Mechanisms of Autophagy Initiation.

Autophagy is the process of cellular self-eating by a double-membrane organelle, the autophagosome. A range of signaling processes converge on two protein complexes to initiate autophagy: the ULK1 (unc51-like autophagy activating kinase 1) protein kinase complex and the PI3KC3-C1 (class III phosphatidylinositol 3-kinase complex I) lipid kinase complex. Some 90% of the mass of these large protein complexes consists of noncatalytic domains and subunits, and the ULK1 complex has essential noncatalytic activities. Structural studies of these complexes have shed increasing light on the regulation of their catalytic and noncatalytic activities in autophagy initiation. The autophagosome is thought to nucleate from vesicles containing the integral membrane protein Atg9 (autophagy-related 9), COPII (coat protein complex II) vesicles, and possibly other sources. In the wake of reconstitution and super-resolution imaging studies, we are beginning to understand how the ULK1 and PI3KC3-C1 complexes might coordinate the nucleation and fusion of Atg9 and COPII vesicles at the start of autophagosome biogenesis.

ULK1 and ULK2 Regulate Stress Granule Disassembly Through Phosphorylation and Activation of VCP/p97.

Disturbances in autophagy and stress granule dynamics have been implicated as potential mechanisms underlying inclusion body myopathy (IBM) and related disorders. Yet the roles of core autophagy proteins in IBM and stress granule dynamics remain poorly characterized. Here, we demonstrate that disrupted expression of the core autophagy proteins ULK1 and ULK2 in mice causes a vacuolar myopathy with ubiquitin and TDP-43-positive inclusions; this myopathy is similar to that caused by VCP/p97 mutations, the most common cause of familial IBM. Mechanistically, we show that ULK1/2 localize to stress granules and phosphorylate VCP, thereby increasing VCP's activity and ability to disassemble stress granules. These data suggest that VCP dysregulation and defective stress granule disassembly contribute to IBM-like disease in Ulk1/2-deficient mice. In addition, stress granule disassembly is accelerated by an ULK1/2 agonist, suggesting ULK1/2 as targets for exploiting the higher-order regulation of stress granules for therapeutic intervention of IBM and related disorders.

Two distinct regulatory pathways govern Cct2-Atg8 binding in the process of solid aggrephagy.

CCT2 serves as an aggrephagy receptor that plays a crucial role in the clearance of solid aggregates, yet the underlying molecular mechanisms by which CCT2 regulates solid aggrephagy are not fully understood. Here we report that the binding of Cct2 to Atg8 is governed by two distinct regulatory mechanisms: Atg1-mediated Cct2 phosphorylation and the interaction between Cct2 and Atg11. Atg1 phosphorylates Cct2 at Ser412 and Ser470, and disruption of these phosphorylation sites impairs solid aggrephagy by hindering Cct2-Atg8 binding. Additionally, we observe that Atg11, an adaptor protein involved in selective autophagy, directly associates with Cct2 through its CC4 domain. Deficiency in this interaction significantly weakens the association of Cct2 with Atg8. The requirement of Atg1-mediated Cct2 phosphorylation and of Atg11 for CCT2-LC3C binding and subsequent aggrephagy is conserved in mammalian cells. These findings provide insights into the crucial roles of Atg1-mediated Cct2 phosphorylation and Atg11-Cct2 binding as key mediators governing the interaction between Cct2 and Atg8 during the process of solid aggrephagy.

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.

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.

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.

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.

Vacuolar protein Tag1 and Atg1-Atg13 regulate autophagy termination during persistent starvation in S. cerevisiae.

Under starvation conditions, cells degrade their own components via autophagy in order to provide sufficient nutrients to ensure their survival. However, even if starvation persists, the cell is not completely degraded through autophagy, implying the existence of some kind of termination mechanism. In the yeast Saccharomyces cerevisiae, autophagy is terminated after 10-12 h of nitrogen starvation. In this study, we found that termination is mediated by re-phosphorylation of Atg13 by the Atg1 protein kinase, which is also affected by PP2C phosphatases, and the eventual dispersion of the pre-autophagosomal structure, also known as the phagophore assembly site (PAS). In a genetic screen, we identified an uncharacterized vacuolar membrane protein, Tag1, as a factor responsible for the termination of autophagy. Re-phosphorylation of Atg13 and eventual PAS dispersal were defective in the Δtag1 mutant. The vacuolar luminal domain of Tag1 and autophagic progression are important for the behaviors of Tag1. Together, our findings reveal the mechanism and factors responsible for termination of autophagy in yeast.

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 kinase regulates autophagosome-vacuole fusion by controlling SNARE bundling.

