RESUMEN
The mechanism that initiates autophagosome formation on the ER in multicellular organisms is elusive. Here, we showed that autophagy stimuli trigger Ca2+ transients on the outer surface of the ER membrane, whose amplitude, frequency, and duration are controlled by the metazoan-specific ER transmembrane autophagy protein EPG-4/EI24. Persistent Ca2+ transients/oscillations on the cytosolic ER surface in EI24-depleted cells cause accumulation of FIP200 autophagosome initiation complexes on the ER. This defect is suppressed by attenuating ER Ca2+ transients. Multi-modal SIM analysis revealed that Ca2+ transients on the ER trigger the formation of dynamic and fusion-prone liquid-like FIP200 puncta. Starvation-induced Ca2+ transients on lysosomes also induce FIP200 puncta that further move to the ER. Multiple FIP200 puncta on the ER, whose association depends on the ER proteins VAPA/B and ATL2/3, assemble into autophagosome formation sites. Thus, Ca2+ transients are crucial for triggering phase separation of FIP200 to specify autophagosome initiation sites in metazoans.
Asunto(s)
Autofagosomas , Calcio , Animales , Autofagosomas/metabolismo , Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Autofagia , Proteínas de Ciclo Celular/metabolismoRESUMEN
The life of eukaryotic cells requires the transport of lipids between membranes, which are separated by the aqueous environment of the cytosol. Vesicle-mediated traffic along the secretory and endocytic pathways and lipid transfer proteins (LTPs) cooperate in this transport. Until recently, known LTPs were shown to carry one or a few lipids at a time and were thought to mediate transport by shuttle-like mechanisms. Over the last few years, a new family of LTPs has been discovered that is defined by a repeating ß-groove (RBG) rod-like structure with a hydrophobic channel running along their entire length. This structure and the localization of these proteins at membrane contact sites suggest a bridge-like mechanism of lipid transport. Mutations in some of these proteins result in neurodegenerative and developmental disorders. Here we review the known properties and well-established or putative physiological roles of these proteins, and we highlight the many questions that remain open about their functions.
Asunto(s)
Proteínas Portadoras , Proteínas , Proteínas Portadoras/química , Proteínas/metabolismo , Transporte Biológico/genética , Membrana Celular/metabolismo , Lípidos/químicaRESUMEN
The biogenesis of mammalian autophagosomes remains to be fully defined. Here, we used cellular and in vitro membrane fusion analyses to show that autophagosomes are formed from a hitherto unappreciated hybrid membrane compartment. The autophagic precursors emerge through fusion of FIP200 vesicles, derived from the cis-Golgi, with endosomally derived ATG16L1 membranes to generate a hybrid pre-autophagosomal structure, HyPAS. A previously unrecognized apparatus defined here controls HyPAS biogenesis and mammalian autophagosomal precursor membranes. HyPAS can be modulated by pharmacological agents whereas its formation is inhibited upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or by expression of SARS-CoV-2 nsp6. These findings reveal the origin of mammalian autophagosomal membranes, which emerge via convergence of secretory and endosomal pathways, and show that this process is targeted by microbial factors such as coronaviral membrane-modulating proteins.
Asunto(s)
Autofagosomas/virología , COVID-19/virología , Autofagia , COVID-19/metabolismo , Sistemas CRISPR-Cas , Línea Celular Tumoral , Retículo Endoplásmico/metabolismo , Endosomas/fisiología , Endosomas/virología , Aparato de Golgi/fisiología , Células HEK293 , Células HeLa , Humanos , Fusión de Membrana , Microscopía Confocal , Fagosomas/metabolismo , Fagosomas/virología , Proteínas Qa-SNARE/biosíntesis , Receptores sigma/biosíntesis , SARS-CoV-2 , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/biosíntesis , Sinaptotagminas/biosíntesis , Receptor Sigma-1RESUMEN
Selective autophagy is the lysosomal degradation of specific intracellular components sequestered into autophagosomes, late endosomes, or lysosomes through the activity of selective autophagy receptors (SARs). SARs interact with autophagy-related (ATG)8 family proteins via sequence motifs called LC3-interacting region (LIR) motifs in vertebrates and Atg8-interacting motifs (AIMs) in yeast and plants. SARs can be divided into two broad groups: soluble or membrane bound. Cargo or substrate selection may be independent or dependent of ubiquitin labeling of the cargo. In this review, we discuss mechanisms of mammalian selective autophagy with a focus on the unifying principles employed in substrate recognition, interaction with the forming autophagosome via LIR-ATG8 interactions, and the recruitment of core autophagy components for efficient autophagosome formation on the substrate.
