RESUMEN
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
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
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.
Asunto(s)
Autofagia , Saccharomyces cerevisiae/citología , Animales , Proteínas Relacionadas con la Autofagia , Humanos , Fagosomas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Quinasas/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismoRESUMEN
Macroautophagy (autophagy) is a highly conserved cellular recycling process involved in degradation of eukaryotic cellular components. During autophagy, macromolecules and organelles are sequestered into the double-membrane autophagosome and degraded in the vacuole/lysosome. Autophagy-related 8 (Atg8), a core Atg protein essential for autophagosome formation, is a marker of several autophagic structures: the pre-autophagosomal structure (PAS), isolation membrane (IM), and autophagosome. Atg8 is conjugated to phosphatidylethanolamine (PE) through a ubiquitin-like conjugation system to yield Atg8-PE; this reaction is called Atg8 lipidation. Although the mechanisms of Atg8 lipidation have been well studied in vitro, the cellular locale of Atg8 lipidation remains enigmatic. Atg3 is an E2-like enzyme that catalyzes the conjugation reaction between Atg8 and PE. Therefore, we hypothesized that the localization of Atg3 would provide insights about the site of the lipidation reaction. To explore this idea, we constructed functional GFP-tagged Atg3 (Atg3-GFP) by inserting the GFP portion immediately after the handle region of Atg3. During autophagy, Atg3-GFP transiently formed a single dot per cell on the vacuolar membrane. This Atg3-GFP dot colocalized with 2× mCherry-tagged Atg8, demonstrating that Atg3 is localized to autophagic structures. Furthermore, we found that Atg3-GFP is localized to the IM by fine-localization analysis. The localization of Atg3 suggests that Atg3 plays an important role in autophagosome formation at the IM.
Asunto(s)
Autofagia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Enzimas Ubiquitina-Conjugadoras/metabolismo , Proteínas Relacionadas con la Autofagia , Proteínas Fluorescentes Verdes/metabolismo , Membranas Intracelulares/enzimología , Microscopía Fluorescente , Fagosomas/enzimología , Fosfatidiletanolaminas , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Vacuolas/enzimologíaRESUMEN
Autophagy is a bulk degradation system mediated by biogenesis of autophagosomes under starvation conditions. In Saccharomyces cerevisiae, a membrane sac called the isolation membrane (IM) is generated from the pre-autophagosomal structure (PAS); ultimately, the IM expands to become a mature autophagosome. Eighteen autophagy-related (Atg) proteins are engaged in autophagosome formation at the PAS. However, the cup-shaped IM was visualized just as a dot by fluorescence microscopy, posing a challenge to further understanding the detailed functions of Atg proteins during IM expansion. In this study, we visualized expanding IMs as cup-shaped structures using fluorescence microscopy by enlarging a selective cargo of autophagosomes, and finely mapped the localizations of Atg proteins. The PAS scaffold proteins (Atg13 and Atg17) and phosphatidylinositol 3-kinase complex I were localized to a position at the junction between the IM and the vacuolar membrane, termed the vacuole-IM contact site (VICS). By contrast, Atg1, Atg8 and the Atg16-Atg12-Atg5 complex were present at both the VICS and the cup-shaped IM. We designate this localization the 'IM' pattern. The Atg2-Atg18 complex and Atg9 localized to the edge of the IM, appearing as two or three dots, in close proximity to the endoplasmic reticulum exit sites. Thus, we designate these dots as the 'IM edge' pattern. These data suggest that Atg proteins play individual roles at spatially distinct locations during IM expansion. These findings will facilitate detailed investigations of the function of each Atg protein during autophagosome formation.
Asunto(s)
Autofagia/fisiología , Fagosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fagosomas/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Macroautophagy/autophagy is the process by which cells degrade their cytoplasmic proteins or organelles in vacuoles to maintain cellular homeostasis under severe environmental conditions. In the yeast Saccharomyces cerevisiae, autophagy-related (Atg) proteins essential for autophagosome formation accumulate near the vacuole to form the dot-shaped phagophore assembly site/pre-autophagosomal structure (PAS). The PAS then generates the phagophore/isolation membrane (PG), which expands to become a closed double-membrane autophagosome. Hereinafter, we refer to the PAS, PG, and autophagosome as autophagy-related structures (ARSs). During autophagosome formation, Atg2 is responsible for tethering the ARS to the endoplasmic reticulum (ER) via ER exit sites (ERESs), and for transferring phospholipids from the ER to ARSs. Therefore, ARS and the ER are spatially close in the presence of Atg2 but are separated in its absence. Because the contact of an ARS with the ER must be established at the earliest stage of autophagosome formation, it is important to know whether the ARS is tethered to the ER. In this study, we developed a rapid and objective method to estimate tethering of the ARS to the ER by measuring the distance between the ARS and ERES under fluorescence microscopy, and found that tethering of the ARS to the ER was lost without Atg1. This method might be useful to predict the tethering activity of Atg2.Abbreviation: ARS, autophagy-related structure; Dautas, automated measurement of the distance between autophagy-related structures and ER exit sites analysis system; ERES, endoplasmic reticulum exit site; PAS, phagophore assembly site/pre-autophagosomal structure; PCR, polymerase chain reaction; PG, phagophore/isolation membrane; prApe1, precursor of vacuolar aminopeptidase I; Qautas, quantitative autophagy-related structure analysis system; SD/CA; synthetic dextrose plus casamino acid medium; WT, wild-type.
