Identification and functional characterization of N-terminally acetylated proteins in Drosophila melanogaster

Abstract

Protein modifications play a major role for most biological processes in living organisms. Amino-terminal acetylation of proteins is a common modification found throughout the tree of life: the N-terminus of a nascent polypeptide chain becomes co-translationally acetylated, often after the removal of the initiating methionine residue. While the enzymes and protein complexes involved in these processes have been extensively studied, only little is known about the biological function of such N-terminal modification events. To identify common principles of N-terminal acetylation, we analyzed the amino-terminal peptides from proteins extracted from Drosophila Kc167 cells. We detected more than 1,200 mature protein N-termini and could show that N-terminal acetylation occurs in insects with a similar frequency as in humans. As the sole true determinant for N-terminal acetylation we could extract the (X)PX rule that indicates the prevention of acetylation under all circumstances. We could show that this rule can be used to genetically engineer a protein to study the biological relevance of the presence or absence of an acetyl group, thereby generating a generic assay to probe the functional importance of N-terminal acetylation. We applied the assay by expressing mutated proteins as transgenes in cell lines and in flies. Here, we present a straightforward strategy to systematically study the functional relevance of N-terminal acetylations in cells and whole organisms. Since the (X)PX rule seems to be of general validity in lower as well as higher eukaryotes, we propose that it can be used to study the function of N-terminal acetylation in all species.

Figure 1. Graphical representation of the datasets using Venn diagrams.

(A) Comparison of dataset generated by COFRADIC and shotgun analysis. A total of 1,102 distinct N-terminal peptides were identified with an overlap of 115 sequences. The COFRADIC approach yielded 835 N-terminal peptides among a total 4,203 distinct peptides identified from 8,402 spectra. In contrast, a classical shotgun approach on Kc cells not using COFRADIC enrichment yielded 382 N-terminal peptides among 19,915 distinct peptides (34,175 spectra) retrieved from Loevenich et al. . (B) Identification of 124 distinct putative, alternative translation initiation sites identified in the COFRADIC and shotgun dataset, respectively. (C) Comparison of N-terminal peptides according to their acetylation status. From 1,226 distinct N-termini 861 were found to be acetylated, 317 non-acetylated with an overlap of 48 identical N-termini showing partial acetylation.

Figure 2. Analysis of translational start sites.

(A) A Frequency analysis of all predicted N-termini present in the Drosophila database BDGP_Release_3.2 revealed the presence of the Cavener consensus sequence C_A/G_A_A/C_ATG ,. The histogram shows the relative frequencies of nucleotides 5′ to the predicted (conventional) initiator codon generating acetylated as well as non-acetylated N-termini starting at position 1 or 2 of the predicted protein sequence. The nucleotide 3′ of the initiator sequence has been proposed to preferentially be a G at +4 for strong initiation but has been shown not to be relevant for Drosophila ,. Interestingly the G at +4 is nevertheless predominant in all cases. (B) Relative frequencies of nucleotides 5′ to the alternative translation initiation sites. The Cavener sequence is conserved showing a shift in sequence preference at position −1 (from A to C), which fully complies with the Cavener consensus sequence (C_A_A_C_ATG) ,.

Figure 3. Schematic drawing of the (X)PX rule.

(A) During translation of a protein with the sequence Met-Sur at its N-terminus (Sur being a small and uncharged amino acid residue, in this case Sur is equal to an Ala residue in green), the iMet will be removed by a methionine aminopeptidase (MAP, brown bubble) and the processed amino-terminus will be acetylated at the alpha amine of the Ala residue by a NAT (green oval). (B and C) A protein with the sequence Met-Pro at its N-terminus (referred to as Pro-X₂, panel B) will undergo iMet cleavage by the methionine aminopeptidase (MAP, brown bubble) and the processed amino-terminus will remain unacetylated. If iMet cleavage is not taking place, proteins with the sequence Met-Pro-X₃ at their mature amino-terminus (panel C) will also remain unacetylated. The Pro residue (red) thus prevents acetylation even if iMet cleavage occurs only partially. (D) Similarly, from a protein having the sequence X₁-Pro-X₃ (X₁ being Ala or Ser, in this case Sur is equal to an Ala residue in green, Pro in red), the iMet will be removed and the processed amino-terminus will remain unacetylated (panel D). Although partial removal of the iMet is rarely observed under these circumstances, the N-terminus with the amino acid sequence M-A-P usually remains unacetylated as also observed by SRM measurements.

Figure 4. Workflow to test the (X)PX rule.

(A) The cDNAs of hyrax (hyx) and cks85A were modified as follows: in the Drosophila hyx cDNA the codon for the secondary Ala was replaced by a Pro to prevent acetylation of the amino-terminus. Similarly, we replaced the codon for the secondary Pro in the Drosophila cks85A cDNA by either a Ser or an Ala to promote acetylation. In addition, all constructs were C-terminally HA-tagged and subjected to the control of the tubulin-1α promoter. The different constructs subsequently express Hyx-A2P-HA, Hyx-Wt-HA, Cks-P2A-HA, Cks-P2S-HA, and Cks-Wt-HA, respectively. (B) To test the (X)PX rule in vitro transient transfections of S2 cells were performed. The tagged proteins were isolated via immunoprecipitation and subjected to mass spectrometry analysis via SRM. As expected, the N-terminus of Hyx-Wt-HA was found to be acetylated in combination with a complete iMet removal. In contrast, the amino terminus of Hyx-A2P-HA was always unmodified but showed partial iMet cleavage (9.1%). For both kinase mutants Cks-P2A-HA as well as Cks-P2S-HA the N-Terminus is always acetylated. For Cks-P2S-HA we also detected the acetylated peptide MSADQIQYSEK caused by an incomplete iMet removal (9.4%). Cks-Wt-HA could not be detected due to toxicity effects of the expressed transgene but has initially been isolated via COFRADIC with a free N-terminus.

Figure 5. In vivo analysis of genetically modified hyrax transgenes.

For an in vivo analysis, the hyrax Hyx-A2P-HA, Hyx-Wt-HA transgenes were integrated into the fly genome using the site-specific phiC31-mediated integration system . Fly genotypes: tub>hyx-wt-HA/CyO; hyx²/TM6b (lanes labeled WT), tub>hyx-A2P-HA/CyO; hyx²/TM6b (lanes labeled A2P), Sp/CyO; hyx²/TM6b (negative controls, lanes labeled hyx). Numbers in lanes represent µg of protein loaded (A, B). (A) Total fly lysates of flies reared at 25°C were subjected to Western blot analysis and revealed identical expression levels for the two transgenes (97% Hyx-Wt-HA in comparison to Hyx-A2P-HA). Western blot analysis of total fly lysates of flies reared at 29°C revealed a 32% reduced amount of Hyx-A2P-HA protein as compared to Hyx-Wt-HA controls. (B) Confocal images of stained wing discs from 3^rd instar larvae of Sp/CyO; hyx²/TM6b (negative controls, opened pinhole), Hyx-Wt-HA/CyO; hyx²/TM6b and Hyx-A2P-HA/CyO; hyx²/TM6b reared at 25°C and 29°C, respectively. At both temperatures, a similar strong nuclear staining was observed for Hyx-Wt-HA and Hyx-A2P-HA and no difference in localization could be detected.