RESUMEN
Misfolded protein aggregation causes disease and aging; autophagy counteracts this by eliminating damaged components, enabling cells to survive starvation. The cytoplasm-to-vacuole targeting pathway in yeast encompasses the aggregation of the premature form of aminopeptidase 1 (prApe1) in cytosol and its sequestration by autophagic proteins into a vesicle for vacuolar transport. We show that the propeptide of Ape1 is important for aggregation and vesicle formation and that it is sufficient for binding to prApe1 and Atg19. Defective aggregation disrupts vacuolar transport, suggesting that aggregate shape is important in vesicle formation, whereas Atg19 binding is not sufficient for vacuolar transport. Aggregation involves hydrophobicity, whereas Atg19 binding requires additional electrostatic interactions. Ape1 dodecamerization may cluster propeptides into trimeric structures, with sufficient affinity to form propeptide hexamers by binding to other dodecamers, causing aggregation. We show that Ape1 aggregates bind Atg19 and Atg8 in vitro; this could be used as a scaffold for an in vitro assay of autophagosome formation to elucidate the mechanisms of autophagy.
Asunto(s)
Aminopeptidasas/metabolismo , Citoplasma/enzimología , Precursores Enzimáticos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Vesículas Secretoras/enzimología , Vacuolas/enzimología , Aminopeptidasas/genética , Familia de las Proteínas 8 Relacionadas con la Autofagia , Proteínas Relacionadas con la Autofagia , Citoplasma/genética , Precursores Enzimáticos/genética , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Unión Proteica , Multimerización de Proteína/fisiología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Vesículas Secretoras/genética , Vacuolas/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
When Pichia pastoris adapts from methanol to glucose growth, peroxisomes are rapidly sequestered and degraded within the vacuole by micropexophagy. During micropexophagy, sequestering membranes arise from the vacuole to engulf the peroxisomes. Fusion of the sequestering membranes and incorporation of the peroxisomes into the vacuole is mediated by the micropexophagy-specific membrane apparatus (MIPA). In this study, we show the P. pastoris ortholog of Atg9, a novel membrane protein is essential for the formation of the sequestering membranes and assembly of MIPA. During methanol growth, GFP-PpAtg9 localizes to multiple structures situated near the plasma membrane referred as the peripheral compartment (Atg9-PC). On glucose-induced micropexophagy, PpAtg9 traffics from the Atg9-PC to unique perivacuolar structures (PVS) that contain PpAtg11, but lack PpAtg2 and PpAtg8. Afterward, PpAtg9 distributes to the vacuole surface and sequestering membranes. Movement of the PpAtg9 from the Atg9-PC to the PVS requires PpAtg11 and PpVps15. PpAtg2 and PpAtg7 are essential for PpAtg9 trafficking from the PVS to the vacuole and sequestering membranes, whereas trafficking of PpAtg9 proceeds independent of PpAtg1, PpAtg18, and PpVac8. In summary, our data suggest that PpAtg9 transits from the Atg9-PC to the PVS and then to the sequestering membranes that engulf the peroxisomes for degradation.
Asunto(s)
Proteínas Fúngicas/metabolismo , Membranas Intracelulares/fisiología , Proteínas de la Membrana/metabolismo , Peroxisomas/fisiología , Pichia/fisiología , Vacuolas/fisiología , Proteínas Fúngicas/genética , Glucosa/metabolismo , Membranas Intracelulares/ultraestructura , Metanol/metabolismo , Microscopía Electrónica de Transmisión , Peroxisomas/ultraestructura , Pichia/metabolismo , Pichia/ultraestructura , Transporte de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Vacuolas/ultraestructuraRESUMEN
Delivery of proteins and organelles to the vacuole by autophagy and the cytoplasm to vacuole targeting (Cvt) pathway involves novel rearrangements of membrane resulting in the formation of vesicles that fuse with the vacuole. The mechanism of vesicle formation and the origin of the membrane are complex issues still to be resolved. Atg18 and Atg21 are proteins essential to vesicle formation and together with Ygr223c form a novel family of phosphoinositide binding proteins that are associated with the vacuole and perivacuolar structures. Their localization requires the activity of Vps34, suggesting that phosphatidylinositol(3)phosphate may be essential for their function. The activity of Atg18 is vital for all forms of autophagy, whereas Atg21 is required for the Cvt pathway but not for nitrogen starvation-induced autophagy. The loss of Atg21 results in the absence of Atg8 from the pre-autophagosomal structure (PAS), which may be ascribed to a reduced rate of conjugation of Atg8 to phosphatidylethanolamine. A similar defect in localization of a second ubiquitin-like conjugate, Atg12-Atg5, suggests that Atg21 may be involved in the recruitment of membrane to the PAS.
