Journal: Cell Death & Disease
Article Title: Arginyltransferase1 drives a mitochondria-dependent program to induce cell death
doi: 10.1038/s41419-025-07917-1
Figure Lengend Snippet: A The left panel shows yeast cells containing Ate1-GFP and Ate1-GFP-Ras2, which were allowed to express for 6 h with 2% galactose induction and then briefly treated with oxidative stressor H 2 O 2 for 10 min before the microscopic images were taken. The regular Ate1-GFP forms puncta-like structures, similar as shown in Fig. , while Ate1-GFP-Ras2 remains localized to the periphery of the cells. The right panel shows quantitative measurement of the distribution of the GFP signal. This was done by calculating the ratio of the mean GFP signal from the plasma membrane (using profile method on Zeiss ZenBlue software; stroke = 1 pixel width) versus the mean GFP throughout the whole cell (on 10 randomly chosen individual yeast cells). Homoscedasticity was determined via Levene’s test, before being analysed via a two-tailed Student’s t -test ( n = 10). Error bars represent S.D. B Scheme illustrating the principle of the reporter for the arginylation activity inside yeast cells. The N-terminal ubiquitin domain of the reporter protein DD-β15-mCherryFP will be promptly removed by endogenous de-ubiquitination (de-Ub) enzymes in the cell, exposing the penultimate peptide DD-β15, which is derived from the N-terminus of mouse β-actin and is known to be arginylated in vivo [ , ]. The arginylated N-terminus can be recognized with a specific antibody anti-RDD . Antibodies for mCherryFP (mChFP) and GFP can be used to probe the levels of the reporter protein and the GFP-fused ATE1, respectively. C To test the arginylation activity of different forms of Ate1 (Ate1-GFP or Ate1-GFP-Ras2), they were expressed in ate1 Δ yeast (to avoid the interference of endogenous ATE1), which was also simultaneously expressing the reporter protein DD-β15-mCherryFP. The arginylation level of the reporter protein was measured as described in ( B ). To avoid potential carryover of antibody signals, the same set of samples were loading twice in different gels for the probing of anti-RDD and anti-mCherry, separately. Pgk1 serves as loading controls for the yeast proteins. The left panel shows representative WB images while the right panel shows quantification from multiple repeats ( n = 4). D Growth test of ate1 Δ yeast cells carrying either the empty expression vector, or the one containing Ate1-GFP or Ate1-GFP-Ras2 was conducted by a serial dilution growth assay on either plate containing glucose or galactose, where the expression of Ate1 is not induced or induced, respectively. E The left panel shows representative Western blots showing the expression of different Ate1 constructs in total cell lysate. The level of Ate1 was probed by its GFP fusion tag and Pgk1 was used as a loading control. The employed Ate1 constructs include the original Ate1-GFP as control (labelled as “cont”), and the forms that are expected to be targeted to the mitochondrial matrix (Mt) or mitochondrial intermembrane space (IMS). The IMS- and Mt- targeting sequences were derived from S. cerevisiae Cytochrome b2 (Cyb2) with or without a deletion of a 19 amino acid(Aa) stop-transfer transmembrane signal as described in published studies [ , , ]. To avoid disrupting mitochondria, the expression of these different Ate1 constructs was achieved by adding 0.5% galactose and incubated for 3 h at 30 °C. The signal of the GFP-fused Ate1 were probed by anti-GFP with Pgk1 as loading controls for total proteins. The right panel shows quantification ( n = 6). F Similar to ( E ), except that the mitochondrial-specific fractions, treated with proteinase K to remove any proteins attached on the outside, were used to prepare the lysates for the measurement of the levels of Ate1 inside mitochondria, with Cmc2 as the loading controls for mitochondrial proteins. The right panel shows quantification ( n = 3). G Growth test of WT W303 yeast cells carrying either the empty expression vector, Ate1-GFP (cont-), the matrix localized Ate1-GFP (Mt-), or the IMS located Ate1-GFP (IMS-) by a serial dilution growth assay on either plate containing galactose or glucose for the induced expression (or not) of Ate1. Note that the concentration of galactose (0.5%) is lower and the cell loading were higher than elsewhere to allow the display of the difference between the different forms of Ate1. Plates were incubated at 30 °C and images were taken after 3 days.
Article Snippet: Western blot blocking reagent was obtained from Roche (catalogue number 75255200) The primary antibodies include: monoclonal mouse anti-GFP (from Roche, clone 7.1 and 13.1, Cat# 11814460001) Rabbit anti-yeast alpha tubulin (Abcam EPR13799 ) anti-yeast- phosphoglycerate kinase1 (Pgk1) (Thermofischer scientific # 459250), Rabbit anti-yeast Cmc2 was a gift from Dr. Antonio Barrientos (University of Miami) mouse anti-Grp78 (SCBT# HDEL Antibody (2E7): sc-53472) HSP70 Monoclonal antibody (Proteintech catalogue# 66183-1) The custom-produced rabbit anti-RDD antibody was ordered from Genscript INC as described in our previous work [ ].