Asunto(s)
Proteínas Reguladoras de la Apoptosis , Proteínas Asociadas a Microtúbulos , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagosomas/metabolismo , Autofagia/genética , Familia de las Proteínas 8 Relacionadas con la Autofagia/genética , Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Mamíferos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismoRESUMEN
During autophagy, vesicle dynamics and cargo recruitment are driven by numerous adaptors and receptors that become tethered to the phagophore through interactions with lipidated ATG8/LC3 decorating the expanding membrane. Most currently described ATG8-binding proteins exploit a well-defined ATG8-interacting motif (AIM, or LC3-interacting region [LIR]) that contacts a hydrophobic patch on ATG8 known as the LIR/AIM docking site (LDS). Here we describe a new class of ATG8 interactors that exploit ubiquitin-interacting motif (UIM)-like sequences for high-affinity binding to an alternative ATG8 interaction site. Assays with candidate UIM-containing proteins together with unbiased screens identified a large collection of UIM-based ATG8 interactors in plants, yeast, and humans. Analysis of a subset also harboring ubiquitin regulatory X (UBX) domains revealed a role for UIM-directed autophagy in clearing non-functional CDC48/p97 complexes, including some impaired in human disease. With this new class of adaptors and receptors, we greatly extend the reach of selective autophagy and identify new factors regulating autophagic vesicle dynamics.
Asunto(s)
Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Autofagia , Proteínas Asociadas a Microtúbulos/metabolismo , Secuencias de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Familia de las Proteínas 8 Relacionadas con la Autofagia/química , Sitios de Unión , Humanos , Proteínas Asociadas a Microtúbulos/química , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de Proteína , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de SecuenciaRESUMEN
Macroautophagy (herein referred to as autophagy) is an evolutionary ancient mechanism that culminates with the lysosomal degradation of superfluous or potentially dangerous cytosolic entities. Over the past 2 decades, the molecular mechanisms underlying several variants of autophagy have been characterized in detail. Accumulating evidence suggests that most, if not all, components of the molecular machinery for autophagy also mediate autophagy-independent functions. Here, we discuss emerging data on the non-autophagic functions of autophagy-relevant proteins.
Asunto(s)
Proteínas Relacionadas con la Autofagia/metabolismo , Autofagia/fisiología , Lisosomas/metabolismo , Animales , HumanosRESUMEN
In squamous cell carcinoma (SCC), macrophages responding to interleukin (IL)-33 create a TGF-ß-rich stromal niche that maintains cancer stem cells (CSCs), which evade chemotherapy-induced apoptosis in part via activation of the NRF2 antioxidant program. Here, we examined how IL-33 derived from CSCs facilitates the development of an immunosuppressive microenvironment. CSCs with high NRF2 activity redistributed nuclear IL-33 to the cytoplasm and released IL-33 as cargo of large oncosomes (LOs). Mechanistically, NRF2 increased the expression of the lipid scramblase ATG9B, which exposed an "eat me" signal on the LO surface, leading to annexin A1 (ANXA1) loading. These LOs promoted the differentiation of AXNA1 receptor+ myeloid precursors into immunosuppressive macrophages. Blocking ATG9B's scramblase activity or depleting ANXA1 decreased niche macrophages and hindered tumor progression. Thus, IL-33 is released from live CSCs via LOs to promote the differentiation of alternatively activated macrophage, with potential relevance to other settings of inflammation and tissue repair.