Asunto(s)
Aminopeptidasas/metabolismo , Autofagia , Endopeptidasas/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Fosfatidilinositoles/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Familia de las Proteínas 8 Relacionadas con la Autofagia , Proteínas Relacionadas con la Autofagia , Membrana Celular/fisiología , Endopeptidasas/genética , Endopeptidasas/metabolismo , Eliminación de Gen , Metabolismo de los Lípidos , Proteínas de la Membrana , Proteínas Asociadas a Microtúbulos/análisis , Fagosomas/química , Fagosomas/fisiología , Procesamiento Proteico-Postraduccional , Transporte de Proteínas/fisiología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/análisis , Proteínas de Saccharomyces cerevisiae/genética , Vacuolas/química , Vacuolas/fisiologíaRESUMEN
To survive extreme environmental conditions, and in response to certain developmental and pathological situations, eukaryotic organisms employ the catabolic process of autophagy. Structures targeted for destruction are enwrapped by double-membrane vesicles, then delivered into the interior of the lysosome/vacuole. Despite the identification of many specific components, the molecular mechanism that directs formation of the sequestering vesicles remains largely unknown. We analyzed the trafficking of Atg23 and the integral membrane protein Atg9 in the yeast Saccharomyces cerevisiae. These components localize both to the pre-autophagosomal structure (PAS) and other cytosolic punctate compartments. We show that Atg9 and Atg23 cycle through the PAS in a process governed by the Atg1-Atg13 signaling complex. Atg1 kinase activity is essential only for retrograde transport of Atg23, while recycling of Atg9 requires additional factors including Atg18 and Atg2. We postulate that Atg9 employs a recycling system mechanistically similar to that used at yeast early and late endosomes.
Asunto(s)
Autofagia/fisiología , Proteínas de la Membrana/metabolismo , Fagosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vesículas Transportadoras/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas Relacionadas con la Autofagia , Compartimento Celular/fisiología , Membrana Celular/metabolismo , Células Cultivadas , Endosomas/metabolismo , Lisosomas/metabolismo , Sustancias Macromoleculares , Modelos Biológicos , Fagosomas/ultraestructura , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transporte de Proteínas/fisiología , Saccharomyces cerevisiae/citología , Vesículas Transportadoras/ultraestructuraRESUMEN
The function of the yeast lysosome/vacuole is critically linked with the morphology of the organelle. Accordingly, highly regulated processes control vacuolar fission and fusion events. Analysis of homotypic vacuole fusion demonstrated that vacuoles from strains defective in the CCZ1 and MON1 genes could not fuse. Morphological evidence suggested that these mutant vacuoles could not proceed to the tethering/docking stage. Ccz1 and Mon1 form a stable protein complex that binds the vacuole membrane. In the absence of the Ccz1-Mon1 complex, the integrity of vacuole SNARE pairing and the unpaired SNARE class C Vps/HOPS complex interaction were both impaired. The Ccz1-Mon1 complex colocalized with other fusion components on the vacuole as part of the cis-SNARE complex, and the association of the Ccz1-Mon1 complex with the vacuole appeared to be regulated by the class C Vps/HOPS complex proteins. Accordingly, we propose that the Ccz1-Mon1 complex is critical for the Ypt7-dependent tethering/docking stage leading to the formation of a trans-SNARE complex and subsequent vacuole fusion.
