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Autophagosome content profiling - STAR Protocol Data Set

ABSTRACT: The ascorbate peroxidase APEX2 is commonly used to study the neighborhood of a protein of interest by proximity-dependent biotinylation. Here, we describe a protocol for sample processing compatible with immunoblotting and mass spectrometry that is suitable to specifically map the content of autophagosomes and potentially other transient vesicles without the need of subcellular fractionation. By combining live-cell biotinylation with proteinase K digestion of cell homogenates, proteins enriched in membrane-protected compartments can be readily enriched and identified.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: Christian Behrends

LAB HEAD: Christian Behrends

PROVIDER: PXD024335 | Pride | 2021-04-06

REPOSITORIES: Pride

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			Action	DRS
	20181026_SK297.raw	Raw
	20181026_SK298.raw	Raw
	20181026_SK299.raw	Raw
	20181026_SK300.raw	Raw
	20181026_SK301.raw	Raw

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Publications

Systematically defining selective autophagy receptor-specific cargo using autophagosome content profiling.

Zellner Susanne S Schifferer Martina M Behrends Christian C

Molecular cell 20210204 6

Autophagy deficiency in fed conditions leads to the formation of protein inclusions highlighting the contribution of this lysosomal delivery route to cellular proteostasis. Selective autophagy pathways exist that clear accumulated and aggregated ubiquitinated proteins. Receptors for this type of autophagy (aggrephagy) include p62, NBR1, TOLLIP, and OPTN, which possess LC3-interacting regions and ubiquitin-binding domains (UBDs), thus working as a bridge between LC3/GABARAP proteins and ubiquitin ...[more]

PMID: 33545068

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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

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

Transcriptional regulation by Pho23 modulates the frequency of autophagosome formation.

2014-04-24 | GSE57031 | GEO

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.

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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.

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Brain-derived autophagosome profiling reveals the engulfment of nucleoid-enriched mitochondrial fragments by basal autophagy in neurons

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Autophagosome Proteomics of HEK293 cells

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2022-01-12 | PXD020367 | Pride

Protocol for cell surface biotinylation of magnetic labeled and captured human peripheral blood mononuclear cells

Project description:Analysis of the surfaceome of a blood cell subset requires cell sorting, followed by surface protein enrichment. Here, we present a protocol combining magnetically activated cell sorting (MACS) and surface biotinylation of the target cell subset from human peripheral blood mononuclear cells (PBMCs). We describe the steps for isolating target cells and their in-column surface biotinylation, followed by isolation and mass spectrometry analysis of biotinylated proteins. The protocol enables in-column surface biotinylation of specific cell subsets with minimal membrane disruption.

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A conserved membrane curvature-generating protein is crucial for autophagosome formation in fission yeast

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