Research ArticlePosttranslational modification

Proteome-Wide Mapping of the Drosophila Acetylome Demonstrates a High Degree of Conservation of Lysine Acetylation

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Science Signaling  26 Jul 2011:
Vol. 4, Issue 183, pp. ra48
DOI: 10.1126/scisignal.2001902

Abstract

Posttranslational modification of proteins by acetylation and phosphorylation regulates most cellular processes in living organisms. Surprisingly, the evolutionary conservation of phosphorylated serine and threonine residues is only marginally higher than that of unmodified serines and threonines. With high-resolution mass spectrometry, we identified 1981 lysine acetylation sites in the proteome of Drosophila melanogaster. We used data sets of experimentally identified acetylation and phosphorylation sites in Drosophila and humans to analyze the evolutionary conservation of these modification sites between flies and humans. Site-level conservation analysis revealed that acetylation sites are highly conserved, significantly more so than phosphorylation sites. Furthermore, comparison of lysine conservation in Drosophila and humans with that in nematodes and zebrafish revealed that acetylated lysines were significantly more conserved than were nonacetylated lysines. Bioinformatics analysis using Gene Ontology terms suggested that the proteins with conserved acetylation control cellular processes such as protein translation, protein folding, DNA packaging, and mitochondrial metabolism. We found that acetylation of ubiquitin-conjugating E2 enzymes was evolutionarily conserved, and mutation of a conserved acetylation site impaired the function of the human E2 enzyme UBE2D3. This systems-level analysis of comparative posttranslational modification showed that acetylation is an anciently conserved modification and suggests that phosphorylation sites may have evolved faster than acetylation sites.

Introduction

Reversible posttranslational modification (PTM) of proteins plays a critical role in cell signaling by regulating protein activity, stability, localization, and protein-protein interactions. Phosphorylation of serine, threonine, and tyrosine is one of the most prevalent and extensively studied PTMs. Reversible acetylation of lysine is another abundant modification with fundamentally important regulatory functions. Acetylation is catalyzed by conserved enzymatic machinery composed of lysine acetyltransferases [KATs, also known as histone acetyltransferases (HATs)] and lysine deacetylases [KDACs, also known as histone deacetylases (HDACs)], which span the phylogenetic tree from bacteria to humans (1). The cellular role of lysine acetylation has been studied for more than 4 decades, and its function in epigenetic regulation of histones is well established. However, acetylation also plays a prominent role outside the nucleus, regulating diverse cellular processes such as protein folding, cytoskeleton dynamics, and cellular metabolism (2). Deregulated lysine acetylation is implicated in several age-associated diseases, including cancer, diabetes, and neurodegenerative disorders. Hence, two KDAC inhibitors are approved for clinical use in cancer therapy, and several others are currently being tested in clinical trials to treat various illnesses, including cancer, muscular dystrophy, Huntington’s disease, and HIV infection (3, 4). Lysine acetylation is thought to regulate energy metabolism in response to calorie restriction and may play an important role in aging. In particular, genetic and biochemical evidence show that sirtuins, NAD+ (nicotinamide adenine dinucleotide)–dependent KDACs, are associated with life-span regulation in several model organisms, including yeast, flies, worms, and mice (5).

Advancements in mass spectrometry (MS)–based proteomics have greatly facilitated the identification of PTMs (6). The number of phosphorylation sites identified by such proteomic studies now numbers in the tens of thousands. Previously, we used high-resolution MS methods combined with efficient enrichment of lysine-acetylated peptides to identify more than 3000 in vivo acetylation sites in human cells (7). Large-scale mapping of cellular PTMs facilitates the study of modified proteins and enables analysis of evolutionary conservation of these sites. Several studies have compared evolutionary conservation of phosphorylation sites (810). Surprisingly, the evolutionary conservation of phosphorylated serine and threonine residues is only marginally, yet significantly, higher than that of unmodified serines and threonines. In contrast to phosphorylation, the evolutionary conservation of other reversible PTMs on a proteome-wide level is unknown, largely because no other PTM has been identified in sufficient numbers and in different organisms to allow such an analysis. The fruit fly Drosophila melanogaster is a well-studied model organism that has been used in a number of high-throughput proteomic screens that identified thousands of phosphorylation sites by MS (1113). Here, we use high-accuracy MS to identify lysine acetylation sites in fruit flies. We compared the Drosophila and human acetylomes to identify acetylation sites that are positionally conserved between these two organisms, which diverged more than 500 million years ago (14). Furthermore, we compared the conservation of lysine acetylation with conservation of phosphorylation to understand the relative conservation of these two different PTMs. We additionally characterized a conserved acetylation site on the N terminus of the human E2 ubiquitin-conjugating enzyme UBE2D3 and show that acetylation reduced its enzymatic function in vitro. Genetic studies of a yeast homolog of UBE2D3, Ubc4, provided evidence for this regulation in vivo as well.

Results

Lysine acetylation in Drosophila

Drosophila acetylation sites were identified by antibody enrichment of lysine-acetylated peptides from cell culture lysate followed by online reversed-phase liquid chromatography–tandem MS (LC-MS/MS), as described previously (7). In five experimental replicates, we identified 1981 unique acetylation sites on 1013 proteins at a false discovery rate of 1% (Table 1 and table S1). The acetylated peptides identified in this study were measured with a high [parts per million (ppm)] mass accuracy (Table 1 and Fig. 1A). Furthermore, most (~85%) acetylated peptides were sequenced with the higher-energy C-trap dissociation (HCD) method in which both intact peptide ions and their fragment ions are detected in the high mass accuracy Orbitrap detector (15). A large fraction of the acetylated peptides were sequenced multiple times, thereby providing additional confidence in these identifications. More than 73% of identifications were supported by two or more sequencing events, and about half of the identifications were supported by more than five independent sequencing events (Fig. 1B). Acetylation neutralizes the positive charge on lysine side chains, preventing peptide cleavage by trypsin and lysyl endopeptidase C (Lys-C). We restricted acetylation site identification to sites that occurred only on internal, miscleaved (not cleaved by trypsin or Lys-C) lysine residues, thereby indicating that these lysine residues were acetylated before trypsin and Lys-C digestion and did not result from subsequent biochemical manipulations. Acetylation sites were most often confidently localized to single internal lysine residues within the peptide sequences, allowing precise mapping of acetylated lysine residues. As a result, the average localization probability of acetylated lysines was 98% in these data.

