RESUMO
Autophagy is initiated by the assembly of multiple autophagy-related proteins that form the phagophore assembly site where autophagosomes are formed. Atg13 is essential early in this process, and a hub of extensive phosphorylation. How these multiple phosphorylations contribute to autophagy initiation, however, is not well understood. Here we comprehensively analyze the role of phosphorylation events on Atg13 during nutrient-rich conditions and nitrogen starvation. We identify and functionally characterize 48 in vivo phosphorylation sites on Atg13. By generating reciprocal mutants, which mimic the dephosphorylated active and phosphorylated inactive state of Atg13, we observe that disrupting the dynamic regulation of Atg13 leads to insufficient or excessive autophagy, which are both detrimental to cell survival. We furthermore demonstrate an involvement of Atg11 in bulk autophagy even during nitrogen starvation, where it contributes together with Atg1 to the multivalency that drives phase separation of the phagophore assembly site. These findings reveal the importance of post-translational regulation on Atg13 early during autophagy initiation, which provides additional layers of regulation to control bulk autophagy activity and integrate cellular signals.
Assuntos
Autofagia , Proteínas de Saccharomyces cerevisiae , Fosforilação , Autofagia/fisiologia , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Transdução de Sinais , Nitrogênio , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Synthetic tethering approaches induced by chemical means offer precise control over protein interactions in cells. They enable the manipulation of when, where, and how proteins interact, making it possible to study their functions, dynamics, and cellular consequences at a molecular level. These methods are versatile, reversible, and adaptable, allowing the dissection of complex cellular processes and the engineering of cellular functions. Here, we describe two chemically induced dimerization systems in the model organism Saccharomyces cerevisiae. Using the autophagy pathway as an example, we show how these approaches can be used to dissect molecular events in cells.
Assuntos
Autofagia , Multimerização Proteica , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
ATG8 proteins form a family of small ubiquitin-like modifiers, well-known for their importance in both macroautophagy and autophagy-independent processes. A unique feature of this protein family is their conjugation to membrane lipids through the covalent attachment of a glycine residue at the C-terminus of ATG8 proteins. Notably, most ATG8 proteins are expressed with additional amino acids at their C-terminus, shielding the key glycine residue. Consequently, lipidation requires the activation of the ATG8 precursors through proteolytic cleavage, known as priming. ATG4 proteases catalyze this priming process, and under physiological conditions, unprimed forms of ATG8 are not detected. This raises the question about the purpose of the C-terminal extension of ATG8 proteins. While the roles of lipidated and free, primed ATG8 proteins have been extensively studied, the potential function of their precursor form or the priming process itself remains largely unexplored. Here, we summarize information from existing literature and our own experiments to contribute to the understanding of these neglected amino acids.
Assuntos
Aminoácidos , Família da Proteína 8 Relacionada à Autofagia , Glicina , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/química , Glicina/metabolismo , Glicina/química , Aminoácidos/metabolismo , Autofagia , HumanosRESUMO
Mitophagy is a specific type of autophagy responsible for the selective elimination of dysfunctional or superfluous mitochondria, ensuring the maintenance of mitochondrial quality control. The initiation of mitophagy is coordinated by the ULK1 kinase complex, which engages mitophagy receptors via its FIP200 subunit. Whether FIP200 performs additional functions in the subsequent later phases of mitophagy beyond this initial step and how its regulation occurs, remains unclear. Our findings reveal that multiple phosphorylation events on FIP200 differentially control the early and late stages of mitophagy. Furthermore, these phosphorylation events influence FIP200's interaction with ATG16L1. In summary, our results highlight the necessity for precise and dynamic regulation of FIP200, underscoring its importance in the progression of mitophagy.
Assuntos
Proteínas Relacionadas à Autofagia , Mitocôndrias , Mitofagia , Fosforilação , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Humanos , Mitocôndrias/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/genética , Células HeLa , Proteínas de Transporte/metabolismoRESUMO
The majority of mitochondrial precursor proteins are imported through the Tom40 ß-barrel channel of the translocase of the outer membrane (TOM). The sorting and assembly machinery (SAM) is essential for ß-barrel membrane protein insertion into the outer membrane and thus required for the assembly of the TOM complex. Here, we demonstrate that the α-helical outer membrane protein Mco6 co-assembles with the mitochondrial distribution and morphology protein Mdm10 as part of the SAM machinery. MCO6 and MDM10 display a negative genetic interaction, and a mco6-mdm10 yeast double mutant displays reduced levels of the TOM complex. Cells lacking Mco6 affect the levels of Mdm10 and show assembly defects of the TOM complex. Thus, this work uncovers a role of the SAMMco6 complex for the biogenesis of the mitochondrial outer membrane.
Assuntos
Proteínas de Membrana Transportadoras , Proteínas de Saccharomyces cerevisiae , Proteínas de Membrana Transportadoras/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas de Transporte/metabolismo , Transporte ProteicoRESUMO
Macroautophagy/autophagy is a conserved catabolic pathway during which cellular material is sequestered within newly formed double-membrane vesicles called autophagosomes and delivered to the lytic compartment of eukaryotic cells for degradation. Autophagosome biogenesis depends on the core autophagy-related (Atg) machinery, and involves a massive supply and remodelling of membranes. To gain insight into the lipid remodelling mechanisms during autophagy, we have systematically investigated whether lipid flippases are required for this pathway in the yeast Saccharomyces cerevisiae. We found that the flippase Drs2, which transfers phosphatidylserine and phosphatidylethanolamine from the lumenal to the cytosolic leaflet of the limiting membrane at the trans-Golgi network, is required for normal progression of autophagy. We also show that Drs2 is important for the trafficking of the core Atg protein Atg9. Atg9 is a transmembrane protein important for autophagosome biogenesis and its anterograde transport from its post-Golgi reservoirs to the site of autophagosome formation is severely impaired in the absence of Drs2. Thus, our results identify a novel autophagy player and highlight that membrane asymmetry regulates early autophagy steps.
RESUMO
This animated movie presents the mechanism of macroautophagy, hereafter autophagy, by showing the molecular features of the formation of autophagosomes, the hallmark organelle of this intracellular catabolic pathway. It is based on our current knowledge and it also illustrates how autophagosomes can recognize and eliminate selected cargoes.