The vast majority of eukaryotic proteins are N-terminally modified by one or more processing enzymes. Enzymes acting on the very first amino acid of a polypeptide include different peptidases, transferases, and ligases. Methionine aminopeptidases excise the initiator methionine leaving the nascent polypeptide with a newly exposed amino acid that may be further modified. N-terminal acetyl-, methyl-, myristoyl-, and palmitoyltransferases may attach an acetyl, methyl, myristoyl, or palmitoyl group, respectively, to the α-amino group of the target protein N-terminus. With the action of ubiquitin ligases, one or several ubiquitin molecules are transferred, and hence, constitute the N-terminal modification. Modifications at protein N-termini represent an important contribution to proteomic diversity and complexity, and are essential for protein regulation and cellular signaling. Consequently, dysregulation of the N-terminal modifying enzymes is implicated in human diseases. We here review the different protein N-terminal modifications occurring co- or post-translationally with emphasis on the responsible enzymes and their substrate specificities.
N-terminal acetylation has been suggested to play a role in the subcellular targeting of proteins, in particular those acetylated by the N-terminal acetyltransferase complex NatC. Based on previous positional proteomics data revealing N-terminal acetylation status and the predicted NAT substrate classes, we selected 13 suitable NatC substrates for subcellular localization studies in Saccharomyces cerevisiae. Fluorescence microscopy analysis of GFP-tagged candidates in the presence or absence of the NatC catalytic subunit Naa30 (Mak3) revealed unaltered localization patterns for all 13 candidates, thus arguing against a general role for the N-terminal acetyl group as a localization determinant. Furthermore, all organelle-localized substrates indicated undisrupted structures, thus suggesting that absence of NatC acetylation does not have a vast effect on organelle morphology in yeast.
N-terminal acetylation is a highly abundant protein modification catalyzed by N-terminal acetyltransferases (NATs) NatA-NatG. The Saccharomyces cerevisiae protein Arl3 depends on interaction with Sys1 for its localization to the Golgi and this targeting strictly requires NatC-mediated N-terminal acetylation of Arl3. We utilized the Arl3 acetylation-dependent localization phenotype as a model system for assessing the functional conservation and in vivo redundancy of several human NATs. The catalytic subunit of human NatC, hNaa30 (Mak3), restored Arl3 localization in the absence of yNaa30, but only in the presence of either yeast or human Naa35 subunit (Mak10). In contrast, hNaa35 was not able to replace its yeast orthologue without the co-expression of hNaa30, suggesting co-evolution of the two NatC subunits. The most recently discovered and organellar human NAT, NatF/Naa60, restored the Golgi localization of Arl3 in the absence of yNaa30. Interestingly, this was also true for hNaa60 lacking its membrane-binding domain whereas hNaa50 did not complement NatC function. This in vivo redundancy reflects NatC and NatF´s overlapping in vitro substrate specificities. The yeast model presented here provides a robust and rapid readout of NatC and NatF activity in vivo, and revealed evolutionary conservation of the NatC complex and redundancy between NatC and NatF.
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