U. of Lleida - Yeast

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Yeast tetYFH1yhb1 experiment 24h_2017-03-28_10-32-03.sky.zip (1 MB)2017-11-0218316218812
Nitric oxide prevents Aft1 activation and metabolic remodeling in Frataxin-deficient yeast

  • Organism: Saccharomyces cerevisiae
  • Instrument: Agilent 6420 Triple Quadrupole Spectrometer
  • SpikeIn: Yes
Yeast frataxin homolog (Yfh1) is the orthologue of human frataxin, a mitochondrial protein whose deficiency causes Friedreich Ataxia. Yfh1 deficiency activates Aft1, a transcription factor governing iron homeostasis in yeast cells. Although the mechanisms causing this activation are not completely understood, it is assumed that it may be caused by iron-sulfur deficiency. However, several evidences indicate that activation of Aft1 occurs in the absence of iron-sulfur deficiency. Besides, Yfh1 deficiency also leads to metabolic remodeling (mainly consisting in a shift from respiratory to fermentative metabolism) and to induction of Yhb1, a nitric oxide (NO) detoxifying enzyme. In this work, we have used conditional Yfh1 mutant yeast strains to investigate the relationship between NO, Aft1 activation and metabolic remodeling. We have observed that NO prevents Aft1 activation caused by Yfh1 deficiency. This phenomenon is not observed when Aft1 is activated by iron scarcity or impaired iron-sulfur biogenesis. In addition, analyzing key metabolic proteins by a targeted proteomics approach, we have observed that NO prevents the metabolic remodeling caused by Yfh1 deficiency. We conclude that Aft1 activation in Yfh1-deficient yeasts is not caused by iron-sulfur deficiency or iron scarcity. Our hypothesis is that Yfh1 deficiency leads to the presence of anomalous iron species that can compromise iron bioavailability and activate a signaling cascade that results in Aft1 activation and metabolic remodeling.
Experiment Description
Yeast cells were resuspended in 50 mM Tris-HCl buffer pH 7,5 plus 1 mM EDTA and a mixture of protease inhibitors and were disrupted using glass beads. After, SDS was added to a final concentration of 2% and protein extracts were vortexed, boiled and centrifuged (12000 rpm 10 min). Proteins were quantified using the Lowry assay and 30 ug were precipitated with cold acetone (9 volumes) and resuspended in 8M urea, 0.1M ammonium bicarbonate. Then, proteins were subjected to reduction by 12 mM DTT and alquilation by 40mM iodoacetamide. Samples were diluted with 0.1 M ammonium bicarbonate to a final concentration of 1.5 M urea and sequencing grade porcine trypsin (Promega) was added to a final enzyme:substrate ratio of 1:100. After digestion, 0,8 ul from a heavy peptide standards mixture was added to the sample. The approximate concentration of each heavy peptide in this mixture was 3,5 uM. Heavy peptides were obtained from JPT (SpikeTidesTM_L). The resulting peptide mix was purified and enriched using C18 columns (Pierce C-18 Spin Columns, Thermo Scientific). Eluted fractions from the C18 column were evaporated using a Concentrator Plus (Eppendorf) and peptides were resuspended in 5% acetonitrile plus 0,1% formic acid. All peptide samples were analyzed on a triple quadrupole spectrometer (Agilent 6420) equipped with an electrospray ion source. Chromatographic separations of peptides were performed on an Agilent 1200 LC system using a Supelco Bioshell A160 Peptide C18 column (1 mm x 15 cm). Peptides (up to 15 micrograms of protein digest) were separated with a linear gradient of acetonitrile/water, containing 0.1% formic acid, at a flow rate of 75 ul/min. A gradient from 5 to 60% acetonitrile in 60 minutes was used. The mass spectrometer was operated in multiple reaction monitoring mode. Transitions were obtained from SRM atlas and imported into Skyline software (MacLean et al, 2010). Each MRM acquisition was performed with Q1 and Q3 operated at unit resolution (0.7 m/z half maximum peak width). Once validated and optimized, the SRM assays were used to quantify all the analyzed peptides using scheduled SRM mode in a single run (retention time window, 120 s; target scan time, 2 sec).
Sample Description
Two yeast strains were used. - BQS201 (also named tetO7-YFH1), in which the YFH1 promoter is replaced by a tet- promoter. - BSS255 (also named tetO7-YFH1 deltaYHB1), which is a nul YHB1 mutant derived from BQS201. Four biological conditions were analyzed: - BQS201 (control condition) - BQS201 plus doxycycline (this drug represses YFH1 expresion). - BQS255 - BQS255 plus doxycycline Yeasts were grown in YPG and doxycycline, where indicated, added for 24 hours.
Created on 11/2/17 1:12 PM