Fig. 1

Identification of in vivo lysine acetylation sites in Drosophila. (A) Relative mass deviation of all acetylated peptide ions. (B) Distribution of the number of peptide fragment (MS2) scans supporting each acetylated peptide identification. The proportion of total acetylated peptide identifications is indicated in black and the number of MS2 scans/peptide identification is shown in red. (C) Sequence properties of Drosophila and human lysine acetylation sites. (D) Acetylated peptides are shorter than expected. Peptide size distribution box plots for the indicated categories of peptides; outer whiskers end at 2% and 98% of data, inner hatch marks show 9% and 91% of data, and outliers are shown as open circles. Statistical significance was calculated with a two-tailed t test assuming unequal variance.

Table 1

Summary of Drosophila acetylome determination.

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To compare the Drosophila and human acetylomes, we performed functional annotation to determine Gene Ontology (GO) terms associated with acetylated proteins. Similarly to the human acetylome, Drosophila acetylation sites were found on a diverse set of cellular proteins with varying molecular functions (fig. S1). We compared the Drosophila and human acetylated sites to nonacetylated lysine residues to reveal any biases that may occur for neighboring amino acids at acetylation sites (Fig. 1C). Although we did not detect a strong bias for a specific acetylation site motif, there were modest biases for flanking amino acid residues that were similar in the Drosophila and human acetylomes. Glycine and glutamic acid occurred most frequently at the −1 position, whereas tyrosine, phenylalanine, and proline occurred frequently at the +1 position. Lysine residues occurred at the −5, −6, +3, +4, +5, and +6 positions, suggesting that acetylation preferentially occurs in lysine-rich regions of proteins.

Because trypsin and Lys-C, which were used to digest protein to peptides in this study, cleave after lysine residues, a bias for flanking lysine residues further suggests that acetylated peptides identified by MS should be smaller, on average, than nonacetylated peptides. We compared the size distribution of acetylated peptides, which were miscleaved and contained internal acetylated lysine residues, with the size distributions of nonacetylated peptides and nonacetylated, miscleaved peptides containing an internal lysine residue (Fig. 1D). We expected that miscleaved peptides would be longer than completely cleaved peptides. However, we found that acetylated peptides were, on average, significantly shorter than nonacetylated peptides. This difference was even greater and more significant when comparing acetylated peptides to nonacetylated, miscleaved peptides. These observations indicate that acetylation occurs in lysine-rich regions of proteins.

Site-level PTM conservation analysis

Functionally important modification sites are more likely to be evolutionarily conserved (10). Thus, identification of evolutionarily conserved acetylation sites may help to identify functionally important sites on proteins. In addition, comparative analysis of phosphorylation and acetylation in Drosophila and humans provides insight into the relative conservation of these PTMs during evolution. To perform PTM conservation analysis, we used a strategy that is similar to that used in studies examining phosphorylation conservation (8, 9). Because only a limited number of tyrosine phosphorylation sites are known in Drosophila, we compared only the conservation of serine and threonine phosphorylation sites. The phosphorylation and acetylation data compared in this study were acquired with a similar high-resolution MS technology and computational platform, thereby providing data sets that are more directly comparable. To use the most current list of protein orthologs and to compare previously published phosphoproteome and acetylome data, we mapped the modified peptides from this study, as well as from previous studies, to the Ensembl database version 59 (August 2010). Protein orthologies and sequence alignments were obtained with the Ensembl Compara multispecies database. Individual modified peptides identified by MS may match to multiple Ensembl entries primarily because (i) the identified peptide sequence is not unique and thus matches multiple distinct proteins derived from distinct genes or (ii) the modified peptide may match to a unique gene, but the Ensembl database contains multiple redundant entries for the same gene. To prevent overcounting of conserved modification sites that map to multiple entries, we defined unique modification sites by the position of the modification site within the identified peptide sequence. Therefore, for our analysis, the number of modified sites used to calculate conservation of acetylation or phosphorylation sites was based on unique entries only. The modifiable amino acids can be conserved at the site level (the amino acid, for example, lysine, but not the PTM, for example, acetylation, is conserved at the same position in the orthologous protein) or at the PTM level (the PTM, for example, acetylated lysine, occurs at the same position in orthologous proteins in both species) (Fig. 2). We examined site-level conservation by comparing the frequency of amino acid conservation at modified sites with the frequency of conservation at unmodified sites. Site conservation was evaluated only in orthologs of proteins that contain the modification, thereby avoiding biases that might arise due to differences in the conservation of amino acids in all proteins compared to modified proteins. We examined PTM-level conservation by determining whether the same PTM was identified at the same position in orthologous proteins in both species. PTM conservation was evaluated only within the group of protein orthologs that were identified as modified in both Drosophila and humans, because it is possible to identify PTM-level conservation only on proteins that are modified by the same PTM.

Fig. 2

Strategy for comparative PTM conservation analysis. The steps used in comparing phosphoserine, phosphothreonine, and acetyllysine conservation in Drosophila (Dm) and humans (Hs) are shown. Numbers of unique modification sites present at each step are shown on the right.

