RESUMO
While the molecular mechanism of autophagy is well studied, the cargoes delivered by autophagy remain incompletely characterized. To examine the selectivity of autophagy cargo, we conducted proteomics on isolated yeast autophagic bodies, which are intermediate structures in the autophagy process. We identify a protein, Hab1, that is highly preferentially delivered to vacuoles. The N-terminal 42 amino acid region of Hab1 contains an amphipathic helix and an Atg8-family interacting motif, both of which are necessary and sufficient for the preferential delivery of Hab1 by autophagy. We find that fusion of this region with a cytosolic protein results in preferential delivery of this protein to the vacuole. Furthermore, attachment of this region to an organelle allows for autophagic delivery in a manner independent of canonical autophagy receptor or scaffold proteins. We propose a novel mode of selective autophagy in which a receptor, in this case Hab1, binds directly to forming isolation membranes during bulk autophagy.
Assuntos
Autofagia , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Vacúolos/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Família da Proteína 8 Relacionada à Autofagia/genética , Transporte Proteico , Proteômica/métodosRESUMO
Clearance of biomolecular condensates by selective autophagy is thought to play a crucial role in cellular homeostasis. However, the mechanism underlying selective autophagy of condensates and whether liquidity determines a condensate's susceptibility to degradation by autophagy remain unknown. Here, we show that the selective autophagic cargo aminopeptidase I (Ape1) undergoes phase separation to form semi-liquid droplets. The Ape1-specific receptor protein Atg19 localizes to the surface of Ape1 droplets both in vitro and in vivo, with the "floatability" of Atg19 preventing its penetration into droplets. In vitro reconstitution experiments reveal that Atg19 and lipidated Atg8 are necessary and sufficient for selective sequestration of Ape1 droplets by membranes. This sequestration is impaired by mutational solidification of Ape1 droplets or diminished ability of Atg19 to float. Taken together, we propose that cargo liquidity and the presence of sufficient amounts of autophagic receptor on cargo are crucial for selective autophagy of biomolecular condensates.
Assuntos
Aminopeptidases/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Receptores de Superfície Celular/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Aminopeptidases/genética , Família da Proteína 8 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Citoplasma/metabolismo , Mutação , Ligação Proteica , Transporte Proteico , Receptores de Superfície Celular/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Solubilidade , Proteínas de Transporte Vesicular/genéticaRESUMO
Many biomolecules undergo liquid-liquid phase separation to form liquid-like condensates that mediate diverse cellular functions1,2. Autophagy is able to degrade such condensates using autophagosomes-double-membrane structures that are synthesized de novo at the pre-autophagosomal structure (PAS) in yeast3-5. Whereas Atg proteins that associate with the PAS have been characterized, the physicochemical and functional properties of the PAS remain unclear owing to its small size and fragility. Here we show that the PAS is in fact a liquid-like condensate of Atg proteins. The autophagy-initiating Atg1 complex undergoes phase separation to form liquid droplets in vitro, and point mutations or phosphorylation that inhibit phase separation impair PAS formation in vivo. In vitro experiments show that Atg1-complex droplets can be tethered to membranes via specific protein-protein interactions, explaining the vacuolar membrane localization of the PAS in vivo. We propose that phase separation has a critical, active role in autophagy, whereby it organizes the autophagy machinery at the PAS.
Assuntos
Autofagossomos/química , Autofagossomos/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagia , Proteínas Relacionadas à Autofagia/química , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/química , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Fosforilação , Mutação Puntual , Ligação Proteica , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismoRESUMO
Macroautophagy is mediated by a unique organelle, the autophagosome, which encloses a portion of cytoplasm for delivery to the lysosome. Autophagosome formation is dynamically regulated by starvation and other stresses and involves complicated membrane reorganization. Since the discovery of yeast Atg-related proteins, autophagosome formation has been dissected at the molecular level. In this review we describe the molecular mechanism of autophagosome formation with particular focus on the function of Atg proteins and the long-standing discussion regarding the origin of the autophagosome membrane.
