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First published online 28 August 2007
doi: 10.1242/jcs.010926

Journal of Cell Science 120, 3279-3288 (2007)
Published by The Company of Biologists 2007
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NO-mediated apoptosis in yeast

Bruno Almeida1, Sabrina Buttner2, Steffen Ohlmeier3, Alexandra Silva1, Ana Mesquita1, Belém Sampaio-Marques1, Nuno S. Osório1, Alexander Kollau4, Bernhard Mayer4, Cecília Leão1, João Laranjinha5, Fernando Rodrigues1, Frank Madeo2 and Paula Ludovico1,*

1 Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal
2 Institute for Molecular Biosciences, Universitätsplatz 2, A-8010 Graz, Austria
3 Proteomics Core Facility, Biocenter Oulu, Department of Biochemistry, University of Oulu, Oulu, Finland
4 Department of Pharmacology and Toxicology, KFUG, Universitätsplatz 2, A-8010 Graz, Austria
5 Faculty of Pharmacy and Center for Neurosciences and Cell Biology, University of Coimbra, 3000 Coimbra, Portugal


Figure 1
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  		Fig. 1. The levels of stress response proteins are increased in both total and purified mitochondrial extracts from H2O2-induced apoptotic cells. Comparison of protein expression levels in total cellular (A) and purified mitochondrial extracts (B) of untreated (control) and H2O2-treated wild-type S. cerevisiae cells. Selected regions of the 2-D gel (isoelectric point/molecular mass) are shown enlarged and the position of altered protein spots are marked with an arrowhead. Putative protein fragments are marked with an asterisk. The apparent isoelectric points and molecular masses of the proteins were calculated with Melanie 3.0 (GeneBio) using identified proteins with known parameters as references.

 

Figure 2
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  		Fig. 2. Yeast cells synthesize NO upon apoptosis induction, which is dependent on L-arginine. (A) NO production in untreated, H2O2-treated and chronologically aged cells was indirectly assessed through measurement of nitrite and nitrate concentrations as described in Materials and Methods. *P<=0.05 versus untreated cells, **P<=0.03 versus 1-day-old cells; t-test, n=3. (B) NO production in untreated and H2O2-treated (1.5 and 2.0 mM) cells assessed by flow cytometric quantification of cells stained with the NO indicator DAF-FM diacetate, in the absence (white area under the peak) or presence (shaded area) of the non-metabolized L-arginine analogue L-NAME. The data are presented in the form of frequency histograms displaying relative fluorescence (x axis) against the number of events analyzed (y axis). (C) Direct measurement of L-arginine-dependent NO production upon H2O2-induced apoptosis. NO production was recorded with a NO-selective electrode (AmiNO-700) upon addition of 4 mM H2O2 to 5x108 wild-type cells (black line) or to wild-type cells pre-incubated with the non-metabolized D-arginine (blue line) or L-NAME (green line). A control experiment without cells was also recorded and is represented as a red line. (D) Rate of NO production is H2O2 dependent. 2 mM or 4 mM of H2O2 was added to 5x108 wild-type cells and NO production assessed using the NO-selective electrode (AmiNO-700). Data presented correspond to the linear part of the NO production curve. Rate of NO production was calculated from the slope.

 

Figure 3
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  		Fig. 3. H2O2-induced apoptotic cells display NOS-like activity. (A) NOS activity assessed in untreated and H2O2-treated wild-type cells. The radioactivity obtained from a negative control consisting of yeast extract boiled for 20 minutes was subtracted from all the samples to remove background radioactivity. Data are expressed as the percentage conversion of L-[3H]arginine to [3H]citrulline. *P<=0.03 versus untreated cells; t-test, n=4. (B) Intracellular amino acid concentrations of untreated (control) and H2O2-treated wild-type cells. *P<=0.05 versus untreated cells; t-test, n=3.