Conservation of lysine acetylation sites was determined by comparing the 1968 Drosophila sites identified here with a previously published data set of 3527 human acetylation sites (7). Conservation of phosphorylated serine and threonine was similarly compared using a data set of 9578 Drosophila (13) and 23,709 human (16) serine and threonine sites. We found PTM conservation (the PTM occurs at the same position in orthologous proteins in both species) at a total of 312 and 343 positions in Drosophila and humans, respectively (Fig. 2 and tables S2 and S3). The number of conserved acetylations in humans is higher because several human proteins can share a single Drosophila ortholog; thus, the absolute number of conserved acetylations is not the same in both species. Although we start with a similar number of phosphorylated threonines (pTs) and acetylated lysines (acK), the number of conserved acetylations is about sixfold higher than the number of conserved phosphothreonines (Fig. 2). These differences were further analyzed in detail by comparing the frequencies of acetylation and phosphorylation conservation at the site level and PTM level. We observed a strong bias toward conservation of lysine residues at sites of acetylation when comparing both Drosophila sites to human orthologs and human sites to Drosophila orthologs (Fig. 3A). Lysine residues were twofold more frequently conserved (P < 1 × 10−6) at sites of acetylation compared to lysine residues at unmodified sites, whereas serine or threonine phosphorylation sites were only marginally or not significantly different. For example, we observed the largest bias toward conservation of phosphorylated residues (1.08-fold) when we compared Drosophila phosphoserine sites to their human orthologs. Phosphorylation site conservation was not significantly higher when allowing for phosphorylated serine and threonine residues to be conserved as either serine or threonine (fig. S2). Thus, the magnitude of acetylated lysine site conservation is significantly higher than conservation of either phosphorylated serine or phosphorylated threonine sites. In addition, the frequency of PTM-level conservation was three- to fourfold higher (P < 1 × 10−6) for lysine acetylation than it was for either serine or threonine phosphorylation (Fig. 3B). These data indicate that acetylation is more highly conserved than phosphorylation, both in the frequency of amino acid conservation at sites of modification and in the frequency of identifying the same PTM at the corresponding position in orthologs.

Fig. 3

Conservation of lysine acetylation. (A) Lysine residues are significantly more frequently conserved at sites of acetylation compared with conservation of serine or threonine residues at sites of phosphorylation. Frequency of amino acid conservation is compared for unmodified [serine (S), threonine (T), and lysine (K)] and modified [phosphoserine (pS), phosphothreonine (pT), and acetyllysine (acK)] amino acids. Drosophila (Dm) sites are compared to human (Hs) orthologs and human sites are compared to Drosophila orthologs. N.S. = not significant. (B) Lysine acetylation is more frequently conserved than is serine or threonine phosphorylation. PTM conservation indicates that the modification occurs at the same position in both species. (C) Acetylated lysine residues are more frequently conserved than unmodified lysine residues when comparing Drosophila or human acetylation sites to zebrafish (D. rerio) and nematode (C. elegans) orthologs. Significant differences (*P = 3.6 × 10−3, **P < 1 × 10−6) refer to the preceding (left) category. Statistical significance in all three panels was calculated with a Fisher exact test.

To further analyze the conservation of lysine acetylation sites, we performed bioinformatics analysis to determine whether lysine residues identified as acetylated in Drosophila and humans were conserved in zebrafish (Danio rerio) and nematodes (Caenorhabditis elegans). The frequency of lysine conservation in zebrafish and nematode orthologs was determined for (i) nonacetylated Drosophila or human lysine residues, (ii) all acetylated Drosophila or human lysine residues, (iii) Drosophila or human acetylated lysines with site conservation, and (iv) Drosophila or human acetylated lysines with PTM-level conservation (Fig. 3C and tables S2 and S3). These data showed that lysines acetylated in Drosophila and humans are more frequently conserved than nonacetylated lysine residues (P < 1 × 10−6). Acetylated lysines with site conservation were significantly (P < 1 × 10−6) more frequently conserved than acetylated lysines in general, and acetylation sites with PTM-level conservation were the most likely to have lysine residues conserved at the same position in zebrafish and nematodes. The strong (up to threefold) bias for lysine conservation in zebrafish and nematodes at sites of conserved acetylation in Drosophila and humans suggests that acetylation is likely to occur at many of these positions in all four species.

A higher degree of conserved acetylation relative to serine and threonine phosphorylation is reflected by a greater relative prevalence of acetylation in prokaryotes, a trend that was inverted in eukaryotic organisms (Fig. 4A). We observed this trend despite more efficient enrichment of phosphorylated peptides compared to acetylated peptides. Furthermore, we found that, in humans, a significantly higher proportion of acetylation, compared to serine and threonine phosphorylation, occurs on mitochondrial proteins (Fig. 4B). Although we found that a similar number of mitochondrial proteins were modified by acetylation or phosphorylation, the serine and threonine phosphoproteome of humans was around fivefold larger than the acetylome (Fig. 4B). Thus, the similar number of phosphorylated and acetylated proteins in mitochondria indicates that acetylation is more prevalent in mitochondria relative to the total cellular distribution of these two modifications. This observation is consistent with the endosymbiotic theory that suggests mitochondria are of prokaryotic origin. Thus, mitochondria, like prokaryotes, have a higher relative prevalence of acetylation than that found in the eukaryotic organisms Drosophila and humans. Together, these data suggest that regulation of protein function by acetylation may be anciently conserved, whereas modification by phosphorylation has been expanding and diversifying over evolutionary time.

Fig. 4

Comparison of acetylation and phosphorylation in prokaryotes, eukaryotes, and mitochondria. (A) The chart shows the numbers of modification sites identified in different organisms in the most comprehensive studies to date. The numbers of identified sites come from the following studies: phosphorylation in E. coli (57), B. subtilis (58), L. lactis (59), H. salinarum (60), H. sapiens (16), and D. melanogaster (13); acetylation in E. coli (18), S. enterica (17), H. sapiens (7), and D. melanogaster (this study). (B) The proportion of modified human proteins that are mitochondrial or the proportion of modified sites on human mitochondrial proteins is shown. Mitochondrial proteins were classified with the gene assignments from the MitoCarta database (http://www.broadinstitute.org/pubs/MitoCarta/index.html). P values were calculated with a Fisher exact test. (C) Acetylation of mitochondrial proteins is proportionally greater than phosphorylation of mitochondrial proteins. The total numbers of modified (phosphorylated or acetylated) mitochondrial and nonmitochondrial proteins are shown.

