Increases in activity of proteasome and papain-like cysteine protease in Arabidopsis autophagy mutants: back-up compensatory effect or cell-death promoting effect?

Abstract Autophagy is essential for protein degradation, nutrient recycling, and nitrogen remobilization. Autophagy is induced during leaf ageing and in response to nitrogen starvation, and is known to play a fundamental role in nutrient recycling for remobilization and seed filling. Accordingly, ageing leaves of Arabidopsis autophagy mutants (atg) have been shown to over-accumulate proteins and peptides, possibly because of a reduced protein degradation capacity. Surprisingly, atg leaves also displayed higher protease activities. The work reported here aimed at identifying the nature of the proteases and protease activities that accumulated differentially (higher or lower) in the atg mutants. Protease identification was performed using shotgun LC-MS/MS proteome analyses and activity-based protein profiling (ABPP). The results showed that the chloroplast FTSH (FILAMENTATION TEMPERATURE SENSITIVE H) and DEG (DEGRADATION OF PERIPLASMIC PROTEINS) proteases and several extracellular serine proteases [subtilases (SBTs) and serine carboxypeptidase-like (SCPL) proteases] were less abundant in atg5 mutants. By contrast, proteasome-related proteins and cytosolic or vacuole cysteine proteases were more abundant in atg5 mutants. Rubisco degradation assays and ABPP showed that the activities of proteasome and papain-like cysteine protease were increased in atg5 mutants. Whether these proteases play a back-up role in nutrient recycling and remobilization in atg mutants or act to promote cell death is discussed in relation to their accumulation patterns in the atg5 mutant compared with the salicylic acid-depleted atg5/sid2 double-mutant, and in low nitrate compared with high nitrate conditions. Several of the proteins identified are indeed known as senescence- and stress-related proteases or as spontaneous cell-death triggering factors.


Supplementary Method 2: Shotgun proteomic analysis
Sample preparation for shotgun proteomics.
Rosette leaves were ground and reduced to a fine powder using liquid nitrogen in a mortar. Three biological replicates from each genotype and nutrition treatment were prepared for the shotgun LC-MS/MS analysis. Leaf total proteins were extracted using a TCA-acetone method (Méchin et al., 2007). Briefly, 100 mg of finely ground leaf powder were homogenized in 1.4 ml of cold precipitating solution (10% TCA, 0.07% β-mercaptoethanol in acetone) and incubated at -20°C for 90 min. After centrifugation (20 000 x g at 40°C, 15 min), the pellets were washed three times with cold acetone containing 0.07% βmercaptoethanol and dried using a speedvacuum system. Pellets were solubilized in 100 µl of ZUT buffer containing 6 M urea, 2 M thiourea, 10 mM DTT, 30 mM Tris-HCl pH 8.8 and 0.1% ZALS (zwitterionic acid labile surfactant, Proteabio, Morgantown, WV, USA) and homogenized for 3 min with a vortex. After centrifugation (14 000 x g at 25°C, 30 min), protein concentrations in the supernatant were assayed using the 2D Quant kit (GE healthcare) using BSA as a standard and following supplier's instructions. For each sample, 10 µl of a 4 µg.µl -1 protein solution was equilibrated for 30 min at room temperature. Proteins were alkylated by incubation in darkness for 1 h at room temperature after addition of 2 µl of 330 mM Iodoacetamide (in 50 mM ammonium bicarbonate). Proteins were then diluted ten times by addition of 90 µl of 50 mM ammonium bicarbonate and digested by addition of 4 µl of a trypsin solution (0.2 g.µl -1 in 50 mM acetic acid) at a 50:1 ratio (soluble proteins/trypsin) and incubation overnight at 37°C. Digestion was stopped by addition of 6 µl of 18.6% trifluoroacetic acid (TFA, final concentration of 1%). Digested proteins were desalted using a Strata TM -XL polymeric reversed phase column (100 µm, Phenomex, Le Pecq, France) according to Duruflé et al., (2017) and solubilized with 2% acetonitrile (ACN) and 0.8% formic acid (FA).
Step 2 was repeated for the eight major ions detected in step 1. Dynamic exclusion was set to 40 s. Only the doubly and triply charged precursor ions were subjected to MS/MS fragmentation.
Xcalibur raw data were transformed to mzXML open source format and centroided using the msconvert software in the ProteoWizard 3.0.3706 package (Kessner et al,. 2008). Protein identification was performed using the X!Tandem Piledriver (version 2015.04.01; www.thegpm.org) by querying MS/MS data against the TAIR10 protein library together with a custom contaminant database (trypsin, keratins). Following parameters were used: one missed trypsin cleavage allowed, alkylation of cysteine and oxidation of methionine were set to static and possible modification, respectively. Precursor mass tolerance was set to 10 ppm and fragment ion mass tolerance was 0.02 Da. A refinement search was added with similar parameters except that the missed cleavage was set to three and possible N-terminal acetylation with peptide signal cleavage was searched. Identified proteins were filtered and grouped using X!Tandem Pipeline (3.4.1) (pappso.inra.fr/bioinfo/xtandempipeline/) (Langella et al., 2017) according to: (1) a minimum of two different peptides required with an E value smaller than 0.01, (2) a protein E value (calculated as the product of unique peptide E values) smaller than 10 −5 . The false discovery rates (FDRs) at peptide and protein level were 0.03% and 0.0%, respectively.
Relative quantification was performed using the MassChroQ software (pappso.inra.fr/bioinfo/masschroq/) (Valot et al., 2011) by peak area integration on extracted ion chromatograms (XICs) within a 10 ppm window, after LC-MS/MS chromatogram alignment and spike filtering. Data were than filtered for shared, unreproducible and uncorrelated peptides. The peptides shared by two or more proteins were removed. Peptides which were quantified in less than 5% of the sample were considered unreproducible and removed. Finally, peptides whose intensity profile deviate from the average profile of the peptides belonging to the same protein (with a coefficient of correlation inferior to 0.7) were removed. Relative protein abundance was thus calculated and defined as the sum of XICs intensities of (1) reproducible peptides, (2) specific peptides and (3) correlated peptides belonging to a same protein. When the peptides of a protein were not present or not reproducibly observed in one or several conditions, spectral counting (SC) was used in place of XICs analysis.