Asunto(s)
Diferenciación Celular , Interleucina-33 , Macrófagos , Células Madre Neoplásicas , Interleucina-33/metabolismo , Animales , Humanos , Ratones , Macrófagos/inmunología , Macrófagos/metabolismo , Células Madre Neoplásicas/inmunología , Células Madre Neoplásicas/metabolismo , Microambiente Tumoral/inmunología , Carcinoma de Células Escamosas/inmunología , Carcinoma de Células Escamosas/metabolismo , Ratones Endogámicos C57BL , Proteínas Relacionadas con la Autofagia/metabolismo , Línea Celular TumoralRESUMEN
Autophagy is the process of cellular self-eating by a double-membrane organelle, the autophagosome. A range of signaling processes converge on two protein complexes to initiate autophagy: the ULK1 (unc51-like autophagy activating kinase 1) protein kinase complex and the PI3KC3-C1 (class III phosphatidylinositol 3-kinase complex I) lipid kinase complex. Some 90% of the mass of these large protein complexes consists of noncatalytic domains and subunits, and the ULK1 complex has essential noncatalytic activities. Structural studies of these complexes have shed increasing light on the regulation of their catalytic and noncatalytic activities in autophagy initiation. The autophagosome is thought to nucleate from vesicles containing the integral membrane protein Atg9 (autophagy-related 9), COPII (coat protein complex II) vesicles, and possibly other sources. In the wake of reconstitution and super-resolution imaging studies, we are beginning to understand how the ULK1 and PI3KC3-C1 complexes might coordinate the nucleation and fusion of Atg9 and COPII vesicles at the start of autophagosome biogenesis.
Asunto(s)
Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Autofagia/genética , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fagosomas/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Homólogo de la Proteína 1 Relacionada con la Autofagia/química , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Vesículas Cubiertas por Proteínas de Revestimiento/ultraestructura , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Fosfatidilinositol 3-Quinasas Clase III/química , Fosfatidilinositol 3-Quinasas Clase III/genética , Células Eucariotas/metabolismo , Células Eucariotas/ultraestructura , Expresión Génica , Regulación de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Fagosomas/ultraestructura , Fosfatidilinositol 3-Quinasa/química , Fosfatidilinositol 3-Quinasa/genética , Unión Proteica , Multimerización de Proteína , Transducción de SeñalRESUMEN
Defects in organellar acidification indicate compromised or infected compartments. Recruitment of the autophagy-related ATG16L1 complex to pathologically neutralized organelles targets ubiquitin-like ATG8 molecules to perturbed membranes. How this process is coupled to proton gradient disruption is unclear. Here, we reveal that the V1H subunit of the vacuolar ATPase (V-ATPase) proton pump binds directly to ATG16L1. The V1H/ATG16L1 interaction only occurs within fully assembled V-ATPases, allowing ATG16L1 recruitment to be coupled to increased V-ATPase assembly following organelle neutralization. Cells lacking V1H fail to target ATG8s during influenza infection or after activation of the immune receptor stimulator of interferon genes (STING). We identify a loop within V1H that mediates ATG16L1 binding. A neuronal V1H isoform lacks this loop and is associated with attenuated ATG8 targeting in response to ionophores in primary murine and human iPSC-derived neurons. Thus, V1H controls ATG16L1 recruitment following proton gradient dissipation, suggesting that the V-ATPase acts as a cell-intrinsic damage sensor.
Asunto(s)
Proteínas Relacionadas con la Autofagia , ATPasas de Translocación de Protón Vacuolares , ATPasas de Translocación de Protón Vacuolares/metabolismo , ATPasas de Translocación de Protón Vacuolares/genética , Humanos , Proteínas Relacionadas con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Animales , Ratones , Unión Proteica , Neuronas/metabolismo , Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Familia de las Proteínas 8 Relacionadas con la Autofagia/genética , Autofagia , Células HEK293 , Células Madre Pluripotentes Inducidas/metabolismo , Gripe Humana/virología , Gripe Humana/metabolismo , Gripe Humana/genética , Ratones Endogámicos C57BL , Transducción de Señal , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Ratones NoqueadosRESUMEN
Mitophagy plays an important role in mitochondrial homeostasis by selective degradation of mitochondria. During mitophagy, mitochondria should be fragmented to allow engulfment within autophagosomes, whose capacity is exceeded by the typical mitochondria mass. However, the known mitochondrial fission factors, dynamin-related proteins Dnm1 in yeasts and DNM1L/Drp1 in mammals, are dispensable for mitophagy. Here, we identify Atg44 as a mitochondrial fission factor that is essential for mitophagy in yeasts, and we therefore term Atg44 and its orthologous proteins mitofissin. In mitofissin-deficient cells, a part of the mitochondria is recognized by the mitophagy machinery as cargo but cannot be enwrapped by the autophagosome precursor, the phagophore, due to a lack of mitochondrial fission. Furthermore, we show that mitofissin directly binds to lipid membranes and brings about lipid membrane fragility to facilitate membrane fission. Taken together, we propose that mitofissin acts directly on lipid membranes to drive mitochondrial fission required for mitophagy.
