Identification and Functional Characterization of N-Terminally Acetylated Proteins in Drosophila melanogaster
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.