Proteome-wide Analysis of Lysine Acetylation Suggests its Broad Regulatory Scope in Saccharomyces cerevisiae
2012; Elsevier BV; Volume: 11; Issue: 11 Linguagem: Inglês
10.1074/mcp.m112.017251
ISSN1535-9484
AutoresPeter Henriksen, Sebastian Wagner, Brian T. Weinert, Satyan Sharma, Giedrė Bačinskaja, Michael Rehman, André H. Juffer, Tobias C. Walther, Michael Lisby, Chunaram Choudhary,
Tópico(s)Ubiquitin and proteasome pathways
ResumoPost-translational modification of proteins by lysine acetylation plays important regulatory roles in living cells. The budding yeast Saccharomyces cerevisiae is a widely used unicellular eukaryotic model organism in biomedical research. S. cerevisiae contains several evolutionary conserved lysine acetyltransferases and deacetylases. However, only a few dozen acetylation sites in S. cerevisiae are known, presenting a major obstacle for further understanding the regulatory roles of acetylation in this organism. Here we use high resolution mass spectrometry to identify about 4000 lysine acetylation sites in S. cerevisiae. Acetylated proteins are implicated in the regulation of diverse cytoplasmic and nuclear processes including chromatin organization, mitochondrial metabolism, and protein synthesis. Bioinformatic analysis of yeast acetylation sites shows that acetylated lysines are significantly more conserved compared with nonacetylated lysines. A large fraction of the conserved acetylation sites are present on proteins involved in cellular metabolism, protein synthesis, and protein folding. Furthermore, quantification of the Rpd3-regulated acetylation sites identified several previously known, as well as new putative substrates of this deacetylase. Rpd3 deficiency increased acetylation of the SAGA (Spt-Ada-Gcn5-Acetyltransferase) complex subunit Sgf73 on K33. This acetylation site is located within a critical regulatory domain in Sgf73 that interacts with Ubp8 and is involved in the activation of the Ubp8-containing histone H2B deubiquitylase complex. Our data provides the first global survey of acetylation in budding yeast, and suggests a wide-ranging regulatory scope of this modification. The provided dataset may serve as an important resource for the functional analysis of lysine acetylation in eukaryotes. Post-translational modification of proteins by lysine acetylation plays important regulatory roles in living cells. The budding yeast Saccharomyces cerevisiae is a widely used unicellular eukaryotic model organism in biomedical research. S. cerevisiae contains several evolutionary conserved lysine acetyltransferases and deacetylases. However, only a few dozen acetylation sites in S. cerevisiae are known, presenting a major obstacle for further understanding the regulatory roles of acetylation in this organism. Here we use high resolution mass spectrometry to identify about 4000 lysine acetylation sites in S. cerevisiae. Acetylated proteins are implicated in the regulation of diverse cytoplasmic and nuclear processes including chromatin organization, mitochondrial metabolism, and protein synthesis. Bioinformatic analysis of yeast acetylation sites shows that acetylated lysines are significantly more conserved compared with nonacetylated lysines. A large fraction of the conserved acetylation sites are present on proteins involved in cellular metabolism, protein synthesis, and protein folding. Furthermore, quantification of the Rpd3-regulated acetylation sites identified several previously known, as well as new putative substrates of this deacetylase. Rpd3 deficiency increased acetylation of the SAGA (Spt-Ada-Gcn5-Acetyltransferase) complex subunit Sgf73 on K33. This acetylation site is located within a critical regulatory domain in Sgf73 that interacts with Ubp8 and is involved in the activation of the Ubp8-containing histone H2B deubiquitylase complex. Our data provides the first global survey of acetylation in budding yeast, and suggests a wide-ranging regulatory scope of this modification. The provided dataset may serve as an important resource for the functional analysis of lysine acetylation in eukaryotes. Lysine acetylation is a dynamic and reversible post-translational modification. Acetylation of lysines on their ε-amino group is catalyzed by lysine acetyltransferases (KATs 1The abbreviations used are:KATlysine acetyl transferaseKDAClysine deacetylaseHAThistone acetyl transferaseHDAChistone deacetylaseLysine0L-lysine 12C6,14N2Lysine8L-lysine 13C6,15N2LTQlinear trap quadrupoleHCDhigher-energy C-trap dissociationSGDSaccharomyces genome databaseFDRfalse discovery rateeggnogevolutionary genealogy of genes: nonsupervised orthology groupKOGeurkaryotic orthology groupeuNOGeukaryotic nonsupervised orthology groupSTRINGSearch Tool for the Retrieval of Interacting Genes/ProteinsSILACstable isotope labeling by amino acids in cell cultureGOBPGO biological processKEGGKyoto encyclopedia of genes and genomesMDmolecular dynamics.1The abbreviations used are:KATlysine acetyl transferaseKDAClysine deacetylaseHAThistone acetyl transferaseHDAChistone deacetylaseLysine0L-lysine 12C6,14N2Lysine8L-lysine 13C6,15N2LTQlinear trap quadrupoleHCDhigher-energy C-trap dissociationSGDSaccharomyces genome databaseFDRfalse discovery rateeggnogevolutionary genealogy of genes: nonsupervised orthology groupKOGeurkaryotic orthology groupeuNOGeukaryotic nonsupervised orthology groupSTRINGSearch Tool for the Retrieval of Interacting Genes/ProteinsSILACstable isotope labeling by amino acids in cell cultureGOBPGO biological processKEGGKyoto encyclopedia of genes and genomesMDmolecular dynamics., also known as histone acetyltrasferases (HATs)), and reversed by lysine deacetylases (KDACs, also known as histone deacetylases (HDACs)) (1Yang X.J. 