Assuntos
Autofagia/fisiologia , Complexos Multiproteicos/metabolismo , Fagossomos/metabolismo , Animais , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Fagossomos/ultraestrutura , Fosfatidilinositol 3-Quinases/metabolismo , Leveduras/citologia , Leveduras/metabolismoRESUMO
The vacuole/lysosome plays essential roles in the growth and proliferation of many eukaryotic cells via the activation of target of rapamycin complex 1 (TORC1). Moreover, the yeast vacuole/lysosome is necessary for progression of the cell division cycle, in part via signaling through the TORC1 pathway. Here, we show that an essential cyclin-dependent kinase, Bur1, plays a critical role in cell cycle progression in cooperation with TORC1. A mutation in BUR1 combined with a defect in vacuole inheritance shows a synthetic growth defect. Importantly, the double mutant, as well as a bur1-267 mutant on its own, has a severe defect in cell cycle progression from G1 phase. In further support that BUR1 functions with TORC1, mutation of bur1 alone results in high sensitivity to rapamycin, a TORC1 inhibitor. Mechanistic insight for Bur1 function comes from the findings that Bur1 directly phosphorylates Sch9, a target of TORC1, and that both Bur1 and TORC1 are required for the activation of Sch9. Together, these discoveries suggest that multiple signals converge on Sch9 to promote cell cycle progression.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Vacúolos , Ciclo Celular/genética , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição , Vacúolos/metabolismoRESUMO
Autophagy is a major cellular degradation pathway that is highly conserved among eukaryotes. The identification of cargos captured by autophagosomes is critical to our understanding of the physiological significance of autophagy in cells, but these studies can be challenging because autophagosomes disintegrate easily. In the yeast Saccharomyces cerevisiae, cells deficient in the vacuolar lipase Atg15 accumulate autophagic bodies (ABs) within the vacuole following the induction of autophagy. As ABs contain cytosolic components including proteins, RNAs, and lipids, their purification allows the identification of material targeted by autophagy for degradation. In this study, we demonstrate a method to purify intact ABs using isolated vacuoles from atg15Δ cells. Taking advantage of the size discrepancy between the vacuoles and ABs, the vacuolar membrane was disrupted by filtration to release ABs. Filtered vacuolar lysates were subjected to density gradient centrifugation to obtain AB fractions. Purified ABs retain membrane integrity and contain autophagic cargos. This technique offers a valuable tool for the identification of the cargos of autophagy, examination of autophagic cargo selectivity, and biochemical characterization of autophagosome membranes.
Assuntos
Autofagossomos , Saccharomyces cerevisiae , Autofagossomos/metabolismo , Autofagia , Fagossomos/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Vacúolos/enzimologia , Vacúolos/metabolismoRESUMO
Autophagy is an intracellular degradation pathway targeting organelles and macromolecules, thereby regulating various cellular functions. Phosphorylation is a key posttranscriptional protein modification implicated in the regulation of biological function including autophagy. Under asynchronous conditions, autophagy activity is predominantly suppressed by mechanistic target of rapamycin (mTOR) kinase, but whether autophagy-related genes (ATG) proteins are phosphorylated differentially throughout the sequential phases of the cell cycle remains unclear. In this issue, Li and colleagues report that cyclin-dependent kinase 1 (CDK1) phosphorylates the ULK complex during mitosis. This phosphorylation induces autophagy and, surprisingly, is shown to drive cell cycle progression. This work reveals a yet-unappreciated role for autophagy in cell cycle progression and enhances our understanding of the specific phase-dependent autophagy regulation during cellular growth and proliferation.
Assuntos
Autofagia , Proteína Quinase CDC2 , Alvo Mecanístico do Complexo 1 de Rapamicina , Mitose , Fosforilação , Serina-Treonina Quinases TORRESUMO
Zinc (Zn) is an essential micronutrient for plant growth. Accordingly, Zn deficiency (-Zn) in agricultural fields is a serious problem, especially in developing regions. Autophagy, a major intracellular degradation system in eukaryotes, plays important roles in nutrient recycling under nitrogen and carbon starvation. However, the relationship between autophagy and deficiencies of other essential elements remains poorly understood, especially in plants. In this study, we focused on Zn due to the property that within cells most Zn is tightly bound to proteins, which can be targets of autophagy. We found that autophagy plays a critical role during -Zn in Arabidopsis (Arabidopsis thaliana). Autophagy-defective plants (atg mutants) failed to grow and developed accelerated chlorosis under -Zn. As expected, -Zn induced autophagy in wild-type plants, whereas in atg mutants, various organelle proteins accumulated to high levels. Additionally, the amount of free Zn2+ was lower in atg mutants than in control plants. Interestingly, -Zn symptoms in atg mutants recovered under low-light, iron-limited conditions. The levels of hydroxyl radicals in chloroplasts were elevated, and the levels of superoxide were reduced in -Zn atg mutants. These results imply that the photosynthesis-mediated Fenton-like reaction, which is responsible for the chlorotic symptom of -Zn, is accelerated in atg mutants. Together, our data indicate that autophagic degradation plays important functions in maintaining Zn pools to increase Zn bioavailability and maintain reactive oxygen species homeostasis under -Zn in plants.
