Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

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

ABSTRACT: 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.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Hela Cell

SUBMITTER: Süleyman Bozkurt

LAB HEAD: Christian Münch

PROVIDER: PXD024419 | Pride | 2022-10-06

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	Basal_1.raw	Raw
	Basal_2.raw	Raw
	Basal_3.raw	Raw
	MG132_1.raw	Raw
	MG132_2.raw	Raw

Items per page:

1 - 5 of 11

Similar Datasets

Nonuple atg8 mutant provides genetic evidence for functional specialization of ATG8 isoforms in Arabidopsis thaliana

Project description:Autophagy is a vital cellular process that maintains organismal health by recycling damaged or superfluous cellular components within autophagosomes. This process is mediated by conserved ATG proteins, which coordinate autophagosome biogenesis and selective cargo degradation. Among these, the ubiquitin-like ATG8 protein plays a central role by linking cargo to the growing autophagosomes through interactions with selective autophagy receptors. Unlike most ATG proteins, which are represented by single or few isoforms, the ATG8 gene family is expanded in vascular plants, yet the extent of functional specialization within ATG8 isoforms remains largely unknown. Using transcriptional and translational reporters in Arabidopsis thaliana, we revealed that ATG8 isoforms are differentially expressed across tissues and form distinct autophagosomes within the same cell. To explore ATG8 specialization, we generated the nonuple Δatg8mutant lacking all nine ATG8 isoforms. The mutant displayed hypersensitivity to carbon and nitrogen starvation, coupled with defects in bulk and selective autophagy as shown by biochemical and ultrastructural analyses. Complementation experiments demonstrated that ATG8A could rescue both carbon and nitrogen starvation phenotypes, whereas ATG8H could only complement carbon starvation. Proximity labeling proteomics further identified isoform-specific interactors under nitrogen starvation, underscoring their functional divergence. These findings provide genetic evidence for the specialization of ATG8 isoforms in plants and lay a foundation for investigating their roles in diverse cell types and stress conditions.

2024-12-11 | GSE283481 | GEO

RAB18 impacts autophagy via lipid droplet-derived lipid transfer and is rescued by ATG9A

Project description:Autophagy is a lysosomal degradation pathway that mediates protein and organelle turnover and maintains cellular homeostasis. Autophagosomes transport cargo to lysosomes and their formation is dependent on an appropriate lipid supply. Here, we show that the knockout of the RAB GTPase RAB18 interferes with lipid droplet (LD) metabolism, resulting in an impaired fatty acid mobilization. The reduced LD-derived lipid availability influences autophagy and provokes adaptive modifications of the autophagy network, which include increased ATG2B expression and ATG12-ATG5 conjugate formation as well as enhanced ATG2B and ATG9A phosphorylation. Phosphorylation of ATG9A directs this transmembrane protein to the site of autophagosome formation and this particular modification is sufficient to rescue autophagic activity under basal conditions in the absence of RAB18. However, it is incapable of enabling an increased autophagy under inductive conditions. Thus, we illustrate the role of RAB18 in connecting LDs and autophagy, further emphasize the importance of LD-derived lipids for the degradative pathway, and characterize an ATG9A phosphorylation-dependent autophagy rescue mechanism as an adaptive response that maintains autophagy under conditions of reduced LD-derived lipid availability.

2020-01-29 | PXD013719 | Pride

TMEM41B is a novel regulator of autophagy and lipid mobilization

Project description:Autophagy maintains cellular homeostasis by targeting damaged organelles, pathogens or misfolded protein aggregates for lysosomal degradation. The autophagic process is initiated by the formation of autophagosomes, which can selectively enclose cargo via autophagy cargo receptors. A machinery of well-characterized autophagy-related proteins orchestrate the biogenesis of autophagosomes, however, the origin of the required membranes is incompletely understood. Here, we applied sensitized pooled CRISPR screens and identified the uncharacterized transmembrane protein TMEM41B as a novel regulator of autophagy. In the absence of TMEM41B, autophagosome biogenesis is stalled, LC3 accumulates at WIPI2- and DFCP1-positive isolation membranes, and lysosomal flux of autophagy cargo receptors and intracellular bacteria is impaired. In addition to defective autophagy, we found that TMEM41B knockout cells display significantly enlarged lipid droplets and reduced mobilization and β-oxidation of fatty acids. TMEM41B localizes to the endoplasmic reticulum (ER) and interacts with SIGMAR1, a chaperone which regulates calcium and lipid transfer at ER contact sites with mitochondria and lipid droplets. Taken together, we propose that TMEM41B is a novel regulator of autophagosome biogenesis and ER lipid mobilization.

