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Transcriptional regulation by Pho23 modulates the frequency of autophagosome formation.

ABSTRACT: Autophagy as a conserved degradation and recycling machinery is important in normal development and physiology, and defects in this process are linked to many kinds of disease. Because too much or too little autophagy can be detrimental, the process must be tightly regulated both temporally and in magnitude. The transcriptional induction and repression of the autophagy-related (ATG) genes is one crucial aspect of this regulation, but the transcriptional regulators that modulate autophagy are not well characterized. In this study, we identified Pho23 as a master transcriptional repressor for autophagy, with transcriptome profiling revealing that ATG9 is one of the key target genes. Physiological studies with a PHO23 null mutant, or with strains expressing modulated levels of Atg9, demonstrate a critical role of this protein as a regulator of autophagosome formation frequency; Atg9 protein levels correlate with the number of autophagosomes generated upon autophagy induction, and the level of autophagy activity.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE57031 | GEO | 2014/04/24

SECONDARY ACCESSION(S): PRJNA245237

REPOSITORIES: GEO

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Transcriptional regulation by Pho23 modulates the frequency of autophagosome formation.

Project description:Autophagy as a conserved degradation and recycling machinery is important in normal development and physiology, and defects in this process are linked to many kinds of disease. Because too much or too little autophagy can be detrimental, the process must be tightly regulated both temporally and in magnitude. The transcriptional induction and repression of the autophagy-related (ATG) genes is one crucial aspect of this regulation, but the transcriptional regulators that modulate autophagy are not well characterized. In this study, we identified Pho23 as a master transcriptional repressor for autophagy, with transcriptome profiling revealing that ATG9 is one of the key target genes. Physiological studies with a PHO23 null mutant, or with strains expressing modulated levels of Atg9, demonstrate a critical role of this protein as a regulator of autophagosome formation frequency; Atg9 protein levels correlate with the number of autophagosomes generated upon autophagy induction, and the level of autophagy activity. WT yeast and pho23 deletion mutants were grown under nutrient rich or nitrogen starvation conditions; gene expression was quantified across these 4 samples.

2014-04-24 | E-GEOD-57031 | biostudies-arrayexpress

The Plant Unique ESCRT Component FREE1 Regulates Autophagosome Closure

Project description:The energy sensor AMP-activated protein kinase (AMPK) can activate autophagy when cellular energy production becomes compromised. However, the degree to which nutrient sensing impinges on the autophagosome closure remains unknown. Here, we provide the mechanism underlying a plant unique protein FREE1, upon autophagy-induced SnRK1α1-mediated phosphorylation, functions as a linkage between ATG conjugation system and ESCRT machinery to regulate the autophagosome closure upon nutrient deprivation. Using high-resolution microscopy, 3D-electron tomography, and protease protection assay, we showed that unclosed autophagosomes accumulated in free1 mutants. Proteomic, cellular and biochemical analysis revealed the mechanistic connection between FREE1 and the ATG conjugation system/ESCRT-III complex in regulating autophagosome closure. Mass spectrometry analysis showed that the evolutionary conserved plant energy sensor SnRK1α1 phosphorylates FREE1 and recruits it to the autophagosomes to promote closure. Mutagenesis of the phosphorylation site on FREE1 caused the autophagosome closure failure. Our findings unveil how cellular energy sensing pathways regulate autophagosome closure to maintain cellular homeostasis.

2023-03-30 | PXD040435 | Pride

Isolation and mass spectrometry analysis of Atg9 vesicles from Saccharomyces cerevisiae

Project description:Autophagy is a eukaryotic bulk degradation pathway that allows cells to degrade potentially harmful cytosolic components or to provide necessary nutrients during starvation. This cargo degradation is achieved by its sequestration within double membrane vesicles termed autophagosomes, which fuse with the vacuole where it is degraded. Atg (autophagy-related) proteins are the main group of proteins involved in this process and, interestingly, Atg9 is the only integral membrane yeast Atg protein absolutely required for autophagy progression. In the cell it resides in Golgi-derived vesicles, which are indispensable for the nucleation of the autophagosome. There is not much known about biochemical properties of these vesicles in particular their protein content apart from Atg9. To address this question and to identify putative interaction partners of Atg9, the Atg9-vesicles were isolated and submitted to mass spectrometry analysis.