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We identified about 4000 unique acetylation sites in this important model organism. Bioinformatic analysis of yeast acetylation sites and comparison with previously identified human and Drosophila acetylation sites indicates that many acetylation sites are evolutionary conserved. Furthermore, quantitative analysis of the Rpd3-regulated acetylation sites identified several nuclear proteins that showed increased acetylation in rpd3 knockout cells. Our results provide a systems-wide view of acetylation in budding yeast, and a rich dataset for functional analysis of acetylation sites in this organism. S. cerevisiae cells from a lysine auxotroph strain (BY4742) were grown in a synthetic complete medium containing "light" lysine (l-lysine 12C6,14N2, or Lysine0) or "heavy" lysine (l-lysine 13C6,15N2, or Lysine8) for more than 10 generations to an absorbance (measured at 600 nm (OD600)) value of ∼0.7. 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The wild-type control strain (BY4742) was grown in media supplemented with "light" lysine whereas rpd3Δ cells were cultured in "heavy" lysine containing media, to an OD600 of ∼0.5. Cells were harvested by centrifugation at 3000 × g for 5 min, washed once in water, and resuspended in lysis buffer (150 mm KAc, 2 mm MgAc, 20 mm Hepes, pH 7.4) supplemented with Protease Inhibitor Mixture (Roche). The cell-suspension was instantly frozen in liquid nitrogen and cells were cryo-grinded using a MM400 ball mill (Retsch) for 2 × 3 min at 25 Hz. Frozen powdered lysates were transferred to 50 ml tubes, thawed, and Triton-X was added to a final concentration of 1%, followed by incubation at 4 °C for 30 min. Extracts were centrifuged twice at 3500 × g for 10 min to remove cellular debris. Protein concentrations were measured using BCA protein assay kit (Thermo Scientific) and lysates were stored at −80 °C until further use. Cell lysates were thawed and 20 mg of proteins from "light" and "heavy" labeled samples were mixed. Proteins were precipitated with 4 volumes of ice-cold acetone. After incubation at −20 °C for more than 1 h, the precipitate was pelleted at 3000 × g for 10 min and the liquid phase was discarded. The protein pellet was dissolved in 8 m urea solution (6 m urea, 2 m thiourea, 10 mm HEPES pH 8.0) and the protein concentration was measured using Bradford assay (BioRad, Hercules, CA). Protein samples were treated with 1 mm DTT for 45 min and subsequently alkylated with 5.5 mm iodoacetamide for 45 min in the dark. Proteins were digested with endoproteinase Lys-C (1 μg Lys-C per 100 μg protein) for 12–16 h at 25 °C. Samples were subsequently diluted with H2O to reduce the urea concentration to 2 m and the pH was adjusted to 8.0 with 1 m ammonium bicarbonate solution. Trypsin endoproteinase was added (1 μg Trypsin per 100 μg protein) and the samples were further digested for 12–16 h at 25 °C. Trypsin activity was stopped by addition of trifluoroacetic acid (TFA) to a final concentration of 1% and the peptide solution was incubated at 4 °C for 1–2 h. The solution was clarified by centrifugation at 3000 × g and peptides were purified using a C18 Sep-Pak cartridges (Waters, Milford, MA). Peptides were eluted from the C18 cartridges with 5 × 2 ml of 40% acetonitrile, 0.1% TFA in H2O. To remove acetonitrile, the sample was freeze-dried. Peptides were redissolved in immune affinity purification buffer (50 mm 3-(N-morpholino)propanesulfonic acid pH 7.2, 10 mm Na3PO4, 50 mm NaCl) and the peptide solution was clarified by centrifugation at 13000 × g for 5 min. Fifty microliters of agarose conjugated anti-acetyllysine antibodies (ImmuneChem Pharmaceuticals Inc., Burnaby, Canada, catalog number ICP0388) were added to the peptide mix (∼20 mg) and incubated on a rotating wheel at 4 °C for 12–16 h. 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Peptide sequences were searched using trypsin specificity and allowing a maximum of two missed cleavages. Carbamidomethylation of cystein was searched as fixed modification and methionine oxidation, N-terminal acetylation, and lysine acetylation were added as variable modifications. MaxQuant presearch determined spectra that result from heavy stable isotope labeling by amino acids in cell culture (SILAC) labeled peptides and searched these with the additional fixed modification of Lys8, whereas spectra with a SILAC state not determined in presearch were searched with Lys8 as additional variable modification. Lysine acetylation sites were required to be located internally within modified peptide sequences. Database searching was performed with 6 ppm mass tolerance for precursor ions and 20 ppm for fragment ions. The false discovery rate (FDR) was estimated using a target-decoy approach (41Elias J.E. Gygi S.P. 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First, all modified yeast peptide sequences were mapped to eggNOG protein sequences and orthology groups. Only peptides matching to sequences in a single orthology group were considered for
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