis.

Atg1-dependent phosphorylation of Vps34 is required for dynamic regulation of the phagophore assembly site and autophagy in Saccharomyces cerevisiae.

Macroautophagy/autophagy is a key catabolic pathway in which double-membrane autophagosomes sequester various substrates destined for degradation, enabling cells to maintain homeostasis and survive under stressful conditions. Several autophagy-related (Atg) proteins are recruited to the phagophore assembly site (PAS) and cooperatively function to generate autophagosomes. Vps34 is a class III phosphatidylinositol 3-kinase, and Atg14-containing Vps34 complex I plays essential roles in autophagosome formation. However, the regulatory mechanisms of yeast Vps34 complex I are still poorly understood. Here, we demonstrate that Atg1-dependent phosphorylation of Vps34 is required for robust autophagy activity in Saccharomyces cerevisiae. Following nitrogen starvation, Vps34 in complex I is selectively phosphorylated on multiple serine/threonine residues in its helical domain. This phosphorylation is important for full autophagy activation and cell survival. The absence of Atg1 or its kinase activity leads to complete loss of Vps34 phosphorylation in vivo, and Atg1 directly phosphorylates Vps34 in vitro, regardless of its complex association type. We also demonstrate that the localization of Vps34 complex I to the PAS provides a molecular basis for the complex I-specific phosphorylation of Vps34. This phosphorylation is required for the normal dynamics of Atg18 and Atg8 at the PAS. Together, our results reveal a novel regulatory mechanism of yeast Vps34 complex I and provide new insights into the Atg1-dependent dynamic regulation of the PAS.Abbreviations: ATG: autophagy-related; BARA: the repeated, autophagy-specific Co-IP: co-immunoprecipitation; GFP: green fluorescent protein; IP-MS: immunoprecipitation followed by tandem mass spectrometry; NTD: the N-terminal domain; PAS: phagophore assembly site; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns3K: phosphatidylinositol 3-kinase; SUR: structurally uncharacterized region; Vps34[KD]: Vps34D731N.

Atg1 modulates mitochondrial dynamics to promote germline stem cell maintenance in Drosophila.

Mitochondrial dynamics (fusion and fission) are necessary for stem cell maintenance and differentiation. However, the relationship between mitophagy, mitochondrial dynamics and stem cell exhaustion needs to be clearly understood. Here we report the multifaceted role of Atg1 in mitophagy, mitochondrial dynamics and stem cell maintenance in female germline stem cells (GSCs) in Drosophila. We found that depletion of Atg1 in GSCs leads to impaired autophagy and mitophagy as measured by reduced formation of autophagosomes, increased accumulation of p62/Ref (2)P and accumulation of damaged mitochondria. Disrupting Atg1 function led to mitochondrial fusion in developing cysts. The fusion resulted from an increase in Marf levels in both GSCs and cysts, and the fusion phenotype could be rescued by overexpression of Drp1 or by depleting Marf via RNAi in Atg1-depleted cyst cells. Interestingly, double knockdown of both Atg1:Drp1 led to the significant loss of germ cells (GCs) as compared to Atg1KD and Drp1KD. Strikingly, Atg1:Marf double knockdown leads to a dramatic loss of GSCs, GCs and a total loss of vitellogenic stages, suggesting a block in oogenesis. Overall, our results demonstrate that Drp1, Marf and Atg1 function together to influence female GSC maintenance, their differentiation into cysts and oogenesis in Drosophila.

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.

Transcriptional regulation of autophagy by RNA polymerase II.

Macroautophagy/autophagy is a catabolic recycling pathway and is tightly regulated by upstream signals. Autophagy genes are quickly upregulated upon stimuli such as nutrition limitation in response to the external environment. However, how the transcriptional activation of autophagy genes occurs is not well understood. We recently found that in yeast, the RNA polymerase II subunit Rpb9 specifically and efficiently upregulates the transcription of the autophagy gene ATG1 with the mediation of Gcn4. Such regulation was shown to be essential for autophagic activities induced by starvation. Furthermore, the function of Rpb9 in autophagy and the activation of ATG1 transcription is conserved in mammalian cells. In conclusion, Rpb9 specifically and positively regulates ATG1 transcription as a key regulator of autophagy.

The crucial role of the regulatory mechanism of the Atg1/ULK1 complex in fungi.