2022-03-03 | PXD020359 | Pride

ULK phosphorylation of STX17 controls autophagosome maturation via FLNA

Project description:Autophagy is a conserved and tightly regulated intracellular quality control pathway. ULK is a key kinase in autophagy initiation, but whether ULK kinase activity also participates in late stages of autophagy remains unknown. Here, we found that the autophagosomal SNARE protein, STX17, is phosphorylated by ULK at residue S289, beyond which it localizes specifically to autophagosomes. Inhibition of STX17 phosphorylation prevents such autophagosome localization. FLNA was then identified as a linker between ATG8 family proteins (ATG8s) and STX17 with essential involvement in STX17 recruitment to autophagosomes. Phosphorylation of STX17 S289 promotes its interaction with FLNA, activating its recruitment to autophagosomes and facilitates autophagosome-lysosome fusion. Disease causative mutations around the ATG8s- and STX17-binding regions of FLNA disrupt its interactions with ATG8s and STX17, inhibiting STX17 recruitment and autophagosome-lysosome fusion. Cumulatively, our study reveals an unexpected role of ULK in autophagosome maturation, uncovers its regulatory mechanism in STX17 recruitment, and highlights a potential association between autophagy and FLNA.

2023-06-18 | PXD043082 | iProX

Autophagosome Proteomics of HEK293 cells

Project description:Pan-caspase inhibitor Z-VAD-fmk acts as an inhibitor of peptide:N-glycanase (NGLY1); an endoglycosidase which cleaves N-linked glycans from glycoproteins exported from the endoplasmic reticulum during ER-associated degradation (ERAD). Pharmacological N-glycanase inhibition by Z-VAD-fmk or siRNA knockdown (KD) induces GFP-LC3 positive puncta in HEK293 cells. Activation of ER stress markers or reactive oxygen species (ROS) induction are not observed. In NGLY1 inhibition or KD, upregulation of autophagosome formation without impairment of autophagic flux are observed. Enrichment and proteomics analysis of autophagosomes after Z-VAD-fmk treatment or NGLY1 KD reveals similar autophagosomal protein content. Autophagy represents a cellular adaptation to NGLY1 inhibition or KD, and ATG13-deficient mouse embryonic fibroblasts (MEFs) show reduced viability under these conditions. In contrast, treatment with pan-caspase inhibitor, Q-VD-OPh does not induce cellular autophagy. Therefore, experiments with Z-VAD-fmk are complicated by the effects of NGLY1 inhibition and Q-VD-OPh represents an alternative caspase inhibitor free from this limitation.

2022-01-12 | PXD020367 | Pride

Autophagy cargo receptors are secreted via extracellular vesicles and particles in response to endolysosomal inhibition or impaired autophagosome maturation

Project description:The endosome-lysosome (endolysosome) system plays central roles in both autophagic degradation and secretory pathways, including the exocytic release of extracellular vesicles and particles (EVPs). Although previous work has revealed important interconnections between autophagy and EVP-mediated secretion, our molecular understanding of these secretory events during endolysosome inhibition remains incomplete. Here, we delineate a secretory autophagy pathway upregulated in response to endolysosomal inhibition that mediates the EVP-associated extracellular release of autophagic cargo receptors, including p62/SQSTM1. This extracellular secretion is highly regulated and critically dependent on multiple ATGs required for the progressive steps of early autophagosome formation as well as Rab27a-dependent exocytosis. Furthermore, the disruption of autophagosome maturation, either due to genetic inhibition of the autophagosome-to-autolyosome fusion machinery or blockade via the SARS-CoV2 viral protein ORF3a, is sufficient to induce robust EVP-associated secretion of autophagy cargo receptors. Finally, we demonstrate that this ATG-dependent, EVP-mediated secretion pathway buffers against the intracellular accumulation of autophagy cargo receptors when classical autophagic degradation is impaired. Based on these results, we propose that secretory autophagy via EVPs functions as an alternate route to clear sequestered material and maintain proteostasis in response to endolysosomal dysfunction or impaired autophagosome maturation.