Asunto(s)
Autofagia , Mitofagia , Animales , Dinámicas Mitocondriales , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Dinaminas/genética , Dinaminas/metabolismo , Lípidos , Mamíferos/metabolismoRESUMEN
Autophagy, a lysosome-mediated degradation process evolutionarily conserved from yeast to mammals, is essential for maintaining cellular homeostasis and combating diverse cellular stresses. Autophagy involves de novo synthesis of a double-membrane autophagosome, sequestration of selected cellular contents, and subsequent delivery of sequestrated contents to the vacuole (in yeasts and plants) or to lysosomes (in animal cells) for degradation and recycling. Genetic studies in unicellular and multicellular model organisms have systematically revealed the molecular machinery, regulation, and function of autophagy in physiological settings. I review genetic studies in model organisms-from yeast to worm to fly-that enable us to not only identify autophagy genes, including ATG genes and the metazoan-specific EPG genes, but also uncover variants of autophagy in developmental contexts, novel regulatory mechanisms, and signaling events involved in mediating systemic autophagy response. Genetic analysis also helps us understand the liquid-liquid phase separation and transition that control autophagic degradation of protein aggregates. The emerging role of autophagy in zebrafish tissue regeneration is also discussed.
Asunto(s)
Saccharomyces cerevisiae , Pez Cebra , Animales , Autofagia/genética , Lisosomas , Transducción de Señal/genética , MamíferosRESUMEN
The migration of neutrophils from the blood circulation to sites of infection or injury is a key immune response and requires the breaching of endothelial cells (ECs) that line the inner aspect of blood vessels. Unregulated neutrophil transendothelial cell migration (TEM) is pathogenic, but the molecular basis of its physiological termination remains unknown. Here, we demonstrated that ECs of venules in inflamed tissues exhibited a robust autophagic response that was aligned temporally with the peak of neutrophil trafficking and was strictly localized to EC contacts. Genetic ablation of EC autophagy led to excessive neutrophil TEM and uncontrolled leukocyte migration in murine inflammatory models, while pharmacological induction of autophagy suppressed neutrophil infiltration into tissues. Mechanistically, autophagy regulated the remodeling of EC junctions and expression of key EC adhesion molecules, facilitating their intracellular trafficking and degradation. Collectively, we have identified autophagy as a modulator of EC leukocyte trafficking machinery aimed at terminating physiological inflammation.
Asunto(s)
Autofagia/fisiología , Células Endoteliales/fisiología , Infiltración Neutrófila/fisiología , Migración Transendotelial y Transepitelial/fisiología , Animales , Quimiotaxis de Leucocito/fisiología , Células Endoteliales/patología , Células Endoteliales de la Vena Umbilical Humana/inmunología , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Inflamación/inmunología , Inflamación/patología , Uniones Intercelulares/fisiología , Ratones , Ratones Endogámicos C57BL , Neutrófilos/fisiologíaRESUMEN
The sequestration of damaged mitochondria within double-membrane structures termed autophagosomes is a key step of PINK1/Parkin mitophagy. The ATG4 family of proteases are thought to regulate autophagosome formation exclusively by processing the ubiquitin-like ATG8 family (LC3/GABARAPs). We discover that human ATG4s promote autophagosome formation independently of their protease activity and of ATG8 family processing. ATG4 proximity networks reveal a role for ATG4s and their proximity partners, including the immune-disease protein LRBA, in ATG9A vesicle trafficking to mitochondria. Artificial intelligence-directed 3D electron microscopy of phagophores shows that ATG4s promote phagophore-ER contacts during the lipid-transfer phase of autophagosome formation. We also show that ATG8 removal during autophagosome maturation does not depend on ATG4 activity. Instead, ATG4s can disassemble ATG8-protein conjugates, revealing a role for ATG4s as deubiquitinating-like enzymes. These findings establish non-canonical roles of the ATG4 family beyond the ATG8 lipidation axis and provide an AI-driven framework for rapid 3D electron microscopy.