2018-08-20 | PXD010207 | Pride

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

The plant-specific COST1 protein balances plant growth and drought tolerance via attenuation of autophagy

Project description:Plants balance their conflicting requirements for growth and stress tolerance via sophisticated pathways and unique genes that control responses to the external environment. We have identified a novel plant-specific gene, COST1(Constitutively Stressed 1), that affects plant growth and negatively regulates drought resistance by manipulating the autophagy pathway. An Arabidopsis cost1 mutant has decreased growth and increased drought tolerance, together with constitutive autophagy and increased expression of drought-response genes. The COST1 protein is degraded upon plant dehydration, and this degradation is blocked by treatment with inhibitors of the 26S proteasome or autophagy pathways. The cost1 mutant drought resistance is dependent on an active autophagy pathway, indicating that COST1 acts through manipulation of autophagy. COST1 co-localizes to autophagosomes with the autophagosome marker ATG8e and the autophagy adaptor NBR1, and physically interacts with ATG8e, indicating a pivotal role in direct regulation of autophagy. We propose a model in which COST1 represses autophagy under optimal conditions, thus allowing plant growth. During drought, COST1 is degraded, enabling activation of autophagy and suppressing growth to enhance drought tolerance.

2020-04-16 | GSE113515 | GEO

Differential centrifugation coupled to affinity purification-mass spectrometry of mcherry-ATG8E

Project description:Autophagosomes are double-membraned vesicles that traffic harmful or unwanted cellular macromolecules to the vacuole for recycling. Although autophagosome biogenesis has been studied extensively, autophagosome maturation, i.e., delivery and fusion with the vacuole, remains largely unknown in plants. To start filling these gaps, we devised a differential centrifugation set up, where we enriched intact autophagosomes and performed affinity purification-mass spectrometry (AP-MS), using ATG8 as a bait. This method enabled the identification of an autophagy adaptor protein.

2022-10-18 | PXD031787 | Pride

GRAMD1C regulates autophagy initiation and mitochondrial bioenergetics

Project description:During autophagy, cytosolic cargo is sequestered into double-membrane vesicles called autophagosomes. The origin and identity of the membranes that form the autophagosome remain to be fully characterized. Here, we investigated the role of cholesterol in starvation-induced autophagy and identify a role for the ER-localized cholesterol transport protein GRAMD1C in the regulation of autophagy and mitochondrial function. We demonstrate that cholesterol depletion leads to a rapid induction of autophagy and a corresponding increased abundance of curved autophagy membranes. We further show that GRAMD1C is a negative regulator of starvation-induced autophagy. Similar to its yeast orthologue, GRAMD1C is recruited to mitochondria through its GRAM domain. Additionally, we find that GRAMD1C depletion leads to increased mitochondrial cholesterol accumulation and mitochondrial oxidative phosphorylation. Finally, we demonstrate that expression of GRAM family genes is linked to clear cell renal carcinoma survival, highlighting the pathophysiological relevance of cholesterol transport proteins.

2022-10-19 | PXD033125 | Pride

Transcriptional regulation by Pho23 modulates the frequency of autophagosome formation.

2014-04-24 | GSE57031 | GEO

: GRAMD1C regulates autophagy initiation and mitochondrial bioenergetics through ER-Mitochondria cholesterol transport

Project description:During autophagy, cytosolic cargo is sequestered into double-membrane vesicles called autophagosomes. The origin and identity of the membranes that form the autophagosome remain to be fully characterized. Here, we investigated the role of cholesterol in starvation-induced autophagy and identify a role for the ER-localized cholesterol transport protein GRAMD1C in the regulation of autophagy and mitochondrial function. We demonstrate that cholesterol depletion leads to a rapid induction of autophagy, possibly caused by a corresponding increased abundance of curved autophagy membranes. We further show that GRAMD1C is a negative regulator of starvation-induced autophagy. Similar to its yeast orthologue, GRAMD1C is recruited to mitochondria through its GRAM domain. Additionally, we find that GRAMD1C depletion leads to increased mitochondrial cholesterol accumulation and mitochondrial oxidative phosphorylation. Finally, we demonstrate that expression of GRAM family genes is linked to clear cell renal carcinoma survival, highlighting the pathophysiological relevance of cholesterol transport proteins.

2022-10-19 | PXD027502 | Pride

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data