2022-03-03 | PXD031527 | Pride

Studies of autophagy pathway during submergence in Arabidopsis thaliana

Project description:Autophagy involves massive degradation of intracellular components and functions as a conserved system that helps cells to adapt to adverse conditions. In Arabidopsis thaliana, submergence induces the transcription of autophagy-related (ATG) genes and the formation of autophagosomes. To study the role of autophagy during submergence, we performed transcriptome analysis with atg5, an autophagy-defective mutant, under submergence conditions. Our data showed that submergence changed the expression profile of DEG in the atg5 versus wild-type.

2021-09-20 | GSE184340 | GEO

Autophagosome content profiling in TBK1-E696K knockin MEFs

Project description:To determine how the mutant TBK1-E696K protein impacts autophagosomes, we performed autophagosome content profiling using protease protection coupled APEX2 proximity proteomics of autophagosomes of homozygous TBK1-E696K knockin and wiltype mouse embryonic fibroblasts (MEFs) transfected with a APEX2-LC3 as previously described in Zellner et al. 2021 Molecular Cell.

2024-03-16 | PXD048795 | Pride

mTOR hyperactivation in Down Syndrome mediates deficits in autophagy induction, autophagosome formation, and mitophagy

Project description:Down syndrome (DS), a complex genetic disorder caused by chromosome 21 trisomy, is associated with mitochondrial dysfunction leading to the accumulation of damaged mitochondria. Here we report that mitophagy, a form of selective autophagy activated to clear damaged mitochondria is deficient in primary human fibroblasts derived from individuals with DS leading to accumulation of damaged mitochondria with consequent increases in oxidative stress. We identified two molecular bases for this mitophagy deficiency: PINK1/PARKIN impairment and abnormal suppression of macroautophagy. First, strongly downregulated PARKIN and the mitophagic adaptor protein SQSTM1/p62 delays PINK1 activation to impair mitophagy induction after mitochondrial depolarization by CCCP or antimycin A plus oligomycin. Secondly, mTOR is strongly hyper-activated, which globally suppresses macroautophagy induction and the transcriptional expression of proteins critical for autophagosome formation such as ATG7, ATG3 and FOXO1. Notably, inhibition of mTOR complex 1 (mTORC1) and complex 2 (mTORC2) using AZD8055 (AZD) restores autophagy flux, PARKIN/PINK initiation of mitophagy, and the clearance of damaged mitochondria by mitophagy. These results recommend mTORC1-mTORC2 inhibition as a promising candidate therapeutic strategy for Down Syndrome.

2019-06-22 | GSE126910 | GEO

Cargo and lipid profiling of isolated autophagosomes upon basal autophagy, starvation, and proteasome inhibition

Project description:Autophagy is responsible for degradation of an extensive portfolio of cytosolic cargoes that are engulfed in autophagosomes to facilitate their transport to lysosomes. Besides basal autophagy, which constantly degrades cellular material, the pathway is dynamically altered by different conditions, resulting in enhanced autophagosome formation and cargo turnover. The extensive profile of autophagosome content as well as the phospholipid composition of human autophagosome membranes remains elusive. Here, we introduce a novel FACS-based approach for isolation of native autophagosomes and confirm the quality and specificity of the preparations. Employing quantitative proteomics and lipidomics, we obtained a profound cargo and lipid profile of autophagosomes isolated upon basal autophagy conditions, nutrient deprivation, and proteasome inhibition. This resource will allow to identify the impact of different autophagy conditions on the degradation of distinct proteins and to dissect the phospholipid composition of the human autophagosome membrane in detail.

2022-10-06 | PXD024419 | Pride

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