A Family of Mammalian E3 Ubiquitin Ligases That Contain the UBR Box Motif and Recognize N-Degrons

Abstract

A subset of proteins targeted by the N-end rule pathway bear degradation signals called N-degrons, whose determinants include destabilizing N-terminal residues. Our previous work identified mouse UBR1 and UBR2 as E3 ubiquitin ligases that recognize N-degrons. Such E3s are called N-recognins. We report here that while double-mutant UBR1^−/− UBR2^−/− mice die as early embryos, the rescued UBR1^−/− UBR2^−/− fibroblasts still retain the N-end rule pathway, albeit of lower activity than that of wild-type fibroblasts. An affinity assay for proteins that bind to destabilizing N-terminal residues has identified, in addition to UBR1 and UBR2, a huge (570 kDa) mouse protein, termed UBR4, and also the 300-kDa UBR5, a previously characterized mammalian E3 known as EDD/hHYD. UBR1, UBR2, UBR4, and UBR5 shared a ∼70-amino-acid zinc finger-like domain termed the UBR box. The mammalian genome encodes at least seven UBR box-containing proteins, which we propose to call UBR1 to UBR7. UBR1^−/− UBR2^−/− fibroblasts that have been made deficient in UBR4 as well (through RNA interference) were significantly impaired in the degradation of N-end rule substrates such as the Sindbis virus RNA polymerase nsP4 (bearing N-terminal Tyr) and the human immunodeficiency virus type 1 integrase (bearing N-terminal Phe). Our results establish the UBR box family as a unique class of E3 proteins that recognize N-degrons or structurally related determinants for ubiquitin-dependent proteolysis and perhaps other processes as well.

ACKNOWLEDGMENTS

We are grateful to Jai Wha Seo for establishment and immortalization of UBR1^−/− UBR2^−/− embryonic fibroblasts, Allen Shearn for anti-HYD antibody, and Song Li and Yong Wan for helpful advice.

This work was in part supported by NIH grant GM69482 and the American Heart Association Award (to Y.T.K), by NIH grants GM31530 and DK39520 and a grant from the Ellison Medical Foundation (to A. Varshavsky), by NIH grant AI47054 (to M.M.), and by NIH grant AI056987 to (L.C.F.M.).