Abstract
The evolutionary conserved N-alpha acetyltransferase Naa40p is among the most selective N-terminal acetyltransferases (NATs) identified to date. Here we identified a conserved N-terminally truncated Naa40p proteoform named Naa40p25 or short Naa40p (Naa40S). Intriguingly, although upon ectopic expression in yeast, both Naa40p proteoforms were capable of restoring N-terminal acetylation of the characterized yeast histone H2A Naa40p substrate, the Naa40p histone H4 substrate remained N-terminally free in human haploid cells specifically deleted for canonical Naa40p27 or 237 amino acid long Naa40p (Naa40L), but expressing Naa40S. Interestingly, human Naa40L and Naa40S displayed differential expression and subcellular localization patterns by exhibiting a principal nuclear and cytoplasmic localization, respectively. Furthermore, Naa40L was shown to be N-terminally myristoylated and to interact with N-myristoyltransferase 1 (NMT1), implicating NMT1 in steering Naa40L nuclear import. Differential interactomics data obtained by biotin-dependent proximity labeling (BioID) further hints to context-dependent roles of Naa40p proteoforms. More specifically, with Naa40S representing the main co-translationally acting actor, the interactome of Naa40L was enriched for nucleolar proteins implicated in ribosome biogenesis and the assembly of ribonucleoprotein particles, overall indicating a proteoform-specific segregation of previously reported Naa40p activities. Finally, the yeast histone variant H2A.Z and the transcriptionally regulatory protein Lge1 were identified as novel Naa40p substrates, expanding the restricted substrate repertoire of Naa40p with two additional members and further confirming Lge1 as being the first redundant yNatA and yNatD substrate identified to date.
Highlights
Believed to occur co-translationally when a protein N-terminus emerges from the ribosomal peptide exit channel, N-terminal acetylation (Nt-acetylation) represents a widespread modification affecting most intracellular proteins in higher eukaryotes [1]
Both yeast (NAT4, subsequently referred to by the alias yNAA40 [12]) [13] and human NAA40 [14] were previously cloned and characterized, and we reported that these orthologs genes encode proteins displaying overlapping substrate specificities [14]
When comparatively analyzing the N-acetylation states of identified histone N-termini originating from control yNAA40, ynaa40∆ and human NAA40 (hNAA40) or hNAA40S yeast, we found that the previously characterized yNaa40p and hNaa40L substrate histone H4 was Nt-acetylated when expressing hNaa40S, while as expected, the N-free form of the Ser-Gly-Gly-Lys- starting N-terminus of yeast H2A (yH2A)—previously identified as being an exclusive yNaa40p substrate—could only be observed in the hNaa40S expressing strain ([14] and data not shown)
Summary
Believed to occur co-translationally when a protein N-terminus emerges from the ribosomal peptide exit channel, N-terminal acetylation (Nt-acetylation) represents a widespread modification affecting most intracellular proteins in higher eukaryotes [1]. In the hNAA40L- HAP1 cell line expressing residual hNaa40S, the complete N-free form of the Ser-Gly-Arg-Gly- H4 N-terminus could be observed, indicating that H4 remained (at least partially) Nt-free (Figure 5B) This observation may indicate that hNaa40p proteoforms may display non-redundant activities, and that expression of hNaa40L is required for (efficient) Nt-acetylation of previously characterized histone substrates. To study the subcellular localization of hNaa40p proteoforms in more detail and to unravel some of the (differential) proteins they interact with and infer different hNaa40p proteoform functionalities, we applied proximity-dependent biotin identification or BioID (Figure 8) For this purpose, a Flag-tagged version of the promiscuous biotin ligase protein BirA* was cloned in frame to the C-terminus of hNaa40L(dTIS>CTC) (permitting only hNaa40L expression) or hNaa40S in a doxycycline-inducible expression vector. Proximal proteins and putative interactors of the long and short proteoform of hNaa40p match dedicated subcellular and segregated molecular functions such as ribosome biogenesis, translation and fatty acid synthesis where hNaa40p has all previously been shown to play a role in
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