 

Figure 4
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  		Fig. 4. Inhibition of NO production by L-NAME protects yeast cells from H2O2, but not from mammalian Bax expression, or acetic acid-induced apoptosis. (A) Comparison of the survival rate of wild-type cells upon H2O2 treatment with or without pre-incubation with L-NAME in order to inhibit NO production. *P<=0.03 versus wild type; t-test, n=3. (B) Comparison of the survival of wild-type cells upon acetic acid-induced apoptosis with or without pre-incubation with L-NAME. (C) Comparison of the survival of yeast cells (strain BY.bax) upon Bax expression for 15 hours (apoptotic inducing conditions), with or without pre-incubation with L-NAME. (D) Epifluorescence and phase-contrast micrographs of untreated and H2O2-treated (1.5 mM) wild-type cells, with or without pre-incubation with L-NAME, stained with dihydrorhodamine 123 (DHR123) as an indicator of high intracellular ROS accumulation. Bars, 5 µm.

 

Figure 5
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  		Fig. 5. NO scavenged by OxyHb is associated with a delay in cell death during chronological life span and to decreased levels of superoxide anion. (A) Growth curve of wild-type cells after addition of indicated concentrations of OxyHb. (B) Survival determined by clonogenicity during chronological aging of wild-type cells with or without addition of OxyHb, at the indicated concentrations on day 0. (C) Quantification (fluorescence) of ROS accumulation using dihydroethidium (DHE) staining during chronological aging of wild-type cells with or without OxyHb treatment.

 

Figure 6
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  		Fig. 6. GAPDH is extensively fragmented in H2O2-induced apoptotic cells. Comparison of protein expression levels in total cellular (A) and purified mitochondrial extracts (B) of untreated (control) and H2O2-treated wild-type cells. Selected regions of the 2-D gel (isoelectric point/molecular mass) are shown enlarged and the position of altered protein spots are marked with an arrowhead. The apparent isoelectric points and molecular masses of the proteins were calculated with Melanie 3.0 (GeneBio) using identified proteins with known parameters as a reference. Putative protein fragments are marked with an asterisk. Tdh3p fragments are numbered 1 to 4. For each Tdh3p and Tdh2p fragment, matched peptides obtained after trypsin digestion and used for identification of the proteins, as well as the amino acids specific for Tdh3p and Tdh2p, are shown in Fig. S1 in supplementary material.

 

Figure 7
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  		Fig. 7. Deletion of GAPDH isoform 2 and 3 prevents H2O2-induced apoptosis. (A) Comparison of the survival of wild-type, {Delta}tdh2 and {Delta}tdh3 cells upon H2O2 treatment, *P<=0.03 versus wild type, t-test, n=3. (B) Epifluorescence and phase-contrast micrographs of untreated and H2O2-treated (1.5 mM) wild-type, {Delta}tdh2 and {Delta}tdh3 cells, stained with dihydrorhodamine 123 (DHR123) as an indicator of high intracellular ROS accumulation. Bar, 5 µm.

 

Figure 8
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  		Fig. 8. GAPDH is S-nitrosated during H2O2-induced apoptosis. (A) Detection of S-nitrosated GAPDH by immunoprecipitation using an anti-CSNO antibody in cell extracts from untreated cells, H2O2-treated (1.0, 1.5 and 2.0 mM) cells and cells treated for 200 minutes with 2 mM of the NO donor diethylenetriamine/NO (DETA/NO). (B) Quantification of band intensity from A by densitometry. Band intensities were normalized to the intensity of IgG bands. Data express the GAPDH/IgG fold change in comparison to control (lane 1). *P<=0.05 versus control, t-test, n=3. (C) Immunoprecipitation of S-nitrosated GAPDH with an anti-CSNO antibody from cellular extracts of untreated, H2O2-treated (1.5 mM), either in the absence or presence of L-NAME, or chronologically aged cultures (2 and 5 days). (D) Quantification of band intensity from C by densitometry. Band intensities were normalized to the intensity of IgG bands. Data express the GAPDH/IgG fold change in comparison to control (lane 1). *P<=0.05 versus control, t-test, n=3.

 

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© The Company of Biologists Ltd 2007