Studies of the N-End Rule Pathway in Saccharomyces cerevisiae

Abstract

Many intracellular proteins are either conditionally or constitutively short-lived, with in vivo half-lives that can be as brief as a few minutes. The regulated and processive degradation of intracellular proteins is carried out largely by the ubiquitin (Ub)-proteasome system (UPS). In eukaryotes, the N-end rule pathway is a part of the UPS. The N-end rule relates the regulation of the in vivo half-life of a protein to the identity of its N-terminal residue. Degradation signals (degrons) that are targeted by the N-end rule pathway include a set called N-degrons. E3 Ub ligases of the N-end rule pathway are called N-recognins. They bind to primary destabilizing N-terminal residues of N-end rule substrates. The N-end rule pathway comprises two major branches, the Arg/N-end rule pathway and the Ac/N-end rule pathway. The Arg/N-end rule branch involves the N-terminal arginylation of protein substrates and also the targeting of specific unmodified N-terminal residues by E3 N-recognins. The S. cerevisiae Arg/N-end rule pathway contains a single N-recognin, Ubr1. The Ub-fusion degradation (UFD) pathway is also a part of the UPS. This pathway recognizes a nonremovable N-terminal Ub moiety of a Ub fusion as a primary degron. My collaborator, Cheol-Sang Hwang, and I demonstrated that the RING-type Ubr1 E3 and the HECT-type Ufd4 E3 interact, both physically and functionally. We showed that the Ubr1-Ufd4 complex targets the S. cerevisiae Mgt1 DNA repair enzyme through a degron near its N-terminus, in addition to mediating the Arg/N-end rule pathway and a part of the UFD pathway as well. We also further characterized the physical interaction between Ubr1 and Ufd4. I also report the discovery of the other branch of the N-end rule pathway, the Ac/N-end rule pathway, which recognizes N-terminally acetylated residues as N-degrons, termed Ac/N-degrons. We showed that Ac/N-degrons are recognized by the Doa10 E3 Ub ligase and apparently by other E3s as well. Given the prevalence of Ac/N-degrons, as nearly 90% of human proteins are Nt-acetylated, we also demonstrated the physiological role of Ac/N-degrons in protein quality, including the regulation of input stoichiometries of subunits in oligomeric proteins.