Asunto(s)
Autofagosomas , Autofagia , Retículo Endoplásmico , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Retículo Endoplásmico/metabolismo , Autofagia/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Autofagosomas/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Vacuolas/metabolismoRESUMEN
The disintegration of cytoplasm-to-vacuole targeting (Cvt) bodies and autophagic bodies in vacuoles is essential to the Cvt pathway and macroautophagy in yeast. Atg15 is a vacuolar lipase required for the degradation of both Cvt and autophagic bodies. However, the molecular mechanism of their degradation by Atg15 remains poorly understood. In a recent study, we showed that recombinant Chaetomium thermophilum Atg15 (CtAtg15) possesses phospholipase activity, and that this activity is significantly elevated by proteolytic cleavage at a site away from the active center. The proteolytic cleavage of CtAtg15 causes a conformational change around the active center, resulting in the active open state. Interestingly, activated CtAtg15 can degrade not only Cvt and autophagic bodies but also organelle membranes. On the basis of these results, we propose an activation mechanism by which Atg15, as an "organellase," functions only in vacuoles.
Asunto(s)
Autofagia , Vacuolas , Vacuolas/metabolismo , Autofagia/fisiología , Fosfolipasas/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Chaetomium/metabolismo , Membranas Intracelulares/metabolismoRESUMEN
Aminopeptidase I (Ape1) is one of the major cargoes of the cytoplasm-to-vacuole targeting (Cvt) pathway, which is a kind of selective autophagy, in Saccharomyces cerevisiae. After synthesis, the Ape1 precursor (prApe1) undergoes phase separation to form liquid droplets, termed Ape1 droplets, in the cytoplasm. In this study, we found that cells expressing prApe1-GFP exhibited temperature-sensitive formation of Ape1 droplets, which affected its transport. Moreover, we showed that endogenous Ape1 transport was defective at high temperatures in various laboratory strains due to the defect in the formation of Ape1 droplets at these temperatures. Finally, we found that gene disruptants showing heat-tolerant growth suppressed the temperature sensitivity of the Ape1 transport. The formation of Ape1 droplets might be an indicator of cytoplasmic integrity at high temperature.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Vacuolas , Vacuolas/metabolismo , Saccharomyces cerevisiae/metabolismo , Temperatura , Autofagia , Citoplasma/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Aminopeptidasas/genética , Aminopeptidasas/metabolismoRESUMEN
Atg15 (autophagy-related 15) is a vacuolar phospholipase essential for the degradation of cytoplasm-to-vacuole targeting (Cvt) bodies and autophagic bodies, hereinafter referred to as intravacuolar/intralysosomal autophagic compartments (IACs), but it remains unknown if Atg15 directly disrupts IAC membranes. Here, we show that the recombinant Chaetomium thermophilum Atg15 lipase domain (CtAtg15(73-475)) possesses phospholipase activity. The activity of CtAtg15(73-475) was markedly elevated by limited digestion. We inserted the human rhinovirus 3C protease recognition sequence and found that cleavage between S159 and V160 was important to activate CtAtg15(73-475). Our molecular dynamics simulation suggested that the cleavage facilitated conformational change around the active center of CtAtg15, resulting in an exposed state. We confirmed that CtAtg15 could disintegrate S. cerevisiae IAC in vivo. Further, both mitochondria and IAC of S. cerevisiae were disintegrated by CtAtg15. This study suggests Atg15 plays a role in disrupting any organelle membranes delivered to vacuoles by autophagy.