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagosomas/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Cisteína Endopeptidasas/metabolismo , Metabolismo de los Lípidos , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Inteligencia Artificial , Autofagosomas/genética , Autofagosomas/ultraestructura , Familia de las Proteínas 8 Relacionadas con la Autofagia/genética , Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Cisteína Endopeptidasas/genética , Células HEK293 , Células HeLa , Humanos , Imagenología Tridimensional , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Microscopía Electrónica de Transmisión , Proteínas Asociadas a Microtúbulos/genética , Mitocondrias/genética , Mitocondrias/ultraestructura , Mitofagia , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Transporte de Proteínas , Transducción de Señal , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Autophagy is a fundamental catabolic process that uses a unique post-translational modification, the conjugation of ATG8 protein to phosphatidylethanolamine (PE). ATG8 lipidation also occurs during non-canonical autophagy, a parallel pathway involving conjugation of ATG8 to single membranes (CASM) at endolysosomal compartments, with key functions in immunity, vision, and neurobiology. It is widely assumed that CASM involves the same conjugation of ATG8 to PE, but this has not been formally tested. Here, we discover that all ATG8s can also undergo alternative lipidation to phosphatidylserine (PS) during CASM, induced pharmacologically, by LC3-associated phagocytosis or influenza A virus infection, in mammalian cells. Importantly, ATG8-PS and ATG8-PE adducts are differentially delipidated by the ATG4 family and bear different cellular dynamics, indicating significant molecular distinctions. These results provide important insights into autophagy signaling, revealing an alternative form of the hallmark ATG8 lipidation event. Furthermore, ATG8-PS provides a specific "molecular signature" for the non-canonical autophagy pathway.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Autofagosomas/metabolismo , Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Autofagia , Proteínas Asociadas a Microtúbulos/metabolismo , Fosfatidilserinas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Autofagosomas/efectos de los fármacos , Autofagosomas/genética , Autofagosomas/patología , Familia de las Proteínas 8 Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Femenino , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Virus de la Influenza A/patogenicidad , Macrólidos/farmacología , Masculino , Ratones , Proteínas Asociadas a Microtúbulos/genética , Monensina/farmacología , Fagocitosis , Fosfatidiletanolaminas/metabolismo , Células RAW 264.7 , Transducción de SeñalRESUMEN
Autophagy is a conserved intracellular degradation pathway exerting various cytoprotective and homeostatic functions by using de novo double-membrane vesicle (autophagosome) formation to target a wide range of cytoplasmic material for vacuolar/lysosomal degradation. The Atg1 kinase is one of its key regulators, coordinating a complex signaling program to orchestrate autophagosome formation. Combining in vitro reconstitution and cell-based approaches, we demonstrate that Atg1 is activated by lipidated Atg8 (Atg8-PE), stimulating substrate phosphorylation along the growing autophagosomal membrane. Atg1-dependent phosphorylation of Atg13 triggers Atg1 complex dissociation, enabling rapid turnover of Atg1 complex subunits at the pre-autophagosomal structure (PAS). Moreover, Atg1 recruitment by Atg8-PE self-regulates Atg8-PE levels in the growing autophagosomal membrane by phosphorylating and thus inhibiting the Atg8-specific E2 and E3. Our work uncovers the molecular basis for positive and negative feedback imposed by Atg1 and how opposing phosphorylation and dephosphorylation events underlie the spatiotemporal regulation of autophagy.
Asunto(s)
Autofagosomas/enzimología , Proteínas Relacionadas con la Autofagia/metabolismo , Autofagia , Proteínas Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Autofagosomas/genética , Familia de las Proteínas 8 Relacionadas con la Autofagia/genética , Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Activación Enzimática , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Fosforilación , Proteínas Quinasas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal , Factores de TiempoRESUMEN
Autophagy sequesters cytoplasmic portions into autophagosomes. While selective cargo is engulfed by elongation of cup-shaped isolation membranes (IMs), the morphogenesis of non-selective IMs remains elusive. Based on recent observations, we propose a novel model for autophagosome morphogenesis wherein active regulation of the IM rim serves the physiological roles of autophagy.