Assuntos
Arabidopsis/metabolismo , Autofagia/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Zinco/deficiência , Zinco/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cloroplastos/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
Autophagy is a fundamental function of eukaryotic cells and is well conserved from yeast to humans. The most remarkable feature of autophagy is the synthesis of double membrane-bound compartments that sequester materials to be degraded in lytic compartments, a process that seems to be mechanistically distinct from conventional membrane traffic. The discovery of autophagy in yeast and the genetic tractability of this organism have allowed us to identify genes that are responsible for this process, which has led to the explosive growth of this research field seen today. Analyses of autophagy-related (Atg) proteins have unveiled dynamic and diverse aspects of mechanisms that underlie membrane formation during autophagy.
Assuntos
Autofagia , Saccharomyces cerevisiae/citologia , Animais , Proteínas Relacionadas à Autofagia , Humanos , Fagossomos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Quinases/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismoRESUMO
Macroautophagy (hereafter referred to as autophagy) degrades various intracellular constituents to regulate a wide range of cellular functions, and is also closely linked to several human diseases. In selective autophagy, receptor proteins recognize degradation targets and direct their sequestration by double-membrane vesicles called autophagosomes, which transport them into lysosomes or vacuoles. Although recent studies have shown that selective autophagy is involved in quality/quantity control of some organelles, including mitochondria and peroxisomes, it remains unclear how extensively it contributes to cellular organelle homeostasis. Here we describe selective autophagy of the endoplasmic reticulum (ER) and nucleus in the yeast Saccharomyces cerevisiae. We identify two novel proteins, Atg39 and Atg40, as receptors specific to these pathways. Atg39 localizes to the perinuclear ER (or the nuclear envelope) and induces autophagic sequestration of part of the nucleus. Atg40 is enriched in the cortical and cytoplasmic ER, and loads these ER subdomains into autophagosomes. Atg39-dependent autophagy of the perinuclear ER/nucleus is required for cell survival under nitrogen-deprivation conditions. Atg40 is probably the functional counterpart of FAM134B, an autophagy receptor for the ER in mammals that has been implicated in sensory neuropathy. Our results provide fundamental insight into the pathophysiological roles and mechanisms of 'ER-phagy' and 'nucleophagy' in other organisms.
Assuntos
Autofagia , Núcleo Celular/metabolismo , Retículo Endoplasmático/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Família da Proteína 8 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Viabilidade Microbiana , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Nitrogênio/deficiência , Nitrogênio/metabolismo , Membrana Nuclear/metabolismo , Fenótipo , Ligação Proteica , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/deficiência , Receptores Citoplasmáticos e Nucleares/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
The biogenesis of double-membrane vesicles called autophagosomes, which sequester and transport intracellular material for degradation in lysosomes or vacuoles, is a central event in autophagy. This process requires a unique set of factors called autophagy-related (Atg) proteins. The Atg proteins assemble to organize the preautophagosomal structure (PAS), at which a cup-shaped membrane, the isolation membrane (or phagophore), forms and expands to become the autophagosome. The molecular mechanism of autophagosome biogenesis remains poorly understood. Previous studies have shown that Atg2 forms a complex with the phosphatidylinositol 3-phosphate (PI3P)-binding protein Atg18 and localizes to the PAS to initiate autophagosome biogenesis; however, the molecular function of Atg2 remains unknown. In this study, we show that Atg2 has two membrane-binding domains in the N- and C-terminal regions and acts as a membrane tether during autophagosome formation in the budding yeast Saccharomyces cerevisiae An amphipathic helix in the C-terminal region binds to membranes and facilitates Atg18 binding to PI3P to target the Atg2-Atg18 complex to the PAS. The N-terminal region of Atg2 is also involved in the membrane binding of this protein but is dispensable for the PAS targeting of the Atg2-Atg18 complex. Our data suggest that this region associates with the endoplasmic reticulum (ER) and is responsible for the formation of the isolation membrane at the PAS. Based on these results, we propose that the Atg2-Atg18 complex tethers the PAS to the ER to initiate membrane expansion during autophagosome formation.