Asunto(s)
Proteínas Fúngicas , Membranas Intracelulares , Fosfolipasas , Chaetomium/enzimología , Chaetomium/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fosfolipasas/química , Fosfolipasas/genética , Fosfolipasas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Dominios Proteicos , Simulación de Dinámica Molecular , Mitocondrias/metabolismo , Membranas Intracelulares/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Estructura Terciaria de Proteína , Modelos Moleculares , Activación EnzimáticaRESUMEN
In Saccharomyces cerevisiae, aminopeptidase I (Ape1p) and α-mannosidase (Ams1p) are known cargoes of selective autophagy. Atg19p has been identified as an Ape1p receptor and targets Ape1p to the preautophagosomal structure (PAS). Under nutrient-rich conditions, transport of Ams1p to the vacuole largely depends on Atg19p. Here, we show that Atg34p (Yol083wp), a homolog of Atg19p, is a receptor for Ams1p transport during autophagy. Atg34p interacted with Ams1p, Atg11p, and Atg8p using distinct domains. Homo-oligomerized Ams1p bound to the Ams1-binding domain of Atg34p; this binding was important for the formation of a higher order complex named the Ams1 complex. In the absence of the interaction of Atg34p with Atg8p, the Ams1 complex was targeted to the preautophagosomal structure but failed to transit to the vacuole, indicating that the interaction of Atg34p with Atg8p is crucial for the Ams1 complex to be enclosed by autophagosomes. Atg34p and Atg19p have similar domain structures and are important for Ams1p transport during autophagy.
Asunto(s)
Autofagia/fisiología , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , alfa-Manosidasa/metabolismo , Aminopeptidasas/genética , Aminopeptidasas/metabolismo , Familia de las Proteínas 8 Relacionadas con la Autofagia , Proteínas Relacionadas con la Autofagia , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Multimerización de Proteína/fisiología , Estructura Terciaria de Proteína , Transporte de Proteínas/fisiología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , alfa-Manosidasa/genéticaRESUMEN
In the yeast Saccharomyces cerevisiae, a precursor form of aminopeptidase I (prApe1) and α-mannosidase (Ams1) are selectively transported to the vacuole through the cytoplasm-to-vacuole targeting pathway under vegetative conditions and through autophagy under starvation conditions. Atg19 plays a central role in these processes by linking Ams1 and prApe1 to Atg8 and Atg11. However, little is known about the molecular mechanisms of cargo recognition by Atg19. Here, we report structural and functional analyses of Atg19 and its paralog, Atg34. A protease-resistant domain was identified in the C-terminal region of Atg19, which was also conserved in Atg34. In vitro pulldown assays showed that the C-terminal domains of both Atg19 and Atg34 are responsible for Ams1 binding; these domains are hereafter referred to as Ams1-binding domains (ABDs). The transport of Ams1, but not prApe1, was blocked in atg19Δatg34Δ cells expressing Atg19(ΔABD), indicating that ABD is specifically required for Ams1 transport. We then determined the solution structures of the ABDs of Atg19 and Atg34 using NMR spectroscopy. Both ABD structures have a canonical immunoglobulin fold consisting of eight ß-strands with highly conserved loops clustered at one side of the fold. These facts, together with the results of a mutational analysis, suggest that ABD recognizes Ams1 using these conserved loops.
Asunto(s)
Autofagia/fisiología , Receptores de Superficie Celular/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacuolas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , alfa-Manosidasa/metabolismo , Aminopeptidasas/química , Aminopeptidasas/genética , Aminopeptidasas/metabolismo , Familia de las Proteínas 8 Relacionadas con la Autofagia , Proteínas Relacionadas con la Autofagia , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Transporte de Proteínas/fisiología , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Vacuolas/química , Vacuolas/genética , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética , alfa-Manosidasa/química , alfa-Manosidasa/genéticaRESUMEN
Autophagy is a cellular degradation system widely conserved among eukaryotes. During autophagy, cytoplasmic materials fated for degradation are compartmentalized in double membrane-bound organelles called autophagosomes. After fusing with the vacuole, their inner membrane-bound structures are released into the vacuolar lumen to become autophagic bodies and eventually degraded by vacuolar hydrolases. Atg15 is a lipase that is essential for disintegration of autophagic body membranes and has a transmembrane domain at the N-terminus and a lipase domain at the C-terminus. However, the roles of the two domains in vivo are not well understood. In this study, we found that the N-terminal domain alone can travel to the vacuole via the multivesicular body pathway, and that targeting of the C-terminal lipase domain to the vacuole is required for degradation of autophagic bodies. Moreover, we found that the C-terminal domain could disintegrate autophagic bodies when it was transported to the vacuole via the Pho8 pathway instead of the multivesicular body pathway. Finally, we identified H435 as one of the residues composing the putative catalytic triad and W466 as an important residue for degradation of autophagic bodies. This study may provide a clue to how the C-terminal lipase domain recognizes autophagic bodies to degrade them.