Functional annotation of conserved acetylation

The evolutionary conservation of Drosophila and human lysines at acetylation sites in diverse organisms suggests that modification of these sites has conserved regulatory roles. To gain insight into the potential functional implications of acetylation, we compared the enrichment of GO terms within the data set of all human genes encoding acetylated proteins to only human genes encoding proteins with conserved acetylation (Fig. 5). We used human genes in this analysis because GO annotation of human genes is greater than the annotation of Drosophila genes and conserved acetylation, by definition, occurred on both the human gene product and its Drosophila ortholog. The most prevalent enrichment occurred in GO terms relating to energy and metabolism, protein folding, translation, ribosomes and RNA processes, chromatin, the ubiquitin-proteasome system, and the actin cytoskeleton (Fig. 5), and these terms covered 89% of the genes encoding proteins with conserved acetylation, thus providing a good overview of the major cellular processes regulated by acetylation.

Fig. 5

Functional annotation of conserved acetylation. Functional annotation terms are derived from the following categories: GO terms “Biological Process,” “Cellular Component,” and “Molecular Function” and Swiss-Prot keywords. Fold enrichment is relative to the expected frequency of the term associated with genes in the entire genome, # indicates the number of genes associated with the term, % indicates the percentage of genes encoding acetylated proteins associated with the term, and the P value indicates the significance of the enrichment of the term.

Acetylation may be functionally important in regulating metabolic pathways (1719), and we identified conserved acetylation sites on several metabolic enzymes that have been previously shown to be regulated by lysine acetylation. These enzymes include glyceraldehyde phosphate dehydrogenase (GAPDH) (17), phosphoglycerate kinase (PGK) (17), acetyl coenzyme A (CoA) synthetase (AceCS) (20, 21), malate dehydrogenase (MDH) (19), and 3-hydroxy-3-methylglutaryl CoA synthase 2 (HMGCS) (22). We found that these metabolic enzymes were often multiply acetylated and contained sites where acetylation was observed in both Drosophila and humans (tables S2 and S3).

The enrichment of terms associated with chromatin and nucleosomes is expected because of the well-established role of acetylation as a histone modification involved in regulation of chromatin structure (23). We identified 26 conserved acetylation sites on histones. Because histone lysine acetylation is well studied, we do not characterize or describe these sites in detail here. Instead, we focused on other acetylated proteins that function in chromatin assembly. Barrier-to-autointegration factor (BAF) is a DNA-bridging protein that is conserved in metazoans (24, 25). We found a conserved acetylation at Lys64 in human BAF. Previous studies have shown that a K64E mutant BAF fails to promote chromatin assembly (26) and has enhanced binding to histone H1 and histone H3 (27). Similar to K64E substitution, acetylation at K64 may affect BAF activity by promoting histone H1 or histone H3 binding, or both.

We identified enrichment of conserved acetylation in several points in the protein synthesis and folding process. We identified 12 conserved acetylations (acetylation found at the same position on both Drosophila and human orthologs) on the chaperone heat shock protein 90 (Hsp90), the highest number of conserved acetylations found on any protein in our analysis. Deacetylation of Hsp90 by HDAC6 promotes glucocorticoid receptor, androgen receptor, and aryl hydrocarbon receptor signaling (2830). In addition, the antitumor activity of some KDAC inhibitors is thought to occur, in part, by modulation of Hsp90 activity (31). The role of acetylation in regulating translation and ribosomal proteins is less clear. However, the prevalence of conserved acetylation sites on proteins involved in these processes may indicate that acetylation has a functionally important role in protein synthesis.

Functional characterization of acetylation of a ubiquitin E2 ligase

Functional annotation of genes encoding acetylated proteins shows that proteins involved in ubiquitylation are often modified by conserved acetylation. Many human class 2 (E2) ubiquitin-conjugating enzymes are acetylated (7), and we found that several of these sites were acetylated on the Drosophila orthologs of these proteins (fig. S3). E2 enzymes bind an E1 enzyme and form an intermediate thioester linkage with activated ubiquitin, and with E3 enzymes coordinate the transfer of ubiquitin to a substrate. We found that conserved acetylation occurred within a specific N-terminal motif on eff (Drosophila)/UBE2D2 (human) and UbcD4 (Drosophila)/UBE2K (human). In humans, three additional E2 ubiquitin-conjugating enzymes, UBE2N, UBE2D3, and UBE2L3, were acetylated within a similar N-terminal motif. We found that acetylation occurred within a characteristic K/R-R-I/L-X-K-E motif (where slashes separate alternative residues in a particular position) that is present on 13 of 36 human E2 enzymes (Fig. 6A). This motif is highly conserved compared with the overall amino acid sequences of the E2 ligases that contain the motif. Amino acid sequence alignment of these 13 E2s revealed nine invariant amino acids, three of which occur within the N-terminal acetylation motif itself (fig. S4). This motif is also conserved in yeast E2s; mutation of either lysine residue within the motif abrogates ubiquitin transfer from the yeast E1, Uba1, to a yeast E2, Ubc1 (32). Furthermore, structural studies of E2-E3 complexes indicate that several amino acid residues within the N terminus of E2s, including the conserved acetylation site lysine, facilitate binding to the E3 enzymes (33). Because we observed conserved acetylation within a specific conserved motif that is present on about one-third of all known human E2 ubiquitin-conjugating enzymes, we decided to further characterize the functional consequence of acetylation at this position.