Autophagy, an evolutionarily conserved cellular degradation pathway in eukaryotes, is hierarchically regulated by autophagy-related genes (Atgs). The Atg1/ULK1 complex is the most upstream factor involved in autophagy initiation. Here，we summarize the recent studies on the structure and molecular mechanism of the Atg1/ULK1 complex in autophagy initiation, with a special focus on upstream regulation and downstream effectors of Atg1/ULK1. The roles of pathogenicity and autophagy aspects in Atg1/ULK1 complexes of various pathogenic hosts, including plants, insects, and humans, are also discussed in this work based on recent research findings. We establish a framework to study how the Atg1/ULK1 complex integrates the signals that induce autophagy in accordance with fungus to mammalian autophagy regulation pathways. This framework lays the foundation for studying the deeper molecular mechanisms of the Atg1 complex in pathogenic fungi.

The RNA polymerase II subunit Rpb9 activates ATG1 transcription and autophagy.

Macroautophagy/autophagy is a conserved process in eukaryotic cells that mediates the degradation and recycling of intracellular substrates. Proteins encoded by autophagy-related (ATG) genes are essentially involved in the autophagy process and must be tightly regulated in response to various circumstances, such as nutrient-rich and starvation conditions. However, crucial transcriptional activators of ATG genes have remained obscure. Here, we identify the RNA polymerase II subunit Rpb9 as an essential regulator of autophagy by a high-throughput screen of a Saccharomyces cerevisiae gene knockout library. Rpb9 plays a crucial and specific role in upregulating ATG1 transcription, and its deficiency decreases autophagic activities. Rpb9 promotes ATG1 transcription by binding to its promoter region, which is mediated by Gcn4. Furthermore, the function of Rpb9 in autophagy and its regulation of ATG1/ULK1 transcription are conserved in mammalian cells. Together, our results indicate that Rpb9 specifically activates ATG1 transcription and thus positively regulates the autophagy process.

Cdc14 plans autophagy for meiotic cell divisions.

The role of meiotic proteasome-mediated degradation has been extensively studied. At the same time, macroautophagy/autophagy only emerged recently as an essential regulator for meiosis progression. Our recent publication showed that autophagy in meiotic cells exhibits a temporal pattern distinct from that in quiescent cells or mitotic cells under prolonged starvation. Importantly, autophagic activity oscillates during meiotic cell divisions, i.e., meiosis I and meiosis II, which can accelerate meiotic progression and increase sporulation efficiency. Our in vitro and in vivo assays revealed that the conserved phosphatase Cdc14 stimulates autophagy initiation during meiotic divisions, specifically in anaphase I and II, when a subpopulation of active Cdc14 relocates to the cytosol and interacts with phagophore assembly sites (PAS) triggering the dephosphorylation of Atg13 to stimulate Atg1 kinase activity and autophagy. Together, our findings reveal a mechanism for the coordination of autophagy activity in the context of meiosis progression.

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.

Atg1 kinase activity links PAS dissolution to balanced Atg8 conjugation.

Atg1 phosphoregulates different steps and factors in autophagy. Schreiber et al. report in Molecular Cell on the cell-free identification of a negative feedback ejection of Atg1 from the pre-autophagosomal structure (PAS), followed by positive feedback recruitment of Atg1 to phagophore-resident Atg8-PE, followed by yet another, negative feedback inhibition of the Atg8 conjugation machinery.

Ci/Gli Phosphorylation by the Fused/Ulk Family Kinases.

Hedgehog (Hh) signaling culminates in the conversion of the latent transcription factor Cubitus interruptus (Ci)/Gli from a repressor form (CiR/GliR) into an activator form (CiA/GliA). While sequential phosphorylation of Ci/Gli by protein kinase A(PKA), glycogen synthase kinase 3 (GSK3), and casein kinase 1 (CK1) is essential for its proteolytic processing that generates CiR/GliR, sequential phosphorylation of Ci/Gli by the Fused (Fu)/Unc-51 like kinase (Ulk) family kinases Fu/Ulk3/Stk36 and CK1 contributes to the formation of CiA/GliA. Fu/Ulk3/Stk36-mediated phosphorylation of Ci/Gli is stimulated by Hh, leading to altered interaction between Ci/Gli and the Hh pathway repressor Sufu. Here we describe both in vitro and in vivo assays that determine Ci/Gli phosphorylation by the Fu/Ulk family kinases and its regulation by Hh.