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The yeast autophagy protease Atg4 is regulated by thioredoxin.

ABSTRACT: Autophagy is a membrane-trafficking process whereby double-membrane vesicles called autophagosomes engulf and deliver intracellular material to the vacuole for degradation. Atg4 is a cysteine protease with an essential function in autophagosome formation. Mounting evidence suggests that reactive oxygen species may play a role in the control of autophagy and could regulate Atg4 activity but the precise mechanisms remain unclear. In this study, we showed that reactive oxygen species activate autophagy in the model yeast Saccharomyces cerevisiae and unraveled the molecular mechanism by which redox balance controls Atg4 activity. A combination of biochemical assays, redox titrations, and site-directed mutagenesis revealed that Atg4 is regulated by oxidoreduction of a single disulfide bond between Cys338 and Cys394. This disulfide has a low redox potential and is very efficiently reduced by thioredoxin, suggesting that this oxidoreductase plays an important role in Atg4 regulation. Accordingly, we found that autophagy activation by rapamycin was more pronounced in a thioredoxin mutant compared with wild-type cells. Moreover, in vivo studies indicated that Cys338 and Cys394 are required for the proper regulation of autophagosome biogenesis, since mutation of these cysteines resulted in increased recruitment of Atg8 to the phagophore assembly site. Thus, we propose that the fine-tuning of Atg4 activity depending on the intracellular redox state may regulate autophagosome formation.

SUBMITTER: Perez-Perez ME

PROVIDER: S-EPMC4502779 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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The yeast autophagy protease Atg4 is regulated by thioredoxin.

Pérez-Pérez María Esther ME Zaffagnini Mirko M Marchand Christophe H CH Crespo José L JL Lemaire Stéphane D SD

Autophagy 20141030 11

Autophagy is a membrane-trafficking process whereby double-membrane vesicles called autophagosomes engulf and deliver intracellular material to the vacuole for degradation. Atg4 is a cysteine protease with an essential function in autophagosome formation. Mounting evidence suggests that reactive oxygen species may play a role in the control of autophagy and could regulate Atg4 activity but the precise mechanisms remain unclear. In this study, we showed that reactive oxygen species activate autop ...[more]

PMID: 25483965

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Project description:Macroautophagy/autophagy is a cellular degradation pathway that delivers cytoplasmic material to lysosomes via double-membrane organelles called autophagosomes. Lipidation of ubiquitin-like LC3/GABARAP proteins on the autophagosome membrane is important for autophagy. The cysteine protease ATG4 executes 2 LC3/GABARAP processing events: priming of newly synthesized pro-LC3/GABARAP to enable subsequent lipidation, and delipidation/deconjugation of lipidated LC3/GABARAP (the exact purpose of which is unclear in mammals). Four ATG4 isoforms (ATG4A to ATG4D) exist in mammals; however, the functional redundancy of these proteins in cells is poorly understood. Here we show that human HAP1 and HeLa cells lacking ATG4B exhibit a severe but incomplete defect in LC3/GABARAP processing and autophagy. By further genetic depletion of ATG4 isoforms using CRISPR-Cas9 and siRNA we uncover that ATG4A, ATG4C and ATGD all contribute to residual priming activity, which is sufficient to enable lipidation of endogenous GABARAPL1 on autophagic structures. We also demonstrate that expressing high levels of pre-primed LC3B in ATG4-deficient cells can rescue a defect in autophagic degradation of the cargo receptor SQSTM1/p62, suggesting that delipidation by human ATG4 is not essential for autophagosome formation and fusion with lysosomes. Overall, our study provides a comprehensive characterization of ATG4 isoform function during autophagy in human cells. Abbreviations: Atg: autophagy-related; baf A1: bafilomycin A1; CASP3: caspase 3; CLEM: correlative light and electron microscopy; CMV: cytomegalovirus; CRISPR: clustered regularly interspaced short palindromic repeats; DKO: double knockout; EGFP: enhanced green fluorescent protein; GABARAP: GABA type A receptor-associated protein; GABARAPL1: GABA type A receptor-associated protein like 1; GABARAPL2: GABA type A receptor-associated protein like 2; GFP: green fluorescent protein; HB: homogenization buffer; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LIR: LC3 interacting region; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MFN2: mitofusin 2; N.A.: numerical aperture; NEM: N-ethylmaleimide; PDHA1: pyruvate dehydrogenase E1 alpha 1 subunit; PLD: phospholipase D; PE: phosphatidylethanolamine; RLUC: Renilla luciferase; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; TKO: triple knockout; ULK1: unc-51 like autophagy activating kinase 1; VCL: vinculin; WT: wild-type.

Dataset Information

The yeast autophagy protease Atg4 is regulated by thioredoxin.

Publications

The yeast autophagy protease Atg4 is regulated by thioredoxin.

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