Fig. 6

Functional characterization of a conserved acetylation site on human UBE2D3 and yeast UBC4. (A) Sequence alignment showing a conserved N-terminal motif that is acetylated on the indicated (orange) lysine residue. Acetylation was observed in 5 of 13 proteins that contain the N-terminal motif (acetylated proteins are marked with red type). An asterisk indicates identical amino acid residues; a double dot indicates conserved amino acid substitutions. In the conserved motif, x represents any amino acid. Abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; and Y, Tyr. (B) Evolutionarily conserved acetylation occurs in the N-terminal motif of human (Hs) UBE2D2, fly (Dm) eff, and yeast (Sc) UBC4. (C) Ubiquitin-conjugating activity of an acetylation-mimetic mutant (K8Q) and an acetylation-deficient mutant (K8R) of UBE2D3 in an in vitro ubiquitylation assay. The chart shows the degree of histone H2A ubiquitylation relative to wild-type (WT) UBE2D3 in three independent experimental replicates. (D) Activity of recombinant UBE2D3 acetylated on Lys8 in an in vitro ubiquitylation assay. The chart shows the degree of histone H2A ubiquitylation relative to WT UBE2D3 in three independent experimental replicates. (E) Rescue of the growth defect of ubc4 deletion mutants exposed to 1 μM cycloheximide (CHX) by WT UBC4, UBC4 K9R, or UBC4 K9Q. All strains exhibited normal growth under nonselective conditions (SC-URA). The black wedge indicates fivefold serial dilution of the indicated yeast strains onto the selective media.

UBE2D2 (human) and eff (Drosophila) have conserved acetylation at Lys8. We immunoprecipitated the Saccharomyces cerevisiae homolog Ubc4 and identified acetylation at the corresponding position, K9 (fig. S5). Thus, we identified conserved acetylation at this position in three evolutionarily divergent organisms (Fig. 5B). In yeast, Ubc4 and Ubc5 are homologous (93% identical) functionally redundant enzymes (34). The human orthologs of Ubc4 and Ubc5, UBE2D2 and UBE2D3, respectively, are also homologous (>97% identical) and functionally redundant (3537). We identified acetylation at the same position in UBE2D3 (fig. S6) and assayed the effects of Lys8 acetylation on auto-ubiquitylation and substrate ubiquitylation by UBE2D3 in vitro.

Glutamine substitution (K to Q) is commonly used to mimic acetylation, whereas arginine substitution (K to R) replaces a lysine with a similarly charged amino acid that cannot be acetylated and does not mimic acetylation, serving as a negative control in genetic and biochemical analysis of acetylation sites. UBE2D3 K8Q substitution caused a modest reduction in both autoubiquitylation and substrate (histone H2A) ubiquitylation, whereas K8R substitution had no effect (Fig. 6C). To directly assay the effect of acetylation at Lys8, we produced site-specifically acetylated recombinant UBE2D3 in Escherichia coli using an orthogonal transfer RNA (tRNA) synthetase/tRNA approach (38, 39). Acetylation of recombinant UBE2D3 was confirmed by MS (fig. S7A) and by immunoblotting with an antibody that recognizes acetyllysine (fig. S7B). Acetylation at Lys8 caused a more pronounced reduction in substrate ubiquitylation than was observed with glutamine substitution (Fig. 6D). Thus, acetylation at this position appears to interfere with the enzymatic activity of UBE2D3. We observed no difference in the efficiency of ubiquitin thiolester formation on wild-type versus acetylated UBE2D3 (fig. S8), suggesting that acetylation may interfere with subsequent reaction steps.

To determine whether acetylation at this position affected the function of this enzyme in vivo, we analyzed the ability of various forms of Ubc4 to rescue growth defects of ubc4 deletion mutants in S. cerevisiae, which exhibit reduced growth on hygromycin B, cycloheximide, or ethanol (40, 41). We rescued a ubc4 deletion strain with an exogenous copy of either wild-type, K9Q, or K9R Ubc4. Surprisingly, the rescue of growth sensitivity was not affected by K9Q substitution on hygromycin B, ethanol, or a low concentration of cycloheximide (0.5 mM) (fig. S9), whereas the K9Q mutant, but not the K9R mutant, caused a modest growth defect on a higher (1 μM) concentration of cycloheximide (Fig. 6E and fig. S9). Although we observed a growth defect in cells containing the K9Q substitution specifically, it occurred only under the most stringent selection. This may reflect the inability of the glutamine substitution to effectively mimic acetylation, which is similar to the results obtained in the in vitro assay with UBE2D3 (Fig. 6, C and D). Alternatively, acetylation of this residue may have a role in fine tuning the activity of Ubc4 in vivo, or Ubc5 may mask the effects.

Discussion

High-resolution mapping of the Drosophila acetylome

The fruit fly D. melanogaster is widely used as a model organism for understanding fundamental biological processes. The role of lysine acetylation is believed to be conserved in Drosophila, but only a limited number (<50) of acetylation sites were known, presenting a major bottleneck in understanding its role in this organism. Here, we report the identification of ~2000 in vivo lysine acetylation sites in Drosophila cells, thereby providing a rich resource of putative regulatory modification sites in this organism. Bioinformatics analysis of the Drosophila acetylome revealed that its properties are similar to that of the human acetylome; acetylation occurs within similar sequence motifs and targets many of the same proteins involved in diverse cellular functions. In addition, our analysis of acetylation site sequences and peptide size distributions indicates that acetylation often occurs in lysine-rich regions of proteins. The large number of acetylation sites in Drosophila suggests that the regulatory scope of this modification is possibly as diverse and complex as in humans. Identification of lysine acetylation sites in Drosophila enables researchers to assay the importance of these sites within a defined genetic system, allowing for characterization of the functional importance of acetylation in a biological context.

