title: Arabidopsis ATG4 cysteine proteases specificity toward ATG8 substrates.
abstract: Macroautophagy (hereafter autophagy) is a regulated intracellular process during which cytoplasmic cargo engulfed by double-membrane autophagosomes is delivered to the vacuole or lysosome for degradation and recycling. Atg8 that is conjugated to phosphatidylethanolamine (PE) during autophagy plays an important role not only in autophagosome biogenesis but also in cargo recruitment. Conjugation of PE to Atg8 requires processing of the C-terminal conserved glycine residue in Atg8 by the Atg4 cysteine protease. The Arabidopsis plant genome contains 9 Atg8 (AtATG8a to AtATG8i) and 2 Atg4 (AtATG4a and AtATG4b) family members. To understand AtATG4's specificity toward different AtATG8 substrates, we generated a unique synthetic substrate C-AtATG8-ShR (citrine-AtATG8-Renilla luciferase SuperhRLUC). In vitro analyses indicated that AtATG4a is catalytically more active and has broad AtATG8 substrate specificity compared with AtATG4b. Arabidopsis transgenic plants expressing the synthetic substrate C-AtAtg8a-ShR is efficiently processed by endogenous AtATG4s and targeted to the vacuole during nitrogen starvation. These results indicate that the synthetic substrate mimics endogenous AtATG8, and its processing can be monitored in vivo by a bioluminescence resonance energy transfer (BRET) assay. The synthetic Atg8 substrates provide an easy and versatile method to study plant autophagy during different biological processes.
authors.affiliation: Department of Plant Sciences; College of Agricultural and Environmental Science; University of California, Davis; Davis, CA USA.
authors.forename: Eunsook
authors.initials: E
authors.lastname: Park
authors.affiliation: Department of Plant Biology and the Genome Center; College of Biological Sciences; University of California, Davis; Davis, CA USA.
authors.forename: Jongchan
authors.initials: J
authors.lastname: Woo
authors.affiliation: Department of Plant Biology and the Genome Center; College of Biological Sciences; University of California, Davis; Davis, CA USA.
authors.forename: S P
authors.initials: SP
authors.lastname: Dinesh-Kumar
mesh_terms: D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D001343:Autophagy / Q000235:genetics; D000071190:Autophagy-Related Protein 8 Family; D000071183:Autophagy-Related Proteins; D060807:Bioluminescence Resonance Energy Transfer Techniques; D057056:Cysteine Proteases / Q000235:genetics / Q000378:metabolism*; D049410:Luciferases, Renilla / Q000235:genetics / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D030821:Plants, Genetically Modified; D011499:Protein Processing, Post-Translational; D013379:Substrate Specificity
chemical_list: C000597073:ATG8 protein, Arabidopsis; D029681:Arabidopsis Proteins; D000071190:Autophagy-Related Protein 8 Family; D000071183:Autophagy-Related Proteins; D008869:Microtubule-Associated Proteins; D049410:Luciferases, Renilla; C587257:Atg4 protein, Arabidopsis; D057056:Cysteine Proteases
keywords: Arabidopsis; AtATG4 cysteine protease; AtATG8; Renillaluciferase; citrine yellow fluorescence protein