Asunto(s)
Proteínas Relacionadas con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/fisiología , Hidrolasas de Éster Carboxílico/metabolismo , Hidrolasas de Éster Carboxílico/fisiología , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Autofagosomas/metabolismo , Autofagia/genética , Proteínas Relacionadas con la Autofagia/genética , Transporte Biológico , Hidrolasas de Éster Carboxílico/genética , Citoplasma/metabolismo , Lipasa/metabolismo , Glicoproteínas de Membrana/genética , Dominios Proteicos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Vacuolas/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Autophagy is a degradation system of cytoplasmic proteins and organelles via formation of double-membrane vesicles called autophagosomes. In the yeast Saccharomyces cerevisiae, autophagosomes are formed via the pre-autophagosomal structure (PAS) in a manner dependent on Atg proteins. Under nutrient-rich condition, Atg9 is recruited to the PAS by binding to Atg11 for the Cvt pathway. However, because Atg9 is recruited to the PAS in atg11Delta cells in starved condition and autophagy is induced, autophagy-specific mechanism for the Atg9 recruitment to the PAS has been assumed. Here, we demonstrate that, in autophagy-inducing condition, Atg9 is recruited to the PAS in a manner dependent on Atg17. Atg9 physically interacts with Atg17 in the presence of rapamycin. This interaction requires Atg1, a protein kinase essential for autophagy. Consistently, the Atg17-dependent PAS localization of Atg9 requires Atg1. However, its kinase activity is dispensable for this process. It rather regulates the equilibrium of assembly and disassembly of Atg9 at the PAS.
Asunto(s)
Autofagia/fisiología , Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Fagosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Autofagia/efectos de los fármacos , Proteínas Relacionadas con la Autofagia , Proteínas Portadoras/efectos de los fármacos , Proteínas de la Membrana/efectos de los fármacos , Fagosomas/efectos de los fármacos , Unión Proteica , Proteínas Quinasas/metabolismo , Transporte de Proteínas/fisiología , Saccharomyces cerevisiae/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/efectos de los fármacos , Sirolimus/farmacologíaRESUMEN
In the yeast Saccharomyces cerevisiae, aminopeptidase I (API), a vacuolar hydrolase, is selectively transported to the vacuole via the autophagosome. API forms a cytosol to vacuole targeting (Cvt) complex in the cytoplasm. The complex is engulfed by the autophagosome under starvation conditions. In this study, the Cvt complex is visualized as a dot in the cytoplasm by fluorescence microscopy with API-GFP. The Cvt complex associates with the preautophagosomal structure (PAS), which plays a central role in autophagosome formation. In a Deltacvt19 mutant, which is specifically defective in API transport, but not in autophagy, the Cvt complex forms normally but never associates with the PAS. This indicates that Cvt19p mediates association between the Cvt complex and the PAS.
Asunto(s)
Aminopeptidasas/metabolismo , Autofagia/fisiología , Fagosomas/metabolismo , Transporte de Proteínas/fisiología , Receptores de Superficie Celular/deficiencia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacuolas/metabolismo , Proteínas de Transporte Vesicular , Autofagia/efectos de los fármacos , Proteínas Relacionadas con la Autofagia , Citosol/efectos de los fármacos , Citosol/metabolismo , Citosol/ultraestructura , Privación de Alimentos/fisiología , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes , Microscopía Fluorescente , Mutación/genética , Fagosomas/efectos de los fármacos , Fagosomas/ultraestructura , Transporte de Proteínas/efectos de los fármacos , Receptores de Superficie Celular/genética , Proteínas Recombinantes de Fusión , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Sirolimus/farmacología , Vesículas Transportadoras/efectos de los fármacos , Vesículas Transportadoras/metabolismo , Vesículas Transportadoras/ultraestructura , Vacuolas/efectos de los fármacos , Vacuolas/ultraestructuraRESUMEN
Autophagy is a primarily non-selective degradation system of cytoplasmic constituents in lysosomes/vacuoles during starvation. In yeast, autophagy is also involved in the selective transport of Ape1, a vacuolar hydrolase, as a biosynthetic route. Ald6, a soluble cytoplasmic enzyme, is preferentially eliminated from cytoplasm via autophagy. However, little is known about the mechanisms of Ald6 targeting to autophagosomes. Here, we show that Lap3, a soluble cytosolic cysteine protease, is spatially associated with Ape1 and selectively transported to the vacuole during nitrogen starvation. The rate of Lap3 transport is much higher than that of Ald6 and is similar to that of Ape1. Moreover, ATG11 and ATG19, essential factors for Ape1 transport, are important for Lap3 transport. Most Lap3 is degraded within a couple of hours in the vacuole in contrast to Ape1; therefore, we conclude that the machinery required for Ape1 biosynthesis is used for selective degradation of Lap3.