Comparative analysis of acetylation and phosphorylation conservation

Large-scale mapping of phosphorylation sites in evolutionarily diverse organisms has led to the analysis of both amino acid conservation and phosphorylation conservation at these sites. It appears that in most cases, phosphorylated serines and threonines are either not conserved or only marginally more conserved than serine and threonine residues that are not phosphorylated (810). Comparison of the serine and threonine phosphoproteomes of humans, flies, worms, and yeast found just 17 sites with phosphorylation conservation in all four species (8). Weak conservation of serine and threonine phosphorylation sites can be attributed to a high frequency of phosphorylation in fast-evolving unstructured regions of proteins (10), resulting in the overall frequency of serine and threonine phosphorylation site conservation appearing similar to that of the corresponding unmodified amino acid. On the basis of the much greater magnitude of acetylation site conservation compared with phosphorylation site conservation seen in our study, we can conclude that a larger proportion of acetylation sites compared to phosphorylation sites are under selective pressure and are likely to be functionally important.

Acetylation of ubiquitin-conjugating enzymes

Posttranslational modification of proteins by ubiquitin and ubiquitin-like (UBL) proteins is an important regulatory mechanism that is conserved from budding yeast to humans. We found that acetylation occurred on a large number of E2 enzymes involved in protein ubiquitylation. So far, only a few functionally important modification sites have been identified in E2s, and these are modified by UBLs. For example, Lys14 of UBE2K is modified by sumoylation (42), whereas Lys92 of UBE2N is ISGylated (43). We find acetylation of both UBE2K and UBE2N on the same lysines that were previously reported to be modified by these UBLs, suggesting that there is direct crosstalk between UBLs and acetylation at these sites. We found that acetylation frequently occurred on a conserved lysine residue in the N-terminal helix of E2 enzymes, and biochemical analysis suggested that acetylation of UBE2D3 at this position impaired its function (Fig. 6D). In addition, we identified acetylation of the corresponding site in the budding yeast ortholog, Ubc4 (Fig. 6B and fig. S5). These data identify acetylation as an evolutionarily conserved modification that targets multiple E2s and suggest that acetylation may regulate the ubiquitylation activity of these proteins. Conserved acetylation also occurs on UBE2I and UFC1, which are responsible for conjugating the UBLs SUMO and Ufm1, respectively. We found conserved acetylation on Lys6 and Lys48 of ubiquitin itself and on Lys6 of the UBL NEDD8, suggesting that acetylation could influence the choice of ubiquitin chain formation by blocking conjugation of ubiquitin or UBLs at these lysines. The idea that acetylation competes with ubiquitylation was demonstrated in a proteomic study identifying lysine ubiquitylation sites in human cells (44). Here, 20% of the identified ubiquitylation sites are also known acetylation sites (44). Thus, acetylation not only competes with ubiquitylation but also may regulate ubiquitylation by affecting the activity of ubiquitin-conjugating and UBL-conjugating enzymes.

In summary, we present a valuable resource for studying the role of lysine acetylation in regulating protein function. Although we have identified many acetylation sites in Drosophila and many conserved sites in humans and Drosophila, their role in regulating the function of these target proteins remains to be explored in detail. We hope that the data presented with this study will be of use to many researchers aiming to understand the functional role of acetylation.

Materials and Methods

Cell culture

Drosophila SL2 cells were grown in suspension culture with EX-CELL 420 serum-free insect cell culture media (SAFC Biosciences) at a cell density of 5 × 106/ml to 10 × 106/ml, 25°C, and shaking at ~120 rpm. Cells were harvested by centrifugation at 1000g, washed once with phosphate-buffered saline (PBS), and stored at −80°C until lysis.

Preparation of Drosophila cell lysates and peptides for MS

Frozen cell pellets containing ~1 × 108 cells were lysed on ice in 2.5 ml of lysis buffer [50 mM tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 0.1% sodium deoxycholate] supplemented with Complete Mini Protease Inhibitor Cocktail (Roche). Cell lysates were incubated on ice for 5 to 10 min before addition of 1/10 volume 5 M NaCl to release chromatin-bound proteins. The lysates were sonicated three times for 30 s at 10 to 15 W on ice and clarified by centrifugation at 16,000g, 15 min, 4°C. Clarified protein supernatant was precipitated overnight at −20°C by addition of 4 volumes of ice-cold acetone. Precipitates were dissolved in urea solution (6 M urea, 2 M thiourea, and 10 mM Hepes, pH 8.0) to a final protein concentration of 5 to 10 mg/ml. In-solution digestion, peptide purification, and acetyllysine peptide enrichment were performed as described (7). Acetylated peptides were fractionated with an Agilent 3100 OFFGEL fractionator (Agilent) (for experiments 1 and 2) or by micro-SCX in a stage-tip format (for experiments 3, 4, and 5) (45). Peptide fractions were purified with reversed-phase StageTips as described (45).

Mass spectrometric analysis

Peptide fractions were analyzed by online nanoflow LC-MS/MS with a Proxeon easy nLC system (Thermo Scientific) connected to an LTQ Orbitrap Velos mass spectrometer. Briefly, the LTQ Orbitrap Velos instrument (46) under Xcalibur 2.1 with LTQ Orbitrap Tune Plus Developers Kit version 2.6.0.1042 software was operated in the data-dependent mode to automatically switch between MS and MS/MS acquisition. Survey full-scan MS spectra [mass/charge ratio (m/z) from 300 to 1700] were acquired in the Orbitrap with a resolution R = 30,000, after accumulation to a “target value” of 1,000,000 ions in the linear ion trap. The 10 most intense ions were sequentially isolated and fragmented by HCD (15), and fragment spectra were recorded in the Orbitrap (for ~90% of the data) or in the linear ion trap by collisionally induced dissociation (CID) at a target value of 5000 or maximum ion injection time of 50 ms (for ~10% of the data). For all measurements with the Orbitrap detector, a lock mass ion from ambient air (m/z 445.120025) was used for internal calibration as described (47). Typical mass spectrometric conditions were spray voltage, 2.2 kV; no sheath and auxiliary gas flow; heated capillary temperature, 175°C. The ion selection threshold was 5000 counts for HCD MS2 and 1000 counts for CID MS2. For CID, an isolation width of 2.0, activation q = 0.25, activation time of 10 ms, and normalized collision energy of 35% were used. For HCD, an isolation width of 4.0, an activation time of 0.1 ms, and normalized collision energy of 40% were used.

