N-acetylation of protein variants. Tandem mass spectra of N-acetylated protein variants from Pma1p and Ura2p are displayed. These proteins were found to be N-acetylated on the amino acids N and D as suggested by the 42 Da mass shifts of the b-ion series compared to the same peptides in their non-acetylated forms, which are displayed in the top row. Fragment ions of the y and b series found in the MS/MS spectra are indicated in black in the tables next to the spectra. Missing ions are marked in grey. For each spectrum the start position of the respective peptide is indicated together with the peptide sequence, protein name, accession and MASCOT score.

Localization of up-regulated phosphoproteins and kinases predicted to be responsible for elevated phosphorylation levels. Panel (A) Bar chart indicating localization of proteins displaying significantly increased phosphorylation levels. The % of proteins being localized in the respective categories was calculated for up-regulated proteins and normalized to the localization distribution determined for all detected proteins. The yellow color highlights proteins of the nucleus and nucleolus. Panel (B) Bar chart indicating kinases predicted to be responsible for the observed elevated phosphorylation sites. The % of phospho-sites being targeted by the respective kinases was calculated for up regulated sites and normalized to the background of detected unchanged phospho-sites, revealing the predominant role of SNF1 in the observed increased phosphorylation in the nat3Δ strain.

Conservation of NatB substrates across species. Panel A displays a bar chart indicating the conservation of NatB targets across species. This analysis was performed using either all 59 identified NatB substrates (black bars) or only the 5 most conserved proteins Arp2p, Bos1p, Erg3p, Rpb5p and Rps28ap (gray bars). NatB substrates are only sporadically conserved in the tree of life with the exception of a few, highly conserved, proteins. The phylogenetic relationship between the species included in this survey is indicated on the left. Panel B shows an alignment of Glc7p with orthologous protein sequences from different species of the fungal kingdom indicating general high conservation at the full-length protein level. The termini, however, are much less conserved including the part that determines N-acetyltransferase substrate specificity.

Differential quantitation of 2560 proteins in the yeast WT/nat3Δ proteome enables identification of NatB substrates and reveals overall increased phosphorylation levels. Panel (A), (C) and (D) display peptide and protein ¹⁵N/¹⁴N ratios (²log transformed) determined in both biological replicates. Data of the two biological replicates are plotted versus each other. In experiment 1 the ΔNat3 strain was labeled with ¹⁴N while WT incorporated the heavy ¹⁵N label. In experiment 2 the isotope labels were reversed. The dashed lines represent a 95% confidence interval indicating high reproducibility of ratio data between biological replicates [32]. The circles indicate the chosen arbitrary thresholds for diminished or elevated protein levels, which were set at a three-fold change. Panel (A) displays ¹⁵N/¹⁴N ratio data of N-acetylated peptides, red colored spots mark N-acetylated peptides displaying the NatB target sequence while the lighter red indicates peptides located outside the 95% confidence. Panel (B) displays ¹⁵N/¹⁴N ratio histograms. The upper histogram shows ratios for all detected N-acetylated peptides not containing the expected NatB substrate sequence. The lower plot illustrates the ratio distribution of N-acetylated peptides containing the expected NatB substrate sequence, namely a methionine at the ultimate and an aspartic acid, glutamic acid or an asparagine in the penultimate position. Individual ratios from the biological replicates were averaged. The insets show frequency plots of the amino acids in the first 5 positions of the N-terminus generated by Weblogo. Panel (C) displays protein ratios as determined from unmodified peptides, with in red again the observed NatB substrate proteins. (D) displays phosphopeptide ratios, irrespective of being NatB substrate or not.

Profiling N-acetylation in yeast. A Venn diagram representation of the overlap between identified N-acetylated proteins carrying the N-acetylation on position 1 or 2, and protein variants detected to display N-acetylation on amino acid position 3 or higher. Sequence logos were calculated for peptides acetylated at position 1 and 2 from the predicted gene-start. For position 1, sequences were devided into peptides that matched the NatB consensus sequence and the rest which is most likely acetylated by other N-acetyltransferases such as NatC. Acetylation in position 2 was found to follow the consensus sequence of NatA. Frequency logos are displayed at the top in the blue frames. The frequency logo for proteins N-acetylated on a amino acid higher than 2 (from the predicted gene-start) are indicated below in the grey panel, revealing no particular consensus sequence for this latter category. Network analysis was performed on these latter protein variants and the three main protein clusters are indicated below the frequency logo. These protein variants were found to be preferentially involved in the proteasome, chaperone network and energy metabolism. Proteins detected to be N-acetylated either in position 1 or 2 and additionally at a position higher than 2 are indicated in blue in the protein clusters. All detected protein variants are given in Additional file 5.

Functional association of proteins increased in phosphorylation. Protein networks illustrating associations between up-regulated phosphoproteins. In the middle the total network is depicted, including a rough functional classification of the mapped proteins. Around this central network are depicted the three most prevalent nuclear protein clusters.