Assuntos
Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Autofagossomos/química , Proteínas Relacionadas à Autofagia/genética , Membranas Intracelulares/metabolismo , Proteínas de Membrana/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Domínios Proteicos , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Autophagy is a conserved intracellular degradation system in eukaryotes. Recent studies have revealed that autophagy can be induced not only by nitrogen starvation but also by many other stimuli. However, questions persist regarding the types of conditions that induce autophagy, as well as the particular kinds of autophagy that are induced under these specific conditions. In experimental studies, abrupt nutrient changes are often used to induce autophagy. In this study, we investigated autophagy induction in batch culture on low-glucose medium, in which growth of yeast (Saccharomyces cerevisiae) cells is clearly reflected exclusively by carbon source state. In this medium, cells pass sequentially through three stages: glucose-utilizing, ethanol-utilizing, and ethanol-depleted phases. Using GFP cleavage assay by immunoblotting methods, fluorescence microscopy, and transmission electron microscopy ultrastructural analysis, we found that bulk autophagy and endoplasmic reticulum-phagy are induced starting at the ethanol-utilizing phase, and bulk autophagy is activated to a greater extent in the ethanol-depleted phase. Furthermore, we found that mitophagy is induced by ethanol depletion. Microautophagy occurred after glucose depletion and involved incorporation of cytosolic components and lipid droplets into the vacuolar lumen. Moreover, we observed that autophagy-deficient cells grow more slowly in the ethanol-utilizing phase and exhibit a delay in growth resumption when they are shifted to fresh medium from the ethanol-depleted phase. Our findings suggest that distinct types of autophagy are induced in yeast cells undergoing gradual changes in carbon source availability.
Assuntos
Autofagia/fisiologia , Carbono/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Gotículas Lipídicas/metabolismo , Saccharomyces cerevisiae/genética , Vacúolos/metabolismoRESUMO
Autophagy is a catabolic process conserved among eukaryotes. Under nutrient starvation, a portion of the cytoplasm is non-selectively sequestered into autophagosomes. Consequently, ribosomes are delivered to the vacuole/lysosome for destruction, but the precise mechanism of autophagic RNA degradation and its physiological implications for cellular metabolism remain unknown. We characterized autophagy-dependent RNA catabolism using a combination of metabolome and molecular biological analyses in yeast. RNA delivered to the vacuole was processed by Rny1, a T2-type ribonuclease, generating 3'-NMPs that were immediately converted to nucleosides by the vacuolar non-specific phosphatase Pho8. In the cytoplasm, these nucleosides were broken down by the nucleosidases Pnp1 and Urh1. Most of the resultant bases were not re-assimilated, but excreted from the cell. Bulk non-selective autophagy causes drastic perturbation of metabolism, which must be minimized to maintain intracellular homeostasis.
Assuntos
Autofagia , Nitrogênio/metabolismo , Estabilidade de RNA , Saccharomyces cerevisiae/metabolismo , Inanição , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Proteínas Relacionadas à Autofagia , Western Blotting , Cromatografia Líquida , Endopeptidases/genética , Endopeptidases/metabolismo , Espectrometria de Massas , Metaboloma , Microscopia de Fluorescência , Ribonucleases/genética , Ribonucleases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismoRESUMO
Degradation of mitochondria via selective autophagy, termed mitophagy, contributes to mitochondrial quality and quantity control whose defects have been implicated in oxidative phosphorylation deficiency, aberrant cell differentiation, and neurodegeneration. How mitophagy is regulated in response to cellular physiology remains obscure. Here, we show that mitophagy in yeast is linked to the phospholipid biosynthesis pathway for conversion of phosphatidylethanolamine to phosphatidylcholine by the two methyltransferases Cho2 and Opi3. Under mitophagy-inducing conditions, cells lacking Opi3 exhibit retardation of Cho2 repression that causes an anomalous increase in glutathione levels, leading to suppression of Atg32, a mitochondria-anchored protein essential for mitophagy. In addition, loss of Opi3 results in accumulation of phosphatidylmonomethylethanolamine (PMME) and, surprisingly, generation of Atg8-PMME, a mitophagy-incompetent lipid conjugate of the autophagy-related ubiquitin-like modifier. Amelioration of Atg32 expression and attenuation of Atg8-PMME conjugation markedly rescue mitophagy in opi3-null cells. We propose that proper regulation of phospholipid methylation is crucial for Atg32-mediated mitophagy.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Mitofagia , Fosfolipídeos/metabolismo , Receptores Citoplasmáticos e Nucleares/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Família da Proteína 8 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Regulação Fúngica da Expressão Gênica , Humanos , Metilação , Mitocôndrias/metabolismo , Transporte Proteico , Saccharomyces cerevisiae/genética , Ativação TranscricionalRESUMO
E1 enzymes activate ubiquitin-like proteins and transfer them to cognate E2 enzymes. Atg7, a noncanonical E1, activates two ubiquitin-like proteins, Atg8 and Atg12, and plays a crucial role in autophagy. Here, we report crystal structures of full-length Atg7 and its C-terminal domain bound to Atg8 and MgATP, as well as a solution structure of Atg8 bound to the extreme C-terminal domain (ECTD) of Atg7. The unique N-terminal domain (NTD) of Atg7 is responsible for Atg3 (E2) binding, whereas its C-terminal domain is comprised of a homodimeric adenylation domain (AD) and ECTD. The structural and biochemical data demonstrate that Atg8 is initially recognized by the C-terminal tail of ECTD and is then transferred to an AD, where the Atg8 C terminus is attacked by the catalytic cysteine to form a thioester bond. Atg8 is then transferred via a trans mechanism to the Atg3 bound to the NTD of the opposite protomer within a dimer.
