Abstract
N-terminal acetylation (Nt-acetylation) by N-terminal acetyltransferases (NATs) is one of the most common protein modifications in eukaryotes. The NatC complex represents one of three major NATs of which the substrate profile remains largely unexplored. Here, we defined the in vivo human NatC Nt-acetylome on a proteome-wide scale by combining knockdown of its catalytic subunit Naa30 with positional proteomics. We identified 46 human NatC substrates, expanding our current knowledge on the substrate repertoire of NatC which now includes proteins harboring Met-Leu, Met-Ile, Met-Phe, Met-Trp, Met-Val, Met-Met, Met-His and Met-Lys N termini. Upon Naa30 depletion the expression levels of several organellar proteins were found reduced, in particular mitochondrial proteins, some of which were found to be NatC substrates. Interestingly, knockdown of Naa30 induced the loss of mitochondrial membrane potential and fragmentation of mitochondria. In conclusion, NatC Nt-acetylates a large variety of proteins and is essential for mitochondrial integrity and function.
Highlights
Protein N-terminal (Nt)1 acetylation is the transfer of an acetyl moiety from acetyl coenzyme A (Ac-CoA) to the N-ter
The NatC complex was originally identified in yeast [18, 19] and we previously described the human NatC complex, which is conserved from yeast both with respect to subunit composition, ribosome binding, and in vitro oligopeptide substrate specificity [19, 20]
Following LC-MS/MS analysis, we identified 1874 unique N termini with a minimum amino acid length of seven originating from 1754 human proteins that were in compliance with the rules of Nt-acetylation [37] and initiator methionine processing, thereby being proxies of translation initiation
Summary
Protein N-terminal (Nt) acetylation is the transfer of an acetyl moiety from acetyl coenzyme A (Ac-CoA) to the N-ter-. NatA-NatF differ both with respect to substrate specificity and subunit composition, where the catalytic subunits are members of the GNAT acetyltransferase superfamily. The NatC complex was originally identified in yeast [18, 19] and we previously described the human NatC complex, which is conserved from yeast both with respect to subunit composition, ribosome binding, and in vitro oligopeptide substrate specificity [19, 20]. Of note is that NatC does not acetylate all proteins matching these N-terminal sequences and typically NatC substrates are less Nt-acetylated as compared with NatA and NatB substrates [1, 25]. GNAT, GCN5-related N-acetyltransferase superfamily; iMet, initiator methionine; NAA#, N-alpha acetyltransferase # (gene/protein); NAT, N-terminal acetyltransferase; z-VAD-fmk, carbobenzoxy-VAD (O-methyl)- fluoromethylketone
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