Asunto(s)
Autofagosomas , Autofagia , Morfogénesis , Autofagosomas/metabolismo , Animales , HumanosRESUMEN
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
Since nuclear envelope breakdown occurs during mitosis in metazoan cells, it has been proposed that macroautophagy must be inhibited to maintain genome integrity. However, repression of macroautophagy during mitosis remains controversial and mechanistic detail limited to the suggestion that CDK1 phosphorylates VPS34. Here, we show that initiation of macroautophagy, measured by the translocation of the ULK complex to autophagic puncta, is repressed during mitosis, even when mTORC1 is inhibited. Indeed, mTORC1 is inactive during mitosis, reflecting its failure to localize to lysosomes due to CDK1-dependent RAPTOR phosphorylation. While mTORC1 normally represses autophagy via phosphorylation of ULK1, ATG13, ATG14, and TFEB, we show that the mitotic phosphorylation of these autophagy regulators, including at known repressive sites, is dependent on CDK1 but independent of mTOR. Thus, CDK1 substitutes for inhibited mTORC1 as the master regulator of macroautophagy during mitosis, uncoupling autophagy regulation from nutrient status to ensure repression of macroautophagy during mitosis.
Asunto(s)
Autofagia/fisiología , Proteína Quinasa CDC2/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Mitosis/fisiología , Células A549 , Línea Celular , Línea Celular Tumoral , Femenino , Células HCT116 , Células HEK293 , Células HT29 , Células HeLa , Humanos , Lisosomas/metabolismo , Masculino , Fosforilación/fisiología , Transducción de Señal/fisiologíaRESUMEN
Aberrant mitophagy has been implicated in a broad spectrum of disorders. PINK1, Parkin, and ubiquitin have pivotal roles in priming mitophagy. However, the entire regulatory landscape and the precise control mechanisms of mitophagy remain to be elucidated. Here, we uncover fundamental mitophagy regulation involving PINK1 and a non-canonical role of the mitochondrial Tu translation elongation factor (TUFm). The mitochondrion-cytosol dual-localized TUFm interacts with PINK1 biochemically and genetically, which is an evolutionarily conserved Parkin-independent route toward mitophagy. A PINK1-dependent TUFm phosphoswitch at Ser222 determines conversion from activating to suppressing mitophagy. PINK1 modulates differential translocation of TUFm because p-S222-TUFm is restricted predominantly to the cytosol, where it inhibits mitophagy by impeding Atg5-Atg12 formation. The self-antagonizing feature of PINK1/TUFm is critical for the robustness of mitophagy regulation, achieved by the unique kinetic parameters of p-S222-TUFm, p-S65-ubiquitin, and their common kinase PINK1. Our findings provide new mechanistic insights into mitophagy and mitophagy-associated disorders.
Asunto(s)
Drosophila melanogaster/crecimiento & desarrollo , Mitocondrias/patología , Proteínas Mitocondriales/metabolismo , Mitofagia , Factor Tu de Elongación Peptídica/metabolismo , Proteínas Quinasas/metabolismo , Animales , Citosol/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Femenino , Células HeLa , Humanos , Masculino , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Factor Tu de Elongación Peptídica/genética , Fosforilación , Dominios y Motivos de Interacción de Proteínas , Proteínas Quinasas/genética , Transporte de Proteínas , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Autophagy eliminates cytoplasmic content selected by autophagy receptors, which link cargo to the membrane-bound autophagosomal ubiquitin-like protein Atg8/LC3. Here, we report a selective autophagy pathway for protein condensates formed by endocytic proteins in yeast. In this pathway, the endocytic protein Ede1 functions as a selective autophagy receptor. Distinct domains within Ede1 bind Atg8 and mediate phase separation into condensates. Both properties are necessary for an Ede1-dependent autophagy pathway for endocytic proteins, which differs from regular endocytosis and does not involve other known selective autophagy receptors but requires the core autophagy machinery. Cryo-electron tomography of Ede1-containing condensates, at the plasma membrane and in autophagic bodies, shows a phase-separated compartment at the beginning and end of the Ede1-mediated selective autophagy route. Our data suggest a model for autophagic degradation of macromolecular protein complexes by the action of intrinsic autophagy receptors.