Asunto(s)
Proteínas Portadoras/metabolismo , Factores de Intercambio de Guanina Nucleótido , Fusión de Membrana/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citología , Vacuolas/metabolismo , Proteínas de Transporte Vesicular , Transporte Biológico , Proteínas Portadoras/genética , Sustancias Macromoleculares , Proteínas de la Membrana/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas SNARE , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Glycine N-methyltransferase (GNMT) is an abundant cytosolic enzyme that catalyses the methylation of glycine into sarcosine, coupled with conversion of the methyl donor, S -adenosylmethionine (AdoMet), into S -adenosylhomocysteine (AdoHcy). GNMT is believed to play a role in monitoring the AdoMet/AdoHcy ratio, and hence the cellular methylation capacity, but regulation of the enzyme itself is not well understood. In the present study, treatment of isolated rat hepatocytes with the protein phosphatase inhibitor okadaic acid, was found to induce an overphosphorylation of GNMT, as shown by proteomic analysis. The analysis comprised two-dimensional gel electrophoretic separation of (32)P-labelled phosphoproteins and identification of individual protein spots by matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry. The identity of GNMT was verified by N-terminal Edman sequencing of tryptic peptides. Chromatographic separation of proteolytic peptides and (32)P-labelled amino acids suggested that GNMT was phosphorylated within a limited region, and only at serine residues. GNMT phosphorylation could be suppressed by naringin, an okadaic acid-antagonistic flavonoid. To assess the possible functional role of GNMT phosphorylation, the effect of okadaic acid on hepatocytic AdoMet and AdoHcy levels was examined, using HPLC separation for metabolite analysis. Surprisingly, okadaic acid was found to have no effect on the basal levels of AdoMet or AdoHcy. An accelerated AdoMet-AdoHcy flux, induced by the addition of methionine (1 mM), was likewise unaffected by okadaic acid. 5-Aminoimidazole-4-carboxamide riboside, an activator of the hepatocytic AMP-activated protein kinase, similarly induced GNMT phosphorylation without affecting AdoMet and AdoHcy levels. Activation of cAMP-dependent protein kinase by dibutyryl-cAMP, reported to cause GNMT phosphorylation under cell-free conditions, also had little effect on hepatocytic AdoMet and AdoHcy levels. Phosphorylation of GNMT would thus seem to play no role in regulation of the intracellular AdoMet/AdoHcy ratio, but could be involved in other GNMT functions, such as the binding of folates or aromatic hydrocarbons.
Asunto(s)
Aminoimidazol Carboxamida/análogos & derivados , Inhibidores Enzimáticos/farmacología , Flavanonas , Flavonoides/farmacología , Glicina/metabolismo , Hepatocitos/efectos de los fármacos , Metiltransferasas/metabolismo , Ácido Ocadaico/farmacología , S-Adenosilhomocisteína/metabolismo , S-Adenosilmetionina/metabolismo , Aminoimidazol Carboxamida/farmacología , Animales , Bucladesina/farmacología , Sistema Libre de Células , Glicina N-Metiltransferasa , Hepatocitos/metabolismo , Fragmentos de Péptidos/química , Fosforilación , Ratas , Ribonucleósidos/farmacologíaRESUMEN
Autophagy, pexophagy, and the Cvt pathway are processes that deliver hydrolytic enzymes and substrates to the yeast vacuole/lysosome via double-membrane cytosolic vesicles. Whereas these pathways operate under different nutritional conditions, they all employ common machinery with only a few specific factors assisting in the choice of the delivery program and the membrane source for the sequestering vesicle. We found that the YKR020w gene product is essential for Cvt vesicle formation but not for pexophagy or induction of autophagy. Autophagosomes in the ykr020wdelta mutant, however, have a reduced size. We demonstrate that Ykr020 is a subunit of the Vps fifty-three tethering complex, composed of Vps52, Vps53, and Vps54, which is required for retrograde traffic from the early endosome back to the late Golgi, and for this reason we named it Vps51. This complex participates in a fusion event together with Tlg1 and Tlg2, two SNAREs also shown to be necessary for Cvt vesicle assembly. In particular, those factors are essential to correctly target the prApe1-Cvt19-Cvt9 complex to the preautophagosomal structure, the site of Cvt vesicle formation.