Peptide identification and computational analysis

Raw data files were processed with MaxQuant software as described (48, 49) (http://www.maxquant.org/). MS and MS2 spectra were searched against the Flybase genome release 5.4 (http://flybase.org). Peptides and proteins were identified by Mascot (Matrix Science) via automated database searching of all tandem mass spectra against an in-house curated target/decoy database (forward and reversed version of the Flybase genome release 5.4) (50). Spectra were initially searched with a mass tolerance of 7 ppm in MS mode, 0.5 dalton in CID MS/MS mode, 0.02 dalton in HCD MS/MS mode, strict trypsin specificity, and allowing up to three missed cleavage sites. Cysteine carbamidomethylation was searched as a fixed modification, whereas N-acetyl protein, oxidized methionine, and acetylation of lysine were searched as variable modifications. Acetyllysine site identifications were filtered to remove C-terminal acetyllysine (we assumed that acetylation of lysine blocks peptide cleavage by Lys-C and trypsin). We additionally filtered for mascot score and posterior error probability (PEP) score to arrive at a false discovery rate of 1%. Site localization probabilities were determined by MaxQuant with a PTM scoring algorithm (51). Sites with localization probability less than 0.9 were not used on the conservation analysis (unless poor localization occurred between an internal lysine and a C-terminal lysine, because we assumed C-terminal acetylation does not normally occur; the internal localization is considered to be correct). Out of a total number of 1981 identified Drosophila acetylation sites, 1968 were used in the conservation analysis.

Analysis of acetylation site sequences

Acetylation site sequence windows, containing six amino acids flanking the acetylation site on each side, were compared with similar-sized sequence windows from nonacetylated lysine residues on proteins identified in each respective acetylation study (Drosophila and human). Proteins identified in these studies include proteins that are not acetylated, because many proteins are identified from nonacetylated contaminating peptides (90 to 95% of peptides are not acetylated). Acetylation site logos were computed with a stand-alone version of iceLogo (52). Comparison of 1981 Drosophila and 3527 human acetylation site sequence windows to 180,380 Drosophila and 217,100 human nonacetylated lysine sequence windows was done.

Functional annotation

Functional annotation was performed with DAVID bioinformatics resource v6.7 [http://david.abcc.ncifcrf.gov/home.jsp (53, 54)]. Lists of genes encoding acetylated proteins or conserved acetylated proteins were compared to the default of all-Drosophila or all-human genes. The following functional annotation term categories were analyzed: GOTERM_BP_FAT, GOTERM_CC_FAT, GOTERM_MF_FAT, and SP_PIR_KEYWORDS. A P value cutoff of 0.01 was chosen (default is 0.05), and fold enrichment was selected; otherwise, all settings were the default value.

Expression of recombinant proteins

Gateway Entry clones of human UBE2N, UBE2D3, and RNF8 open reading frames (ORFs) were obtained from the Invitrogen Ultimate ORF collection (Invitrogen). The coding sequences were shuffled into the pDEST-15 vector (Invitrogen) or pET-55-DEST (Novagen) and transformed into BL21 (DE3) cells. Expression of recombinant proteins was induced by addition of 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) at 30°C for 6 hours. Proteins were purified from bacterial lysates with Glutathione Sepharose (GE Healthcare) and eluted with 20 mM reduced glutathione (Sigma-Aldrich) or with StrepTactin Sepharose (IBA) and eluted with 5 mM d-desthiobiotin (Sigma-Aldrich). The purity and concentration of proteins were determined by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and Bradford assay. Expression and purification of recombinant acetylated proteins were performed as previously described (38, 39). Coding sequences of pyIT, AcKRS, and glutathione S-transferase (GST)–UBE2D3 were amplified by polymerase chain reaction (PCR) and cloned into pETDuet-1 (Novagen). The resulting vector was transformed into E. coli BL21 (DE3). Expression of proteins was induced by addition of 1 mM IPTG in the presence of 5 mM Nε-acetyl-l-lysine (Sigma-Aldrich) at 30°C for 6 hours. Detection of recombinant protein by Western blot was performed with a polyclonal rabbit antibody against UBE2D3 (ProteinTech Group, 11677-1-AP) and a polyclonal rabbit antibody against acetyllysine (Cell Signaling Technology).

In vitro ubiquitylation assays

FLAG-ubiquitin and His-tagged E1 enzyme (UBE1) were obtained from Boston Biochem. Histone H2A was obtained from New England Biolabs. The reaction mix containing 5 μg of FLAG-ubiquitin, 250 ng of E1, 500 ng of E2, 500 ng of E3 enzyme, and 500 ng of the substrate protein in assay buffer [25 mM tris (pH 7.5), 50 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol (DTT), and 5 mM adenosine 5′-triphosphate (ATP)] was incubated for 1 hour at 30°C. The reaction was quenched by addition of SDS-PAGE sample buffer, and the complete reaction mix was resolved by SDS-PAGE. Ubiquitinated proteins were detected by immunoblotting with the M2 antibody that recognizes FLAG (Sigma-Aldrich) or an antibody that recognizes H2A (Cell Signaling Technology).

