N-terminal acetylome analyses and functional insights of the N-terminal acetyltransferase NatB

Abstract

Protein N-terminal acetylation (Nt-acetylation) is an important mediator of protein function, stability, sorting, and localization. Although the responsible enzymes are thought to be fairly well characterized, the lack of identified in vivo substrates, the occurrence of Nt-acetylation substrates displaying yet uncharacterized N-terminal acetyltransferase (NAT) specificities, and emerging evidence of posttranslational Nt-acetylation, necessitate the use of genetic models and quantitative proteomics. NatB, which targets Met-Glu-, Met-Asp-, and Met-Asn-starting protein N termini, is presumed to Nt-acetylate 15% of all yeast and 18% of all human proteins. We here report on the evolutionary traits of NatB from yeast to human and demonstrate that ectopically expressed hNatB in a yNatB-Δ yeast strain partially complements the natB-Δ phenotypes and partially restores the yNatB Nt-acetylome. Overall, combining quantitative N-terminomics with yeast studies and knockdown of hNatB in human cell lines, led to the unambiguous identification of 180 human and 110 yeast NatB substrates. Interestingly, these substrates included Met-Gln-N-termini, which are thus now classified as in vivo NatB substrates. We also demonstrate the requirement of hNatB activity for maintaining the structure and function of actomyosin fibers and for proper cellular migration. In addition, expression of tropomyosin-1 restored the altered focal adhesions and cellular migration defects observed in hNatB-depleted HeLa cells, indicative for the conserved link between NatB, tropomyosin, and actin cable function from yeast to human.

Actin polymerization and cell motility are affected by NAA80-mediated posttranslational N-terminal acetylation of actin

“…Usually, the identity of the substrate amino acids at position 1 and 2 are the major determinants for enzyme binding of the other known NATs [11]. Another factor limiting the cellular substrate pool of NAA80 is the presence of NatB which efficiently acetylate all Met-starting acidic N-termini in a co-translational manner (such as the premature N-terminus of actin), and yeast depleted for NatB are lacking Nt-acetylation at these substrates [12]. However, expression of human NAA80 in a yeast NatB deletion strain partially restored the Nt-acetylation of NatB substrates [10].…”

N-terminal acetylation promotes synaptonemal complex assembly in C. elegans

“…The fact that residual/partial N-terminal acetylation of NatB substrates is still detected in NatB mutants suggests a redundancy between NatB and other NATs, which has also been observed in other organisms (Van Damme et al 2012). Redundancies between NATs may serve as a Figure 5.…”

“…These groups of termini are also defined as NatB substrates in budding yeast and humans (Van Damme et al 2012), suggesting a conservation of N-terminal acetylation specificity. However, partial acetylation of NatB substrates was still observed in the null mutants (27%-80%), suggesting potential redundancies between NatB and other NATs, consistent with previous observations in yeast (Van Damme et al 2012). Importantly, the decrease in N-terminal acetylation observed in natb-1 mutants mimicked that of cra-1 mutants, consistent with the hypothesis that CRA-1 and NATB-1 are the constitutive subunits of the NatB complex in worms.…”

A Saccharomyces cerevisiae Model Reveals In Vivo Functional Impairment of the Ogden Syndrome N-Terminal Acetyltransferase NAA10 Ser37Pro Mutant

“…In addition, a putative hNatE complex has been described (9 -10). Except for NatF, which is only expressed in higher eukaryotes (1), the substrate specificity profiles of the NatA-E complexes seem to be conserved among eukaryotes (5)(6)(7)(8)(9)(11)(12)(13).…”