Figure 4.

The levels of Ate1 protein and global arginylation activity are upregulated during stress. (a) A scheme illustrating how DD-β15-GFP is used as the reporter of arginylation activity. The fusion protein containing a stretch of 15 amino acids starting with two aspartic acids (D) derived from the N terminus of mammalian β-actin, a known substrate of arginylation.³¹ This peptide is fused with an N-terminal ubiquitin, which is cleaved co-translationally by endogenous de-ubiquitylation enzymes in eukaryotic system and leaves the aspartic acids as the new N terminus. The arginylation state of this reporter can be probed with an anti-RDD antibody, which only reacts with the arginylated form of the reporter protein. A C-terminal GFP tag is used to facilitate the detection of steady-state level of the reporter protein by immunoblotting with anti-GFP antibody. (b) The arginylation level of DD-β15-GFP expressed in either WT or ate1Δ yeast was examined with anti-RDD antibody, which only shows a visible signal in the WT cells. An antibody for GFP was used to probe the total protein level of the DD-β15-GFP. PGK was used as a loading control for total yeast cellular proteins. (c) Illustrative scheme (left panel) and representative immunoblots (right panel) showing the arginylation activity in cell lysates prepared from yeast exposed to 1 M NaCl stress for increasing times. The lysates were then mixed with the recombinant protein DD-β15-GFP prepared from ate1Δ yeast for an in-lysate arginylation reaction for 10 min at RT. The arginylation level of the reporter protein was detected by immunoblotting with anti-RDD antibody. The steady-state level of the reporter protein was probed with anti-GFP. An anti-3-phosphoglycerate kinase (PGK) antibody was also used as a loading control. (d) On the left, a scheme illustrating the domain structure of the ‘in locus' GFP-fused Ate1, which is driven be the endogenous ATE1 promoter at the native chromosome locus (Chromosome VII) in the yeast (termed ‘endo: Ate1-GFP'). The right panels present immunoblots showing the steady-state levels of ‘endo: Ate1-GFP' in yeast treated with increasing concentrations of different stressors: H₂O₂ (left) or NaCl (right). Tubulin or PGK was used as loading controls. (e) WT MEFs were exposed to increased concentrations of H₂O₂ for 30 h. The lysates from all these cells, as well as untreated ATE1-KO MEFs (as a control), were then mixed with the recombinant protein DD-β15-GFP purified from ate1Δ yeast for an in-lysate arginylation reaction for 45 min at RT. The arginylation level of the reporter protein was detected by immunoblotting with anti-RDD antibody. The steady-state level of the reporter protein was probed with anti-GFP. Actin antibody was used as a loading control. The graph on the right side shows quantification from four independent repeats. (f) Left: representative immunoblots showing the levels of endogenous Ate1 proteins in MEFs treated with increasing concentrations of H₂O₂ for 30 h, detected by a specific antibody for mouse Ate1 (Millipore, clone 6F11) recognizing all four major Ate1-splicing variants. Actin was used as loading controls. Right: quantification of the endogenous Ate1 level in MEFs treated with H₂O₂ from three independent repeats. The Ate1 level was calculated by normalization with actin loading control, and then further normalized to the level at non-stressing condition (0 μM H₂O₂). In all above figures, error bars represent S.E.M. and statistical significance was calculated by Student's t-test