Assuntos
Proteínas Associadas aos Microtúbulos/química , Proteínas de Saccharomyces cerevisiae/química , Trifosfato de Adenosina/metabolismo , Proteína 7 Relacionada à Autofagia , Família da Proteína 8 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Sítios de Ligação , Cristalografia por Raios X , Ativação Enzimática , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Moleculares , Complexos Multienzimáticos , Mutação , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Multimerização Proteica , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-Atividade , Fatores de Tempo , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
Autophagy is a conserved process in which cytoplasmic components are sequestered for degradation in the vacuole/lysosomes in eukaryotic cells. Autophagy is induced under a variety of starvation conditions, such as the depletion of nitrogen, carbon, phosphorus, zinc, and others. However, apart from nitrogen starvation, it remains unclear how these stimuli induce autophagy. In yeast, for example, it remains contentious whether autophagy is induced under carbon starvation conditions, with reports variously suggesting both induction and lack of induction upon depletion of carbon. We therefore undertook an analysis to account for these inconsistencies, concluding that autophagy is induced in response to abrupt carbon starvation when cells are grown with glycerol but not glucose as the carbon source. We found that autophagy under these conditions is mediated by nonselective degradation that is highly dependent on the autophagosome-associated scaffold proteins Atg11 and Atg17. We also found that the extent of carbon starvation-induced autophagy is positively correlated with cells' oxygen consumption rate, drawing a link between autophagy induction and respiratory metabolism. Further biochemical analyses indicated that maintenance of intracellular ATP levels is also required for carbon starvation-induced autophagy and that autophagy plays an important role in cell viability during prolonged carbon starvation. Our findings suggest that carbon starvation-induced autophagy is negatively regulated by carbon catabolite repression.
Assuntos
Autofagia , Carbono/metabolismo , Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Meios de Cultura , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática , Glucose/metabolismo , Glicerol/metabolismo , Oxigênio/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
Autophagy is a bulk degradation process conserved from yeast to mammals. To examine the roles of autophagy in cellular metabolism, we generated autophagy-defective (atg) mutants in the X2180-1B strain background. We compared the growth of wild-type (WT) and atg cells in minimal (synthetic dextrose, SD) and rich (yeast extract/peptone/dextrose, YEPD) medium, and we found that mutations in the core autophagy machinery result in defects in the diauxic shift, the transition from fermentation to respiratory growth upon glucose depletion, specifically in SD. Furthermore, we confirmed that autophagy was induced prior to the diauxic shift, implying that it plays a role in this process. In YEPD, atg mutants grew normally, so we assumed that the insufficiency of certain nutrients in SD was responsible for the defects. We ultimately identified iron, which is a necessary cofactor for respiratory activity, as the nutrient required for the diauxic shift in atg mutants. Indeed, atg mutants exhibited defects in respiration, which was rescued by supplementation with iron. Based on these data, we hypothesized that autophagy is involved in iron recycling during the diauxic shift. smf3Δfet5Δ or smf3Δftr1Δ cells, which are unable to export iron from the vacuole, also exhibit defects in the diauxic shift, so iron released from the vacuole is important for the shift in SD medium. Finally, we observed that smf3Δfet5Δ cells accumulated nearly twice as much vacuolar iron as smf3Δfet5Δatg2Δ cells, suggesting that autophagy is involved in iron recycling by the vacuolar transport and degradation of iron-containing cargos.