Techniques: Clinical Proteomics, Membrane, Software, Two Tailed Test, Activity Assay, Ubiquitin Proteomics, Derivative Assay, In Vivo, Expressing, Plasmid Preparation, Serial Dilution, Growth Assay, Western Blot, Construct, Control, Incubation, Concentration Assay
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![A The left panel shows yeast cells containing Ate1-GFP and Ate1-GFP-Ras2, which were allowed to express for 6 h with 2% galactose induction and then briefly treated with oxidative stressor H 2 O 2 for 10 min before the microscopic images were taken. The regular Ate1-GFP forms puncta-like structures, similar as shown in Fig. , while Ate1-GFP-Ras2 remains localized to the periphery of the cells. The right panel shows quantitative measurement of the distribution of the GFP signal. This was done by calculating the ratio of the mean GFP signal from the plasma membrane (using profile method on Zeiss ZenBlue software; stroke = 1 pixel width) versus the mean GFP throughout the whole cell (on 10 randomly chosen individual yeast cells). Homoscedasticity was determined via Levene’s test, before being analysed via a two-tailed Student’s t -test ( n = 10). Error bars represent S.D. B Scheme illustrating the principle of the reporter for the arginylation activity inside yeast cells. The N-terminal ubiquitin domain of the reporter protein DD-β15-mCherryFP will be promptly removed by endogenous de-ubiquitination (de-Ub) enzymes in the cell, exposing the penultimate peptide DD-β15, which is derived from the N-terminus of mouse β-actin and is known to be arginylated in vivo [ , ]. The arginylated N-terminus can be recognized with a specific antibody anti-RDD . Antibodies for mCherryFP (mChFP) and GFP can be used to probe the levels of the reporter protein and the GFP-fused ATE1, respectively. C To test the arginylation activity of different forms of Ate1 (Ate1-GFP or Ate1-GFP-Ras2), they were expressed in ate1 Δ yeast (to avoid the interference of endogenous ATE1), which was also simultaneously expressing the reporter protein DD-β15-mCherryFP. The arginylation level of the reporter protein was measured as described in ( B ). To avoid potential carryover of antibody signals, the same set of samples were loading twice in different gels for the probing of anti-RDD and anti-mCherry, separately. Pgk1 serves as loading controls for the yeast proteins. The left panel shows representative WB images while the right panel shows quantification from multiple repeats ( n = 4). D Growth test of ate1 Δ yeast cells carrying either the empty expression vector, or the one containing Ate1-GFP or Ate1-GFP-Ras2 was conducted by a serial dilution growth assay on either plate containing glucose or galactose, where the expression of Ate1 is not induced or induced, respectively. E The left panel shows representative Western blots showing the expression of different Ate1 constructs in total cell lysate. The level of Ate1 was probed by its GFP fusion tag and Pgk1 was used as a loading control. The employed Ate1 constructs include the original Ate1-GFP as control (labelled as “cont”), and the forms that are expected to be targeted to the mitochondrial matrix (Mt) or mitochondrial intermembrane space (IMS). The IMS- and Mt- targeting sequences were derived from S. cerevisiae Cytochrome b2 (Cyb2) with or without a deletion of a 19 amino acid(Aa) stop-transfer transmembrane signal as described in published studies [ , , ]. To avoid disrupting mitochondria, the expression of these different Ate1 constructs was achieved by adding 0.5% galactose and incubated for 3 h at 30 °C. The signal of the GFP-fused Ate1 were probed by anti-GFP with Pgk1 as loading controls for total proteins. The right panel shows quantification ( n = 6). F Similar to ( E ), except that the mitochondrial-specific fractions, treated with proteinase K to remove any proteins attached on the outside, were used to prepare the lysates for the measurement of the levels of Ate1 inside mitochondria, with Cmc2 as the loading controls for mitochondrial proteins. The right panel shows quantification ( n = 3). G Growth test of WT W303 yeast cells carrying either the empty expression vector, Ate1-GFP (cont-), the matrix localized Ate1-GFP (Mt-), or the IMS located Ate1-GFP (IMS-) by a serial dilution growth assay on either plate containing galactose or glucose for the induced expression (or not) of Ate1. Note that the concentration of galactose (0.5%) is lower and the cell loading were higher than elsewhere to allow the display of the difference between the different forms of Ate1. Plates were incubated at 30 °C and images were taken after 3 days.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_7888/pmc12357888/pmc12357888__41419_2025_7917_Fig3_HTML.jpg)