The retained N-terminal methionine (Met) residue of a nascent protein is often N-terminally acetylated (Nt-acetylated). Removal of N-terminal Met by Met-aminopeptidases frequently leads to Nt-acetylation of the resulting N-terminal Ala, Val, Ser, Thr and Cys residues. Although a majority of eukaryotic proteins, for example, more than 80% of human proteins, are cotranslationally Nt-acetylated, the function of this extensively studied modification is largely unknown. Here we found, using the yeast Saccharomyces cerevisiae, that the Nt-acetylated Met residue could act as a degradation signal (degron), targeted by the Doa10 ubiquitin ligase. Moreover, Doa10 also recognized the Nt-acetylated Ala, Val, Ser, Thr and Cys residues. Several examined proteins of diverse functions contained these N-terminal degrons, termed AcN-degrons, which comprise a prevalent class of degradation signals in cellular proteins.

SUMMARY Nα-terminal acetylation of cellular proteins was recently discovered to create specific degradation signals, termed Ac/N-degrons and targeted by the Ac/N-end rule pathway. We show that Hcn1, a subunit of the APC/C ubiquitin ligase, contains an Ac/N-degron that is repressed by Cut9, another APC/C subunit and the ligand of Hcn1. Cog1, a subunit of the Golgi-associated COG complex, is also shown to contain an Ac/N-degron. Cog2 and Cog3, direct ligands of Cog1, can repress this degron. The subunit decoy technique was used to show that the long-lived endogenous Cog1 is destabilized and destroyed via its activated (unshielded) Ac/N-degron if the total level of Cog1 increased in a cell. Hcn1 and Cog1 are the first examples of protein regulation through the physiologically relevant transitions that shield and unshield natural Ac/N-degrons. This mechanistically straightforward circuit can employ the demonstrated conditionality of Ac/N-degrons to regulate subunit stoichiometries and other aspects of protein quality control.

SUMMARY The Arg/N-end rule pathway targets for degradation proteins that bear specific unacetylated N-terminal residues while the Ac/N-end rule pathway targets proteins through their Nα-terminally acetylated (Nt-acetylated) residues. Here we show that Ubr1, the ubiquitin ligase of the Arg/N-end rule pathway, recognizes unacetylated N-terminal methionine if it is followed by a hydrophobic residue. This capability of Ubr1 expands the range of substrates that can be targeted for degradation by the Arg/N-end rule pathway, because virtually all nascent cellular proteins bear N-terminal methionine. We identified Msn4, Sry1, Arl3, and Pre5 as examples of normal or misfolded proteins that can be destroyed through the recognition of their unacetylated N-terminal methionine. Inasmuch as proteins bearing the Nt-acetylated N-terminal methionine residue are substrates of the Ac/N-end rule pathway, the resulting complementarity of the Arg/N-end rule and Ac/N-end rule pathways enables the elimination of protein substrates regardless of acetylation state of N-terminal methionine in these substrates.

Copper-and zinc-containing superoxide dismutase (Cu/ZnSOD) is suspected to be one of the anti-oxidant enzymes and virulence determinants active in some pathogenic micro-organisms. To elucidate the role of Cu/ZnSOD in the major human fungal pathogen Candida albicans, its gene, designated SOD1, was disrupted by the URA-blaster technique. The resulting sod1/sod1 mutant showed delayed hyphal growth on Spider medium but could still form hyphae on other solid media or in liquid media, particularly in response to serum. Moreover, the sod1/sod1 mutant was more sensitive to menadione, a redoxcycling agent, than the isogenic wild-type strain, although it still showed an adaptive oxidative stress response. Furthermore, the sod1/sod1 mutant cells exhibited slow growth in minimal medium when compared to the wild-type cells, but their growth was restored by the addition of lysine to the medium. Interestingly, C. albicans cells lacking Cu/ZnSOD showed increased susceptibility to macrophage attack and had attenuated virulence in mice. Thus, these results suggest that Cu/ZnSOD is required for the protection of C. albicans against oxidative stresses and for the full virulence of the organism to be expressed.

Substrates of the N-end rule pathway are recognized by the Ubr1 E3 ubiquitin ligase through their destabilizing N-terminal residues. Our previous work showed that the Ubr1 E3 and the Ufd4 E3 co-target an internal degron of the Mgt1 DNA repair protein. Ufd4 is an E3 of the ubiquitin-fusion degradation (UFD) pathway that recognizes an N-terminal ubiquitin moiety. Here we report that the RING-type Ubr1 E3 and the HECT-type Ufd4 E3 interact, both physically and functionally. Although Ubr1 can recognize and polyubiquitylate an N-end rule substrate in the absence of Ufd4, the Ubr1-Ufd4 complex is more processive in that it produces a longer substrate-linked polyubiquitin chain. Conversely, Ubr1 can function as a polyubiquitylation-enhancing component of the Ubr1-Ufd4 complex in its targeting of UFD substrates. We also found that Ubr1 can recognize the N-terminal ubiquitin moiety. These and related advances unify two proteolytic systems that have been studied separately over two decades.