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
1, also known as histone acetyltrasferases (HATs)), and reversed by lysine deacetylases (KDACs, also known as histone deacetylases (HDACs)) (
1). The enzymatic machinery involved in lysine acetylation is evolutionary conserved in all forms of life (
2–
4). The role of acetylation has been extensively studied in the regulation of gene expression via modification of histones (
5). Acetylation also plays important roles in controlling cellular metabolism (
6–
10), protein folding (
11), and sister chromatid cohesion (
12). Furthermore, acetylation has been implicated in regulating the beneficial effects of calorie restriction (
13), a low nutrient diet without starvation, and aging. Based on these findings, it is proposed that the functional roles of acetylation in these processes are evolutionary conserved from yeast to mammals.Advancements in mass spectrometry (MS)-based proteomics have greatly facilitated identification of thousands of post-translational modification (PTM) sites in eukaryotic cells (
14–
18). Proteome-wide mapping of PTM sites can provide important leads for analyzing the functional relevance of individual sites and a systems-wide view of the regulatory scope of post-translational modifications. Also, large-scale PTM datasets are an important resource for the in silico analysis of PTMs, which can broaden the understanding of modification site properties and their evolutionary trajectories.The budding yeast
Saccharomyces cerevisiae is a commonly used unicellular eukaryotic model organism. Yeast has been used in many pioneering “-omics” studies, including sequencing of the first eukaryotic genome (
19), systems-wide genetic interactions analysis (
20,
21), MS-based comprehensive mapping of a eukaryotic proteome (
22), and proteome-wide analysis of protein-protein interactions (
23,
24). In addition,
S. cerevisiae has been extensively used to study the molecular mechanisms of acetylation. Many lysine acetyltransferases and deacetylases were discovered in this organism (
2,
25), and their orthologs were subsequently identified in higher eukaryotes. Furthermore, the functional roles of many well-studied acetylation sites on histones are conserved from yeast to mammals. Recent data from human and
Drosophila cells show that acetylation is present on many highly conserved metabolic enzymes (
26–
28). However, only a few dozen yeast acetylation sites are annotated in the Uniprot database. Given the presence of a well-conserved and elaborate acetylation machinery in yeast, we reasoned that many more acetylation sites exist in this organism that remained to be identified.Here we used high resolution mass spectrometry-based proteomics to investigate the scope of acetylation in
S. cerevisiae. 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.
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