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 ϳ70-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.

The ATE1-encoded Arg-transferase mediates conjugation of Arg to N-terminal Asp, Glu, and Cys of certain eukaryotic proteins, yielding N-terminal Arg that can act as a degradation signal for the ubiquitin-dependent N-end rule pathway. We have previously shown that mouse ATE1 ؊/؊ embryos die with defects in heart development and angiogenesis. Here, we report that the ATE1 Arg-transferase mediates the in vivo degradation of RGS4 and RGS5, which are negative regulators of specific G proteins whose functions include cardiac growth and angiogenesis. The proteolysis of these regulators of G protein signaling (RGS) proteins was perturbed either by hypoxia or in cells lacking ubiquitin ligases UBR1 and͞or UBR2. Mutant RGS proteins in which the conserved Cys-2 residue could not become N-terminal were long-lived in vivo. We propose a model in which the sequential modifications of RGS4, RGS5, and RGS16 (N-terminal exposure of their Cys-2, its oxidation, and subsequent arginylation) act as a licensing mechanism in response to extracellular and intracellular signals before the targeting for proteolysis by UBR1 and UBR2. We also show that ATE1 ؊/؊ embryos are impaired in the activation of extracellular signal-regulated kinase mitogen-activated protein kinases and in the expression of G protein-induced downstream effectors such as Jun, cyclin D1, and ␤-myosin heavy chain. These results establish RGS4 and RGS5 as in vivo substrates of the mammalian N-end rule pathway and also suggest that the O2-ATE1-UBR1͞UBR2 proteolytic circuit plays a role in RGS-regulated G protein signaling in the cardiovascular system. ATE1 R-transferase ͉ G protein signaling ͉ oxidation ͉ ubiquitin ͉ UBR T he ubiquitin (Ub)-dependent N-end rule pathway relates the in vivo half-life of a protein to the identity of its N-terminal residue (1) (Fig. 1A). We previously identified the mouse ATE1 gene encoding Arg-transferase, which conjugates Arg to Nterminal Asp, Glu, and Cys of engineered N-end rule substrates (2, 3), yielding N-terminal Arg that can act as an essential component of N-degron (N-terminal degradation signal). Ndegrons can be recognized by Ub ligases (E3s) for protein ubiquitylation. Mouse ATE1 Ϫ/Ϫ embryos died with cardiovascular defects, including ventricular hypoplasia, ventricular septal defect, and impaired late angiogenesis (3), suggesting that the ATE1-dependent proteolysis of unknown substrate(s) is a crucial regulatory mechanism for myocardial growth and blood vessel integrity͞maturation. One aim of this study was to identify in vivo ATE1 substrates that are important for cardiovascular functions.We previously reported that RGS4, a G protein-specific GTPase-activating protein (4), is N-terminally arginylated and degraded by the Ub system in ATP-supplemented reticulocyte extract and that its in vitro degradation is inhibited by the Arg-Ala dipeptide inhibitor of the N-end rule pathway (5). However, previous attempts to verify the functional connection between RGS4 and the N-end rule pathway in vivo (in mammalian cells) were unsuccessful....

The S. cerevisiae proteasome comprises a 19-subunit regulatory particle (RP) and 28-subunit core particle (CP). To be degraded, substrates must cross the CP-RP interface, a site of complex conformational changes and regulatory events. This interface includes two aligned heteromeric rings: the six ATPase (Rpt) subunits of the RP and the seven α subunits of the CP. Rpt C-termini bind intersubunit cavities of the α ring, thus directing CP gating and proteasome assembly. We used crosslinking to map the Rpt C-termini to the α subunit pockets. This reveals an unexpected asymmetry: one side of the ring shows 1:1 contacts of Rpt2–α4, Rpt6–α3, and Rpt3–α2, whereas, on the opposite side, the Rpt1, Rpt4, and Rpt5 tails each crosslink to multiple α pockets. Rpt-CP crosslinks are all sensitive to nucleotide, implying that ATP hydrolysis drives dynamic alterations at the CP-RP interface.