Asunto(s)
Endosomas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacuolas/metabolismo , Proteínas de Transporte Vesicular , Transporte Biológico , Proteínas de la Membrana/análisis , Subunidades de Proteína/metabolismo , Proteínas de Saccharomyces cerevisiae/análisisRESUMEN
The proper functioning of eukaryotic organelles is largely dependent on the specific packaging of cargo proteins within transient delivery vesicles. The cytoplasm to vacuole targeting (Cvt) pathway is an autophagy-related trafficking pathway whose cargo proteins, aminopeptidase I and alpha-mannosidase, are selectively transported from the cytoplasm to the lysosome-like vacuole in yeast. This study elucidates a molecular mechanism for cargo specificity in this pathway involving four discrete steps. The Cvt19 receptor plays a central role in this process: distinct domains in Cvt19 recognize oligomerized cargo proteins and link them to the vesicle formation machinery via interaction with Cvt9 and Aut7. Because autophagy is the primary mechanism for organellar turnover, these results offer insights into physiological processes that are critical in cellular homeostasis, including specific packaging of damaged or superfluous organelles for lysosomal delivery and breakdown.
Asunto(s)
Autofagia/fisiología , Citoplasma/metabolismo , Fagosomas/metabolismo , Transporte de Proteínas/fisiología , Receptores de Superficie Celular/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacuolas/metabolismo , Proteínas de Transporte Vesicular , Aminopeptidasas/genética , Aminopeptidasas/metabolismo , Proteínas Relacionadas con la Autofagia , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Compartimento Celular/fisiología , Citoplasma/efectos de los fármacos , Citoplasma/ultraestructura , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes , Manosidasas/genética , Manosidasas/metabolismo , Mutación/genética , Péptidos/metabolismo , Fagosomas/efectos de los fármacos , Fagosomas/ultraestructura , Estructura Secundaria de Proteína/fisiología , Estructura Terciaria de Proteína/fisiología , Transporte de Proteínas/efectos de los fármacos , Proteínas Recombinantes de Fusión , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal/fisiología , Vesículas Transportadoras/efectos de los fármacos , Vesículas Transportadoras/metabolismo , Vesículas Transportadoras/ultraestructura , Vacuolas/efectos de los fármacos , Vacuolas/ultraestructura , alfa-ManosidasaRESUMEN
Mon1 and Ccz1 were identified from a gene deletion library as mutants defective in the vacuolar import of aminopeptidase I (Ape1) via the cytoplasm to vacuole targeting (Cvt) pathway. The mon1Delta and ccz1Delta strains also displayed defects in autophagy and pexophagy, degradative pathways that share protein machinery and mechanistic features with the biosynthetic Cvt pathway. Further analyses indicated that Mon1, like Ccz1, was required in nearly all membrane-trafficking pathways where the vacuole represented the terminal acceptor compartment. Accordingly, both deletion strains had kinetic defects in the biosynthetic delivery of resident vacuolar hydrolases through the CPY, ALP, and MVB pathways. Biochemical and microscopy studies suggested that Mon1 and Ccz1 functioned after transport vesicle formation but before (or at) the fusion step with the vacuole. Thus, ccz1Delta and mon1Delta are the first mutants identified in screens for the Cvt and Apg pathways that accumulate precursor Ape1 within completed cytosolic vesicles. Subcellular fractionation and co-immunoprecipitation experiments confirm that Mon1 and Ccz1 physically interact as a stable protein complex termed the Ccz1-Mon1 complex. Microscopy of Ccz1 and Mon1 tagged with a fluorescent marker indicated that the Ccz1-Mon1 complex peripherally associated with a perivacuolar compartment and may attach to the vacuole membrane in agreement with their proposed function in fusion.