N-terminal methionine excision of proteins creates tertiary destabilizing N-degrons of the Arg/N-end rule pathway

Abstract

All organisms begin protein synthesis with methionine (Met). The resulting initiator Met of nascent proteins is irreversibly processed by Met aminopeptidases (MetAPs). N-terminal (Nt) Met excision (NME) is an evolutionarily conserved and essential process operating on up to two-thirds of proteins. However, the universal function of NME remains largely unknown. MetAPs have a well-known processing preference for Nt-Met with Ala, Ser, Gly, Thr, Cys, Pro, or Val at position 2, but using CHX-chase assays to assess protein degradation in yeast cells, as well as protein-binding and RT-qPCR assays, we demonstrate here that NME also occurs on nascent proteins bearing Met-Asn or Met-Gln at their N termini. We found that the NME at these termini exposes the tertiary destabilizing Nt residues (Asn or Gln) of the Arg/N-end rule pathway, which degrades proteins according to the composition of their Nt residues. We also identified a yeast DNA repair protein, MQ-Rad16, bearing a Met-Gln N terminus, as well as a human tropomyosin-receptor kinase-fused gene (TFG) protein, MN-TFG, bearing a Met-Asn N terminus as physiological, MetAP-processed Arg/N-end rule substrates. Furthermore, we show that the loss of the components of the Arg/N-end rule pathway substantially suppresses the growth defects of naa20⌬ yeast cells lacking the catalytic subunit of NatB Nt acetylase at 37°C. Collectively, the results of our study reveal that NME is a key upstream step for the creation of the Arg/N-end rule substrates bearing tertiary destabilizing residues in vivo. Virtually all polypeptides emerging from ribosomes begin with methionine (Met), as this is the residue dictated by the translation initiation codons. The initiator N-terminal (Nt-) 2 Met of nascent polypeptides is co-translationally and irreversibly excised by ribosome-bound Met-aminopeptidases (MetAPs) if it includes a penultimate residue with a small and uncharged side chain (Ala, Gly, Ser, Cys, Thr, Pro, or Val) (1). Evolutionarily conserved Nt-Met (Nt-M) excision (NME) is applied to more than 50% of all nascent proteins, thus profoundly contributing to Nt-proteome diversity, enzyme activity, cell growth and viability, free Met or GSH homeostasis, etc. Nonetheless, the universal role of this massive NME process remains largely elusive (1-3). In ϳ60% of nascent yeast proteins and 80% of nascent human proteins, the retained Nt-Met (Nt-M) and neo-Nt residues after NME very frequently undergo N␣-terminal acetylation (Ntacetylation) (4). The Nt-M acetylation state of nascent proteins can be a primary decision-making step for the retention or removal of Nt-M, as it dynamically competes with NME on ribosomes by blocking MetAPs via steric hindrance (1, 4, 5). Moreover, numerous lines of evidence have uncovered that Nt-acetylation substantially contributes to protein stability, activity, folding, localization, and interaction, thus affecting cell proliferation, apoptosis, development, etc. (4, 6). Furthermore, Nt-acetylation elicits specific protein degradation signals (degrons), which are reco...

Does N‐Terminal Protein Acetylation Lead to Protein Degradation?

“…Importantly, it was observed that Map1‐mediated proteolytic processing of MN N‐termini exposes the asparagine amino acid residue at the substrate N‐terminal side of the target protein, which is subsequently targeted for proteasomal degradation via the action of the classical Arg/N‐end rule pathway (Figure B) . In line with the previous observation, recent work unveiled the Nt‐excision of Met adjacent to Asn (and Gln), thus creating substrates for this classical Arg/N‐end rule pathway …”

Recognition of nonproline N-terminal residues by the Pro/N-degron pathway

“…Among the routes to N-degrons are cleavages of proteins by proteases that include Met-aminopeptidases (MetAPs), caspases, and calpains. These and many other nonprocessive proteases function as initial targeting components of N-degron pathways by cleaving a prosubstrate protein and exposing a destabilizing Nt-residue in the resulting C-terminal (Ct) fragment (37,57,69,70,(74)(75)(76)(77)(78)(79)(80).…”

The plant N‐degron pathways of ubiquitin‐mediated proteolysis

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