NatF Contributes to an Evolutionary Shift in Protein N-Terminal Acetylation and Is Important for Normal Chromosome Segregation

Petra Van Damme,Rui G Martinho,Ana Pimenta-Marques,Kristine Hole,Kris Gevaert,Thomas Arnesen,Joël Vandekerckhove,Kenny Helsens,Michael Snyder

doi:10.1371/journal.pgen.1002169

Abstract

N-terminal acetylation (N-Ac) is a highly abundant eukaryotic protein modification. Proteomics revealed a significant increase in the occurrence of N-Ac from lower to higher eukaryotes, but evidence explaining the underlying molecular mechanism(s) is currently lacking. We first analysed protein N-termini and their acetylation degrees, suggesting that evolution of substrates is not a major cause for the evolutionary shift in N-Ac. Further, we investigated the presence of putative N-terminal acetyltransferases (NATs) in higher eukaryotes. The purified recombinant human and Drosophila homologues of a novel NAT candidate was subjected to in vitro peptide library acetylation assays. This provided evidence for its NAT activity targeting Met-Lys- and other Met-starting protein N-termini, and the enzyme was termed Naa60p and its activity NatF. Its in vivo activity was investigated by ectopically expressing human Naa60p in yeast followed by N-terminal COFRADIC analyses. hNaa60p acetylated distinct Met-starting yeast protein N-termini and increased general acetylation levels, thereby altering yeast in vivo acetylation patterns towards those of higher eukaryotes. Further, its activity in human cells was verified by overexpression and knockdown of hNAA60 followed by N-terminal COFRADIC. NatF's cellular impact was demonstrated in Drosophila cells where NAA60 knockdown induced chromosomal segregation defects. In summary, our study revealed a novel major protein modifier contributing to the evolution of N-Ac, redundancy among NATs, and an essential regulator of normal chromosome segregation. With the characterization of NatF, the co-translational N-Ac machinery appears complete since all the major substrate groups in eukaryotes are accounted for.

Highlights

N-terminal acetylation (N-Ac) is a common modification of proteins, but its general role has remained rather enigmatic
Fruit fly and human proteomes, it is evident that the general distribution of N-termini is largely unaltered between the different classes, ‘NatA’, ‘NatB’, ‘NatC’ and ‘Other’ (Figure 1A)
Besides the general difference of the amino acid usage in yeast versus human N-termini in agreement with recent observations [34], the occurrence of (Met-)Ala- N-termini increased from 8% in yeast to 23% in humans, while Met-Glu- Ntermini increased from 5% to 10%

Summary

Introduction

N-terminal acetylation (N-Ac) is a common modification of proteins, but its general role has remained rather enigmatic. Three major NAT complexes conserved from yeast to humans are thought to be responsible for the majority of N-terminal acetylation events: NatA, NatB and NatC [15]. NatB and NatC potentially acetylate Met- Ntermini when the second residue is either acidic or hydrophobic respectively [19,20,21]. NatD was described to acetylate the Ser- N-termini of histones 2A and 4 in vitro and in vivo [22], while no such activity has yet been presented for higher eukaryotes. NatE is another NAT of which the in vitro activity was described for the human hNaa50p towards some Met-LeuN-termini [23], but direct evidence of in vivo activity is still lacking.

Methods

Results

Conclusion