Figure 5
Naa50/San N-terminally acetylates Scc1.
The N-terminal phenylalanine and tyrosine residues of Scc1 are highly conserved. The Scc1 N-terminal sequence (MFY-) is efficiently Nt-acetylated in vitro by Naa50/San. (A) Orthologs of Scc1 from 15 distinct species representative of the eukaryotic tree of life were retrieved using reciprocal bidirectional protein BLAST analysis. N-terminal sequences were aligned using the Geneious R7 software. The N-terminal second residue phenylalanine (F) and third residue tyrosine (Y) are highly conserved across the eukaryotic tree of life (see asterisks). The height of the letters represents the overall relative degree of residue conservation. (B) Wild-type Drosophila Naa50/San efficiently N-terminally acetylated in vitro Scc1 N-terminal peptide (MFY-) and a previously defined Naa50/San substrate MLG- peptide (positive control). In contrast, Naa50/San did not efficiently N-terminally acetylate a Naa10 substrate peptide SES- (negative control), nor the non-acetylatable N-terminal peptide MPY-. A catalytically dead-version of Naa50/San (SanR84A Y124F) failed to efficiently N-terminally acetylate all tested peptides. Both wild type Naa50/San and Naa50/SanR84A Y124F were heterologously expressed in E. coli (see methods). All measurements were done in the linear range of enzymatic activity (data not shown).