Autophagy mediates the degradation of cytoplasmic material. Upon autophagy induction, autophagosomes form a sealed membrane around the cargo and fuse with the lytic compartment to release the cargo for degradation. In order to avoid premature fusion of immature autophagosomal membranes with the lytic compartment, this process needs to be tightly regulated. Several factors mediating autophagosome-vacuole fusion have recently been identified. In budding yeast, autophagosome-vacuole fusion requires the R-SNARE Ykt6 on the autophagosome, together with the three Q-SNAREs Vam3, Vam7, and Vti1 on the vacuole. However, how these SNAREs are regulated during the fusion process is poorly understood. In this study, we investigate the regulation of Ykt6. We found that Ykt6 is directly phosphorylated by Atg1 kinase, which keeps this SNARE in an inactive state. Ykt6 phosphorylation prevents SNARE bundling by disrupting its interaction with the vacuolar SNAREs Vam3 and Vti1, thereby preventing premature autophagosome-vacuole fusion. These findings shed new light on the regulation of autophagosome-vacuole fusion and reveal a further step in autophagy controlled by the Atg1 kinase.

Deficiency of ULK1/ATG1 in the follicle cells disturbs ER homeostasis and causes defective chorion deposition in the vector Rhodnius prolixus.

In insects, synthesis and deposition of the chorion (eggshell) are performed by the professional secretory follicle cells (FCs) that surround the oocytes in the course of oogenesis. Here, we found that ULK1/ATG1, an autophagy-related protein, is highly expressed in the FCs of the Chagas-Disease vector Rhodnius prolixus, and that parental RNAi silencing of ULK1/ATG1 results in oocytes with abnormal chorion ultrastructure and FCs presenting expanded rough ER membranes as well as increased expression of the ER chaperone BiP3, both indicatives of ER stress. Silencing of LC3/ATG8, another essential autophagy protein, did not replicate the ULK1/ATG1 phenotypes, whereas silencing of SEC16A, a known partner of the noncanonical ULK1/ATG1 function in the ER exit sites phenocopied the silencing of ULK1/ATG1. Our findings point to a cooperated function of ULK1/ATG1 and SEC16A in the FCs to complete choriogenesis and provide additional in vivo phenotype-based evidence to the literature of the role of ULK1/ATG1 in the ER in a professional secretory cell.

Regulation of ATG and Autophagy Initiation.

ATG is involved in multiple processes of autophagosome formation, including the initial phase of autophagy. The mammalian autophagy complex-ULK1 complex is composed of ULK1, FIP200, ATG13 and ATG101, and the yeast autophagy initiation complex-ATG1 complex is composed of ATG1, ATG13, ATG17, ATG29 and ATG31. After this complex is activated, it binds and phosphorylates ATG9 on the vesicles. Then PI3KC3-C1 (yeast: ATG34: ATG15: ATG6: ATG14 or mammal animal: ATG34: ATG15: BECN1: ATG14L) is recruited to the PAS. Further, ATG12-ATG5-ATG16 complex is localized on PAS (Yeast) or localized on the outer surface of the membrane (mammal) and makes binding of ATG8 (LC3) with PE to form ATG8-PE complex, promoting autophagic membrane elongation, closure and formation autophagosome and autophagosome lysosome.

Spodoptera litura autophagy-related protein 1 interacts with autophagy-related protein 5 and enhances its degradation.

It is known that the autophagy-related protein 1 (ATG1) plays critical roles in the regulation of autophagy in mammals and yeast, whereas the function of ATG1 in lepidopteran insects is not well elucidated. Here Spodoptera litura ATG1 (SlATG1) and its interactions with other ATG proteins were characterized. Alternative splicing of SlAtg1 produced at least four transcript variants. Over-expression and RNA interference knockdown of SlAtg1 demonstrated that SlATG1 enhanced autophagy. SlATG1A-Green fluorescent protein (GFP) tagged localized in the cytoplasm and formed some punctuate dots, which were colocalized with red fluorescent protein mCherry tagged Spodoptera exigua ATG5 (SeATG5). SlATG1A-GFP over-expression reduced the nuclear abundance of mCherry-SeATG5 but increased its cytoplasmic abundance. Pull-down, co-immunoprecipitation and bimolecular fluorescence complementation assays showed that SlATG1A bound to SeATG5 through the N-terminus of SlATG1A. The over-expression of FLAG epitope tagged SlATG1A significantly increased the accumulation of the cleaved GFP from GFP-SeATG5, suggesting the enhanced degradation of GFP-SeATG5. In addition, we confirmed that the interactions of SlATG1 with other autophagy-related proteins were conserved. These results provide the first evidence that ATG1 interacts with ATG5 and enhances its degradation in lepidopteran insect cells, which may have important physiological functions.

Crocetin shifts autophagic cell survival to death of breast cancer cells in chemotherapy.