Asunto(s)
Aminopeptidasas/metabolismo , Autofagia , Cisteína Endopeptidasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Aminopeptidasas/genética , Proteínas Relacionadas con la Autofagia , Cisteína Endopeptidasas/genética , Proteasas de Cisteína , Proteínas de Unión al ADN/genética , Nitrógeno/metabolismo , Proteínas Quinasas/metabolismo , Transporte de Proteínas , Receptores de Superficie Celular/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/genética , Vacuolas/enzimología , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Nutrient starvation induces autophagy to degrade cytoplasmic materials in the vacuole/lysosomes. In the yeast, Saccharomyces cerevisiae, Atg17, Atg29, and Atg31/Cis1 are specifically required for autophagosome formation by acting as a scaffold complex essential for pre-autophagosomal structure (PAS) organization. Here, we show that these proteins constitutively form an Atg17-Atg29-Atg31 ternary complex, in which phosphorylated Atg31 is included. Reconstitution analysis of the ternary complex in E. coli indicates that the three proteins are included in equimolar amounts in the complex. The molecular mass of a monomeric Atg17-Atg29-Atg31 complex is calculated at 97kDa; however, analytical ultracentrifugation shows that the molecular mass of the ternary complex is 198kDa, suggesting a dimeric complex. We propose that this ternary complex acts as a functional unit for autophagosome formation.
Asunto(s)
Autofagia , Proteínas Portadoras/metabolismo , Fagosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Proteínas Relacionadas con la Autofagia , Proteínas Portadoras/química , Citosol/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , UltracentrifugaciónRESUMEN
Autophagy - the degradation of organelles and cytoplasmic material - occurs through dynamic rearrangements of cellular membrane structures. Following the induction of autophagy, newly formed autophagosomes transfer cytosolic materials to the lysosome or vacuole for degradation. The autophagosome is an organelle destined for degradation, suggesting that the membrane structure is formed de novo many times. The autophagosome is formed through the nucleation, assembly and elongation of membrane structures. The concerted action of several Apg/Aut/Cvt proteins around a characteristic subcellular structure (the preautophagosomal structure) is the key to understanding this novel type of membrane-formation process.
Asunto(s)
Autofagia , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Citosol/metabolismo , Retículo Endoplásmico/metabolismo , Lisosomas/metabolismo , Modelos Biológicos , Nucleosomas/metabolismo , Saccharomyces cerevisiae/fisiologíaRESUMEN
Saccharomyces cerevisiae alpha-mannosidase (Ams1) is a cargo protein that is transported to the vacuole by the cytoplasm-to-vacuole targeting (Cvt) pathway during conditions of growth and by autophagy during conditions of starvation. After transport to the vacuole, Ams1 functions as a resident hydrolase. Ams1 has been overexpressed in the methylotrophic yeast Pichia pastoris, purified and crystallized in two crystal forms. Form I belongs to space group P2(1), with unit-cell parameters a = 145.7, b = 127.7, c = 164.0 A, beta = 101.5 degrees . Form II belongs to space group I222 or I2(1)2(1)2(1), with unit-cell parameters a = 127.9, b = 163.7, c = 291.5 A. Diffraction data were collected from these crystals to a resolution of 3.3 A for form I and of 2.6 A for form II using synchrotron radiation.
Asunto(s)
Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas de Transporte Vesicular/metabolismo , alfa-Manosidasa/metabolismo , Autofagia/fisiología , Cristalización , Cristalografía por Rayos X , Recolección de Datos , Pichia/genética , Transporte de Proteínas , Rotación , Saccharomyces cerevisiae/fisiología , Estadística como Asunto , Sincrotrones , Vacuolas/metabolismo , Difracción de Rayos X , alfa-Manosidasa/aislamiento & purificaciónRESUMEN
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 degradation process accompanied by dynamic membrane organization. In the yeast, Saccharomyces cerevisiae, about 30 ATG (autophagy-related) genes have been identified as important genes for autophagy. Among them, 17 are indispensable for formation of the autophagosome, an organelle enclosed by a double lipid bilayer during starvation-induced autophagy. Recently, a central structure for autophagosome generation, termed the pre-autophagosomal structure, was identified. Despite intensive study, many questions regarding the mechanisms underlying autophagosome formation remain unanswered. In this review, we will give an overview of recent studies on the mechanisms of autophagosome formation and discuss these unresolved questions.