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 Δ 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 .