The chemotherapy with fluorouracil is not always effective, in which some breast cancer cells may survive the fluorouracil treatment through enhanced autophagy. Crocetin is the major constituent of saffron, a Chinese traditional herb, which has recently found to have multiple pharmacological effects, including anticancer. However, the effects of Crocetin on the outcome of fluorouracil therapy for breast cancer have not been studied. Here, we showed that fluorouracil treatment inhibited the growth of breast cancer cells, in either a Cell Counting Kit-8 assay or an MTT assay. Inhibition of autophagy further suppressed breast cancer cell growth, suggesting that the breast cancer cells increased autophagic cell survival during fluorouracil treatment. However, Crocetin significantly increased the suppressive effects of fluorouracil on breast cancer cell growth, without affecting either cell apoptosis or autophagy. Inhibition of autophagy at the presence of Crocetin partially abolished the suppressive effects on breast cancer cell growth, suggesting that Crocetin may increase autophagic cell death in fluorouracil-treated breast cancer cells. Furthermore, Crocetin decreased Beclin-1 levels but increased ATG1 levels in fluorouracil-treated breast cancer cells. Together, these data suggest that Crocetin may shift autophagic cell survival to autophagic cell death in fluorouracil-treated breast cancer cells, possibly through modulation of the expression of ATG1 and Beclin-1.

Autophagy-independent function of Atg1 for apoptosis-induced compensatory proliferation.

BACKGROUND: ATG1 belongs to the Uncoordinated-51-like kinase protein family. Members of this family are best characterized for roles in macroautophagy and neuronal development. Apoptosis-induced proliferation (AiP) is a caspase-directed and JNK-dependent process which is involved in tissue repair and regeneration after massive stress-induced apoptotic cell loss. Under certain conditions, AiP can cause tissue overgrowth with implications for cancer. RESULTS: Here, we show that Atg1 in Drosophila (dAtg1) has a previously unrecognized function for both regenerative and overgrowth-promoting AiP in eye and wing imaginal discs. dAtg1 acts genetically downstream of and is transcriptionally induced by JNK activity, and it is required for JNK-dependent production of mitogens such as Wingless for AiP. Interestingly, this function of dAtg1 in AiP is independent of its roles in autophagy and in neuronal development. CONCLUSION: In addition to a role of dAtg1 in autophagy and neuronal development, we report a third function of dAtg1 for AiP.

Protein aggregation propensity is a crucial determinant of intracellular inclusion formation and quality control degradation.

Protein aggregation is linked to many pathological conditions, including several neurodegenerative diseases. The aggregation propensities of proteins are thought to be controlled to a large extent by the physicochemical properties encoded in the primary sequence. We have previously exploited a set of amyloid ß peptide (Aß42) variants exhibiting a continuous gradient of intrinsic aggregation propensities to demonstrate that this rule applies in vivo in bacteria. In the present work we have characterized the behavior of these Aß42 mutants when expressed in yeast. In contrast to bacteria, the intrinsic aggregation propensity is gated by yeast, in such a way that this property correlates with the formation of intracellular inclusions only above a specific aggregation threshold. Proteins displaying solubility levels above this threshold escape the inclusion formation pathway. In addition, the most aggregation-prone variants are selectively cleared by the yeast quality control degradation machinery. Thus, both inclusion formation and proteolysis target the same aggregation-prone variants and cooperate to minimize the presence of these potentially dangerous species in the cytosol. The demonstration that sorting to these pathways in eukaryotes is strongly influenced by protein primary sequence should facilitate the development of rational approaches to predict and hopefully prevent in vivo protein deposition.

The Multifaceted Interactions of Dictyostelium Atg1 with Mitochondrial Function, Endocytosis, Growth, and Development.

Autophagy is a degradative recycling process central to the maintenance of homeostasis in all eukaryotes. By ensuring the degradation of damaged mitochondria, it plays a key role in maintaining mitochondrial health and function. Of the highly conserved autophagy proteins, autophagy-related protein 1 (Atg1) is essential to the process. The involvement of these proteins in intracellular signalling pathways, including those involving mitochondrial function, are still being elucidated. Here the role of Atg1 was investigated in the simple model organism Dictyostelium discoideum using an atg1 null mutant and mutants overexpressing or antisense-inhibiting atg1. When evaluated against the well-characterised outcomes of mitochondrial dysfunction in this model, altered atg1 expression resulted in an unconventional set of phenotypic outcomes in growth, endocytosis, multicellular development, and mitochondrial homeostasis. The findings here show that Atg1 is involved in a tightly regulated signal transduction pathway coordinating energy-consuming processes such as cell growth and multicellular development, along with nutrient status and energy production. Furthermore, Atg1's effects on energy homeostasis indicate a peripheral ancillary role in the mitochondrial signalling network, with effects on energy balance rather than direct effects on electron transport chain function. Further research is required to tease out these complex networks. Nevertheless, this study adds further evidence to the theory that autophagy and mitochondrial signalling are not opposing but rather linked, yet strictly controlled, homeostatic mechanisms.

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.

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.

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.