RESUMEN
The mechanisms governing autophagy of proteins and organelles have been well studied, but how other cytoplasmic components such as RNA and polysaccharides are degraded remains largely unknown. In this study, we examine autophagy of glycogen, a storage form of glucose. We find that cells accumulate glycogen in the cytoplasm during nitrogen starvation and that this carbohydrate is rarely observed within autophagosomes and autophagic bodies. However, sequestration of glycogen by autophagy is observed following prolonged nitrogen starvation. We identify a yet-uncharacterized open reading frame, Yil024c (herein Atg45), as encoding a cytosolic receptor protein that mediates autophagy of glycogen (glycophagy). Furthermore, we show that, during sporulation, Atg45 is highly expressed and is associated with an increase in glycophagy. Our results suggest that cells regulate glycophagic activity by controlling the expression level of Atg45.
RESUMEN
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.
Asunto(s)
Autofagia , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Vacuolas/metabolismo , Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Familia de las Proteínas 8 Relacionadas con la Autofagia/genética , Transporte de Proteínas , Proteómica/métodosRESUMEN
Autophagy is a lysosomal/vacuolar delivery system that degrades cytoplasmic material. During autophagy, autophagosomes deliver cellular components to the vacuole, resulting in the release of a cargo-containing autophagic body (AB) into the vacuole. AB membranes must be disrupted for degradation of cargo to occur. The lipase Atg15 and vacuolar proteases Pep4 and Prb1 are known to be necessary for this disruption and cargo degradation, but the mechanistic underpinnings remain unclear. In this study, we establish a system to detect lipase activity in the vacuole and show that Atg15 is the sole vacuolar phospholipase. Pep4 and Prb1 are required for the activation of Atg15 lipase function, which occurs following delivery of Atg15 to the vacuole by the MVB pathway. In vitro experiments reveal that Atg15 is a phospholipase B of broad substrate specificity that is likely implicated in the disruption of a range of membranes. Further, we use isolated ABs to demonstrate that Atg15 alone is able to disrupt AB membranes.
Asunto(s)
Autofagosomas , Proteínas Relacionadas con la Autofagia , Autofagia , Fosfolipasas , Vacuolas , Lipasa , Membrana CelularRESUMEN
In autophagy, a membrane cisterna called the isolation membrane expands, bends, becomes spherical, and closes to sequester cytoplasmic constituents into the resulting double-membrane vesicle autophagosome for lysosomal/vacuolar degradation. Here, we discover a mechanism that allows the isolation membrane to expand with a large opening to ensure non-selective cytoplasm sequestration within the autophagosome. A sorting nexin complex that localizes to the opening edge of the isolation membrane plays a critical role in this process. Without the complex, the isolation membrane expands with a small opening that prevents the entry of particles larger than about 25 nm, including ribosomes and proteasomes, although autophagosomes of nearly normal size eventually form. This study sheds light on membrane morphogenesis during autophagosome formation and selectivity in autophagic degradation.
Asunto(s)
Autofagosomas , Autofagia , Citosol , Macroautofagia , RibosomasRESUMEN
Although light is essential for photosynthesis, it has the potential to elevate intracellular levels of reactive oxygen species (ROS). Since high ROS levels are cytotoxic, plants must alleviate such damage. However, the cellular mechanism underlying ROS-induced leaf damage alleviation in peroxisomes was not fully explored. Here, we show that autophagy plays a pivotal role in the selective removal of ROS-generating peroxisomes, which protects plants from oxidative damage during photosynthesis. We present evidence that autophagy-deficient mutants show light intensity-dependent leaf damage and excess aggregation of ROS-accumulating peroxisomes. The peroxisome aggregates are specifically engulfed by pre-autophagosomal structures and vacuolar membranes in both leaf cells and isolated vacuoles, but they are not degraded in mutants. ATG18a-GFP and GFP-2×FYVE, which bind to phosphatidylinositol 3-phosphate, preferentially target the peroxisomal membranes and pre-autophagosomal structures near peroxisomes in ROS-accumulating cells under high-intensity light. Our findings provide deeper insights into the plant stress response caused by light irradiation.
Asunto(s)
Macroautofagia , Peroxisomas , Especies Reactivas de Oxígeno/metabolismo , Peroxisomas/metabolismo , Autofagia/fisiología , Hojas de la Planta/metabolismoRESUMEN
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.