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/fisiología , Factores de Intercambio de Guanina Nucleótido , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/fisiología , Vacuolas/metabolismo , Proteínas de Transporte Vesicular , Transporte Biológico , Proteínas Portadoras/genética , Membrana Celular/metabolismo , Clonación Molecular , Citoplasma/metabolismo , Epítopos/metabolismo , Eliminación de Gen , Immunoblotting , Microscopía Fluorescente , Modelos Biológicos , Mutación , Sistemas de Lectura Abierta , Pruebas de Precipitina , Regiones Promotoras Genéticas , Unión Proteica , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/metabolismo , Fracciones Subcelulares/metabolismo , Factores de TiempoRESUMEN
Autophagy is a catabolic membrane-trafficking mechanism involved in cell maintenance and development. Most components of autophagy also function in the cytoplasm to vacuole targeting (Cvt) pathway, a constitutive biosynthetic pathway required for the transport of aminopeptidase I (Ape1). The protein components of autophagy and the Cvt pathway include a putative complex composed of Apg1 kinase and several interacting proteins that are specific for either the Cvt pathway or autophagy. A second required complex includes a phosphatidylinositol (PtdIns) 3-kinase and associated proteins that are involved in its activation and localization. The majority of proteins required for the Cvt and autophagy pathways localize to a perivacuolar pre-autophagosomal structure. We show that the Cvt13 and Cvt20 proteins are required for transport of precursor Ape1 through the Cvt pathway. Both proteins contain phox homology domains that bind PtdIns(3)P and are necessary for membrane localization to the pre-autophagosomal structure. Functional phox homology domains are required for Cvt pathway function. Cvt13 and Cvt20 interact with each other and with an autophagy-specific protein, Apg17, that interacts with Apg1 kinase. These results provide the first functional connection between the Apg1 and PtdIns 3-kinase complexes. The data suggest a role for PtdIns(3)P in the Cvt pathway and demonstrate that this lipid is required at the pre-autophagosomal structure.
Asunto(s)
Autofagia , Citoplasma/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas/metabolismo , Vacuolas/metabolismo , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Unión Proteica , Proteínas/química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Under starvation conditions, the majority of intracellular degradation occurs at the lysosome or vacuole by the autophagy pathway. The cytoplasmic substrates destined for degradation are packaged inside unique double-membrane transport vesicles called autophagosomes and are targeted to the lysosome/vacuole for subsequent breakdown and recycling. Genetic analyses of yeast autophagy mutants, apg and aut, have begun to identify the molecular machinery as well as indicate a substantial overlap with the biosynthetic cytoplasm to vacuole targeting (Cvt) pathway. Transport vesicle formation is a key regulatory step of both pathways. In this study, we characterize the putative compartment from which both autophagosomes and the analogous Cvt vesicles may originate. Microscopy analyses identified a perivacuolar membrane as the resident compartment for both the Apg1-Cvt9 signaling complex, which mediates the switching between autophagic and Cvt transport, and the autophagy/Cvt-specific phosphatidylinositol 3-kinase complex. Furthermore, the perivacuolar compartment designates the initial site of membrane binding by the Apg/Cvt vesicle component Aut7, the Cvt cargo receptor Cvt19, and the Apg conjugation machinery, which functions in the de novo formation of vesicles. Biochemical isolation of the vesicle component Aut7 and density gradient analyses recapitulate the microscopy findings although also supporting the paradigm that components required for vesicle formation and packaging concentrate at subdomains within the donor membrane compartment.