Assuntos
Autofagia/fisiologia , Glicólise/fisiologia , Ferro/metabolismo , Consumo de Oxigênio/fisiologia , Saccharomyces cerevisiae/metabolismo , Deleção de Genes , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Zinc is an essential nutrient for all forms of life. Within cells, most zinc is bound to protein. Because zinc serves as a catalytic or structural cofactor for many proteins, cells must maintain zinc homeostasis under severely zinc-deficient conditions. In yeast, the transcription factor Zap1 controls the expression of genes required for uptake and mobilization of zinc, but to date the fate of existing zinc-binding proteins under zinc starvation remains poorly understood. Autophagy is an evolutionarily conserved cellular degradation/recycling process in which cytoplasmic proteins and organelles are sequestered for degradation in the vacuole/lysosome. In this study, we investigated how autophagy functions under zinc starvation. Zinc depletion induced non-selective autophagy, which is important for zinc-limited growth. Induction of autophagy by zinc starvation was not directly related to transcriptional activation of Zap1. Instead, TORC1 inactivation directed zinc starvation-induced autophagy. Abundant zinc proteins, such as Adh1, Fba1, and ribosomal protein Rpl37, were degraded in an autophagy-dependent manner. But the targets of autophagy were not restricted to zinc-binding proteins. When cellular zinc is severely depleted, this non-selective autophagy plays a role in releasing zinc from the degraded proteins and recycling zinc for other essential purposes.
Assuntos
Autofagia , Saccharomyces cerevisiae/metabolismo , Zinco/metabolismo , Álcool Desidrogenase/genética , Álcool Desidrogenase/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteólise , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Autophagy is an evolutionarily conserved intracellular vacuolar process. Since Christian de Duve first coined the term 'autophagy' in 1963, it had not been well understood at the molecular level until much later, due to limitations in biochemical approaches and/or morphological approaches posed by electron microscopy. An important milestone was achieved with the isolation and identification of autophagy-related (ATG) genes by genetic screening using yeast Saccharomyces cerevisiae. ATG genes are well conserved in most eukaryotic organisms, which allowed the subsequent isolation of ATG gene-knockouts in plants. From the phenotypic analyses of the autophagy-defective plants, the physiological roles of autophagy have been predicted. However, in some cases, all the phenotypes cannot be simply explained by defects in autophagy. Therefore, in order to fully understand the physiological implications of plant autophagy, it is quite important to elucidate the molecular mechanisms involved in each process in macro-/micro-autophagy. Although, until recently, our understanding of the molecular mechanisms of plant autophagy was lagging compared to similar research in yeast and animals, current studies have made many great advances in the plant research field. In this review, we discuss current knowledge of the molecular mechanisms of plant autophagy, from autophagy-induction/autophagosome-formation to vacuolar degradation, comparing these to processes in yeast and mammals. We also review aspects of plant autophagy research that require further investigation in the future.
Assuntos
Autofagossomos/fisiologia , Autofagia/fisiologia , Células Vegetais/fisiologia , Vacúolos/metabolismo , Proteínas de Plantas/metabolismoRESUMO
During autophagosome formation, autophagosome-related (Atg) proteins are recruited hierarchically to organize the preautophagosomal structure (PAS). Atg13, which plays a central role in the initial step of PAS formation, consists of two structural regions, the N-terminal HORMA (from Hop1, Rev7, and Mad2) domain and the C-terminal disordered region. The C-terminal disordered region of Atg13, which contains the binding sites for Atg1 and Atg17, is essential for the initiation step in which the Atg1 complex is formed to serve as a scaffold for the PAS. The N-terminal HORMA domain of Atg13 is also essential for autophagy, but its molecular function has not been established. In this study, we searched for interaction partners of the Atg13 HORMA domain and found that it binds Atg9, a multispanning membrane protein that exists on specific cytoplasmic vesicles (Atg9 vesicles). After the Atg1 complex is formed, Atg9 vesicles are recruited to the PAS and become part of the autophagosomal membrane. HORMA domain mutants, which are unable to interact with Atg9, impaired the PAS localization of Atg9 vesicles and exhibited severe defects in starvation-induced autophagy. Thus, Atg9 vesicles are recruited to the PAS via the interaction with the Atg13 HORMA domain. Based on these findings, we propose that the two distinct regions of Atg13 play crucial roles in distinct steps of autophagosome formation: In the first step, Atg13 forms a scaffold for the PAS via its C-terminal disordered region, and subsequently it recruits Atg9 vesicles via its N-terminal HORMA domain.