Asunto(s)
Autofagosomas , Saccharomyces cerevisiae , Autofagosomas/metabolismo , Autofagia , Fagosomas/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Vacuolas/enzimología , Vacuolas/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Vacuolas , Ciclo Celular/genética , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción , Vacuolas/metabolismoRESUMEN
Autophagosome biogenesis is an essential feature of autophagy. Lipidation of Atg8 plays a critical role in this process. Previous in vitro studies identified membrane tethering and hemi-fusion/fusion activities of Atg8, yet definitive roles in autophagosome biogenesis remained controversial. Here, we studied the effect of Atg8 lipidation on membrane structure. Lipidation of Saccharomyces cerevisiae Atg8 on nonspherical giant vesicles induced dramatic vesicle deformation into a sphere with an out-bud. Solution NMR spectroscopy of Atg8 lipidated on nanodiscs identified two aromatic membrane-facing residues that mediate membrane-area expansion and fragmentation of giant vesicles in vitro. These residues also contribute to the in vivo maintenance of fragmented vacuolar morphology under stress in fission yeast, a moonlighting function of Atg8. Furthermore, these aromatic residues are crucial for the formation of a sufficient number of autophagosomes and regulate autophagosome size. Together, these data demonstrate that Atg8 can cause membrane perturbations that underlie efficient autophagosome biogenesis.
Asunto(s)
Autofagosomas/metabolismo , Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Autofagia/fisiología , Membrana Celular/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Familia de las Proteínas 8 Relacionadas con la Autofagia/química , Familia de las Proteínas 8 Relacionadas con la Autofagia/genética , Nanoestructuras , Resonancia Magnética Nuclear Biomolecular , Fosfatidiletanolaminas/química , Conformación Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Vacuolas/metabolismoRESUMEN
Synthesis and degradation of cellular constituents must be balanced to maintain cellular homeostasis, especially during adaptation to environmental stress. The role of autophagy in the degradation of proteins and organelles is well-characterized. However, autophagy-mediated RNA degradation in response to stress and the potential preference of specific RNAs to undergo autophagy-mediated degradation have not been examined. In this study, we demonstrate selective mRNA degradation by rapamycin-induced autophagy in yeast. Profiling of mRNAs from the vacuole reveals that subsets of mRNAs, such as those encoding amino acid biosynthesis and ribosomal proteins, are preferentially delivered to the vacuole by autophagy for degradation. We also reveal that autophagy-mediated mRNA degradation is tightly coupled with translation by ribosomes. Genome-wide ribosome profiling suggested a high correspondence between ribosome association and targeting to the vacuole. We propose that autophagy-mediated mRNA degradation is a unique and previously-unappreciated function of autophagy that affords post-transcriptional gene regulation.
Asunto(s)
Autofagia/genética , Estabilidad del ARN/genética , Ribosomas/genética , Saccharomyces cerevisiae/genética , Vacuolas/genética , Northern Blotting , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , RNA-Seq/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacuolas/metabolismoRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
The molecular function of Atg9, the sole transmembrane protein in the autophagosome-forming machinery, remains unknown. Atg9 colocalizes with Atg2 at the expanding edge of the isolation membrane (IM), where Atg2 receives phospholipids from the endoplasmic reticulum (ER). Here we report that yeast and human Atg9 are lipid scramblases that translocate phospholipids between outer and inner leaflets of liposomes in vitro. Cryo-EM of fission yeast Atg9 reveals a homotrimer, with two connected pores forming a path between the two membrane leaflets: one pore, located at a protomer, opens laterally to the cytoplasmic leaflet; the other, at the trimer center, traverses the membrane vertically. Mutation of residues lining the pores impaired IM expansion and autophagy activity in yeast and abolished Atg9's ability to transport phospholipids between liposome leaflets. These results suggest that phospholipids delivered by Atg2 are translocated from the cytoplasmic to the luminal leaflet by Atg9, thereby driving autophagosomal membrane expansion.
Asunto(s)
Autofagosomas/química , Proteínas Relacionadas con la Autofagia/química , Proteínas de la Membrana/química , Fosfolípidos/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Proteínas de Transporte Vesicular/química , Autofagosomas/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Sitios de Unión , Transporte Biológico , Microscopía por Crioelectrón , Expresión Génica , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Fosfolípidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteolípidos/química , Proteolípidos/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteína Fluorescente RojaRESUMEN
The mechanism and function of autophagy as a highly-conserved bulk degradation pathway are well studied, but the physiological role of autophagy remains poorly understood. We show that autophagy is involved in the adaptation of Saccharomyces cerevisiae to respiratory growth through its recycling of serine. On respiratory media, growth onset, mitochondrial initiator tRNA modification and mitochondrial protein expression are delayed in autophagy defective cells, suggesting that mitochondrial one-carbon metabolism is perturbed in these cells. The supplementation of serine, which is a key one-carbon metabolite, is able to restore mitochondrial protein expression and alleviate delayed respiratory growth. These results indicate that autophagy-derived serine feeds into mitochondrial one-carbon metabolism, supporting the initiation of mitochondrial protein synthesis and allowing rapid adaptation to respiratory growth.