Thiolester formation assay

FLAG-ubiquitin and His-tagged E1 enzyme (UBE1) were obtained from Boston Biochem. The reaction mix containing 5 μg of FLAG-ubiquitin, 250 ng of E1, and 500 of ng E2 in assay buffer [25 mM tris (pH 7.5), 50 mM NaCl, 5 mM MgCl2, and 5 mM ATP] was incubated for the indicated time points at 30°C and quenched by addition of SDS-PAGE sample buffer. The sample was split into two equal portions, half of which was resolved by SDS-PAGE under nonreducing conditions, and the remaining half was resolved under reducing conditions in the presence of DTT to demonstrate the instability of the UBE2D3-ubiquitin thiolester linkage. Thiolester-induced mobility shifts were visualized by immunoblotting with a polyclonal rabbit antibody against UBE2D3 (ProteinTech Group, 11677-1-AP).

Yeast

Yeast strains were grown under standard conditions. ubc4 mutant yeast were transformed with cloned genomic fragments of the UBC4 gene, including ~500 base pairs (bp) upstream and downstream from the ORF, thus containing the endogenous promoter and regulatory elements. Genomic DNA fragments were amplified from wild-type yeast and cloned into the Bam H1 and Sal 1 sites of pRS316 plasmid. Site-specific mutations were introduced with a standard QuikChange site-directed mutagenesis approach. All clones, including parent clones and mutations, were verified by DNA sequencing. Green fluorescent protein (GFP)–tagged UBC4 was grown to mid-log phase in 500 ml of YPD (yeast extract, peptone, and dextrose) medium and cells were lysed using CelLytic Y reagent (Sigma-Aldrich) according to the manufacturer’s protocol. UBC4-GFP was enriched using GFP-Trap_A (ChromoTek), washed three times with CelLytic Y reagent, and eluted with 2× Laemmli sample buffer. Protein was separated on a 4 to 12% tris-glycine gel (Novex/Invitrogen) and visualized with Colloidal Blue Staining Kit (Novex/Invitrogen). Peptides were recovered with a standard in-gel digestion protocol (55). Growth on selective media was assayed by fivefold serial dilution of freshly prepared, mid-log phase cultures on both control and selective media. Yeast were grown at 30°C. Yeast strains used were (i) BY4741-ubc4KO (YSC1021-555493), MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 ubc4::Kan (Open Biosystems) and (ii) UBC4-GFP (American Type Culture Collection 201388), MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 (Invitrogen) (56). Primers used to clone the UBC4 genomic coding region were forward (ggc ggc gga tcc GCA TGA TAA ATT CGA ACC CAT TCC) and reverse (gga gga gtc gac CTC CTT AGT ACA GTG TAA TGC TCC). Primers used to introduce site-specific mutations in UBC4 were K9Q forward (GTC TTC TTC TAA ACG TAT TGC Tca aGA ACT AAG TGA TCT AGA AAG GT), K9Q reverse (ACC TTT CTA GAT CAC TTA GTT Ctt gAG CAA TAC GTT TAG AAG AAG AC), K9R forward (ACC TTT CTA GAT CAC TTA GTT Ctt gAG CAA TAC GTT TAG AAG AAG AC), and K9R reverse (ACC TTT CTA GAT CAC TTA GTT Ctt gAG CAA TAC GTT TAG AAG AAG AC).

Supplementary Materials

www.sciencesignaling.org/cgi/content/full/4/183/ra48/DC1

Fig. S1. Functional annotation of Drosophila and human acetylomes.

Fig. S2. Conservation of serine and threonine phosphorylation sites to either serine or threonine.

Fig. S3. Summary of acetylation sites identified in E2-conjugating enzymes.

Fig. S4. Sequence alignment of human E2 ubiquitin-conjugating enzymes.

Fig. S5. Identification of UBC4 Lys9 acetylation in S. cerevisiae.

Fig. S6. Identification of in vivo UBE2D3 Lys8 acetylation in human cells.

Fig. S7. Confirmation of Lys8 acetylation in recombinant UBE2D3 purified from E. coli.

Fig. S8. Effect of UBE2D3 Lys8 acetylation on ubiquitin thiolester formation.

Fig. S9. Rescue of growth sensitivity in S. cerevisiae ubc4 mutant cells.

Definitions of the columns for Tables S1 to S3

Details regarding data availability

Table S1. List of Drosophila in vivo acetylation sites.

Table S2. Drosophila acetylated lysine conservation.

Table S3. Human acetylated lysine conservation.

References and Notes

  1. Acknowledgments: We thank the members of the Departments of Proteomics and Disease Systems Biology at the Center for Protein Research (CPR) for their helpful discussions and the Protein Science and Technology unit at the CPR for providing recombinant RNF8. Funding: The CPR is funded by a grant from the Novo Nordisk Foundation. This work was funded by the European Commission’s 7th Framework Programme grants Proteomics Research Infrastructure Maximizing knowledge EXchange and access (XS) (INFRASTRUCTURES-F7-2010-262067/PRIME-XS) and by the Lundbeck Foundation (R48-A4649). S.A.W. is supported by a postdoctoral grant from the Danish Council for Independent Research (FSS: 10-083519). Author contributions: B.T.W. and C.C. conceived the project; B.T.W., S.A.W., and P.H. performed the experiments and analyzed the data; H.H. performed PTM conservation analysis; W.R.L. provided the plasmid vectors for expression of recombinant acetylated UBE2D3; J.V.O. provided the idea of comparing peptide length distributions; L.J.J. supervised the PTM site conservation analysis; and C.C. supervised the entire project. B.T.W. and C.C. wrote the manuscript. S.A.W., H.H., P.H., J.V.O., and L.J.J. read and commented on the manuscript. Competing interests: L.J.J. is a cofounder and scientific adviser of Intomics A/S; however, this company was not involved in this study. The other authors declare that they have no competing interests. Data availability: The data associated with this manuscript may be downloaded from ProteomeCommons.org (see Supplementary Materials for hashes).
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