Asunto(s)
Adaptación Fisiológica , Autofagia/fisiología , Proteínas Mitocondriales/biosíntesis , Saccharomyces cerevisiae/fisiología , Carbono/metabolismo , Respiración de la Célula/fisiología , Mitocondrias/metabolismo , Biosíntesis de Proteínas/fisiología , ARN de Transferencia/metabolismo , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina/metabolismoRESUMEN
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.
Asunto(s)
Autofagia , Proteína Quinasa CDC2 , Diana Mecanicista del Complejo 1 de la Rapamicina , Mitosis , Fosforilación , Serina-Treonina Quinasas TORRESUMEN
The mechanisms underlying turnover of the nuclear pore complex (NPC) and the component nucleoporins (Nups) are still poorly understood. In this study, we found that the budding yeast Saccharomyces cerevisiae triggers NPC degradation by autophagy upon the inactivation of Tor kinase complex 1. This degradation largely depends on the selective autophagy-specific factor Atg11 and the autophagy receptor-binding ability of Atg8, suggesting that the NPC is degraded via receptor-dependent selective autophagy. Immunoelectron microscopy revealed that NPCs embedded in nuclear envelope-derived double-membrane vesicles are sequestered within autophagosomes. At least two pathways are involved in NPC degradation: Atg39-dependent nucleophagy (selective autophagy of the nucleus) and a pathway involving an unknown receptor. In addition, we found the interaction between Nup159 and Atg8 via the Atg8-family interacting motif is important for degradation of this nucleoporin not assembled into the NPC. Thus, this study provides the first evidence for autophagic degradation of the NPC and Nups, which we term "NPC-phagy" and "nucleoporinophagy."
Asunto(s)
Familia de las Proteínas 8 Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/genética , Autofagia/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Proteínas de Complejo Poro Nuclear/genética , Poro Nuclear/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/genética , Autofagosomas/efectos de los fármacos , Autofagosomas/metabolismo , Autofagia/efectos de los fármacos , Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Regulación Fúngica de la Expresión Génica , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Microscopía Inmunoelectrónica , Poro Nuclear/efectos de los fármacos , Poro Nuclear/ultraestructura , Proteínas de Complejo Poro Nuclear/metabolismo , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Proteolisis/efectos de los fármacos , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Sirolimus/farmacología , Proteínas de Transporte Vesicular/metabolismoRESUMEN
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.
Asunto(s)
Autofagosomas/química , Autofagosomas/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Autofagia , Proteínas Relacionadas con la Autofagia/química , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/química , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Fosforilación , Mutación Puntual , Unión Proteica , Proteínas Quinasas/química , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Vacuolas/metabolismoRESUMEN
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.
Asunto(s)
Arabidopsis/metabolismo , Autofagia/fisiología , Especies Reactivas de Oxígeno/metabolismo , Zinc/deficiencia , Zinc/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cloroplastos/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
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.
Asunto(s)
Aminopeptidasas/metabolismo , Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Autofagia , Receptores de Superficie Celular/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacuolas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Aminopeptidasas/genética , Familia de las Proteínas 8 Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/genética , Citoplasma/metabolismo , Mutación , Unión Proteica , Transporte de Proteínas , Receptores de Superficie Celular/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Solubilidad , Proteínas de Transporte Vesicular/genéticaRESUMEN
A key event in autophagy is autophagosome formation, whereby the newly synthesized isolation membrane (IM) expands to form a complete autophagosome using endomembrane-derived lipids. Atg2 physically links the edge of the expanding IM with the endoplasmic reticulum (ER), a role that is essential for autophagosome formation. However, the molecular function of Atg2 during ER-IM contact remains unclear, as does the mechanism of lipid delivery to the IM. Here we show that the conserved amino-terminal region of Schizosaccharomyces pombe Atg2 includes a lipid-transfer-protein-like hydrophobic cavity that accommodates phospholipid acyl chains. Atg2 bridges highly curved liposomes, thereby facilitating efficient phospholipid transfer in vitro, a function that is inhibited by mutations that impair autophagosome formation in vivo. These results suggest that Atg2 acts as a lipid-transfer protein that supplies phospholipids for autophagosome formation.
Asunto(s)
Autofagosomas/metabolismo , Retículo Endoplásmico/metabolismo , Schizosaccharomyces/metabolismo , Autofagia/fisiología , Proteínas Relacionadas con la Autofagia/metabolismo , Proteínas Portadoras/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Liposomas/metabolismo , Fosfolípidos/metabolismoRESUMEN
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.