No abstract

Substrates of the ubiquitin-dependent N-end rule pathway include proteins with destabilizing N-terminal residues. UBR1 ؊/؊ mice, which lacked the pathway's ubiquitin ligase E3␣, were viable and retained the N-end rule pathway. The present work describes the identification and analysis of mouse UBR2, a homolog of UBR1. We demonstrate that the substrate-binding properties of UBR2 are highly similar to those of UBR1, identifying UBR2 as the second E3 of the mammalian N-end rule pathway. UBR2 ؊/؊ mouse strains were constructed, and their viability was found to be dependent on both gender and genetic background. In the strain 129 (inbred) background, the UBR2 ؊/؊ genotype was lethal to most embryos of either gender. In the 129/B6 (mixed) background, most UBR2 ؊/؊ females died as embryos, whereas UBR2 ؊/؊ males were viable but infertile, owing to the postnatal degeneration of the testes. The gross architecture of UBR2 ؊/؊ testes was normal and spermatogonia were intact as well, but UBR2 ؊/؊ spermatocytes were arrested between leptotene/zygotene and pachytene and died through apoptosis. A conspicuous defect of UBR2 ؊/؊ spermatocytes was the absence of intact synaptonemal complexes. We conclude that the UBR2 ubiquitin ligase and, hence, the N-end rule pathway are required for male meiosis and spermatogenesis and for an essential aspect of female embryonic development.

Substrates of the N-end rule pathway include proteins with destabilizing N-terminal residues. Three of them, Asp, Glu, and (oxidized) Cys, function through their conjugation to Arg, one of destabilizing N-terminal residues that are recognized directly by the pathway's ubiquitin ligases. The conjugation of Arg is mediated by arginyltransferase, encoded by ATE1. Through its regulated degradation of specific proteins, the arginylation branch of the N-end rule pathway mediates, in particular, the cardiovascular development, the fidelity of chromosome segregation, and the control of signaling by nitric oxide. We show that mouse ATE1 specifies at least six mRNA isoforms, which are produced through alternative splicing, encode enzymatically active arginyltransferases, and are expressed at varying levels in mouse tissues. We also show that the ATE1 promoter is bidirectional, mediating the expression of both ATE1 and an oppositely oriented, previously uncharacterized gene. In addition, we identified GRP78 (glucose-regulated protein 78) and protein-disulfide isomerase as putative physiological substrates of arginyltransferase. Purified isoforms of arginyltransferase that contain the alternative first exons differentially arginylate these proteins in extract from ATE1 ؊/؊ embryos, suggesting that specific isoforms may have distinct functions. Although the N-end rule pathway is apparently confined to the cytosol and the nucleus, and although GRP78 and protein-disulfide isomerase are located largely in the endoplasmic reticulum, recent evidence suggests that these proteins are also present in the cytosol and other compartments in vivo, where they may become N-end rule substrates.A protein substrate of the ubiquitin (Ub) 2 -proteasome system, which controls the levels of many intracellular proteins, is conjugated to Ub through the action of ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-protein ligase (E3) (1-7). The term "Ub ligase" denotes either an E2-E3 holoenzyme or its E3 component. The selectivity of ubiquitylation is mediated largely by E3, which recognizes a degradation signal (degron) of a substrate (1, 3, 8 -12). The E3 Ub ligases are an exceptionally large family, with more than 500 distinct E3s in a mammal (5,(12)(13)(14). A ubiquitylated protein bears a covalently linked poly-Ub chain and is targeted for processive degradation by the 26 S proteasome (15)(16)(17)(18)(19). Ub has other functions as well, including nonproteolytic ones (20,21).An essential determinant of one class of degrons, called N-degrons, is a destabilizing N-terminal residue of a substrate. The set of destabilizing residues in a given cell type yields a rule, called the N-end rule, which relates the in vivo half-life of a protein to the identity of its N-terminal residue (1,(22)(23)(24)(25)(26). In eukaryotes, the N-degron consists of three determinants: a destabilizing N-terminal residue of a protein substrate, its internal Lys residue(s) (the site of formation of a poly-Ub chain), and a conformationally fl...

c‐MOS, a MAP kinase kinase kinase, is a regulator of oocyte maturation. The concentration of c‐MOS is controlled in part through its conditional degradation. Previous studies proposed the ‘second‐codon rule’, according to which the N‐terminal proline (Pro) of c‐MOS is a destabilizing residue that targets c‐MOS for degradation. We analyzed the degradation signal (degron) of c‐MOS in Xenopus oocytes, found it to be a portable degron, and demonstrated that, contrary to the model above, the N‐terminal Pro residue of c‐MOS is entirely dispensable for its degradation if Ser‐2 (encoded Ser‐3) of c‐MOS is replaced by a small non‐phosphorylatable residue such as Gly. The dependence of c‐MOS degradation on N‐terminal Pro is shown to be caused by a Pro‐mediated downregulation of the net phosphorylation of Ser‐2, a modification that halts c‐MOS degradation in oocytes. Thus, the N‐terminal Pro residue of c‐MOS is not a recognition determinant for a ubiquitin ligase, in agreement with earlier evidence that Pro is a stabilizing residue in the N‐end rule.

Previous studies have shown that a highly conserved lysyl residue (Lys 419) near the C-terminus of Anabaena ADP-glucose pyrophosphorylase is involved in the binding of 3-P-glycerate, the allosteric activator [Charng, Y., Iglesias, A. A., & Preiss, J. (1994) J. Biol. Chem. 269, 24107-24113]. Phosphopyridoxylation of the K419R mutant enzyme modified another conserved lysyl residue (Lys382), suggesting that this residue might be also located within the activator-binding site [Charng, Y., Iglesias, AA., & Preiss, J. (1994) J. Biol. Chem. 269, 24107-24113]. Site-directed mutagenesis of Lys382 of the Anabaena enzyme was performed to determine the role of this residue. Replacing Lys382 with either arginine, alanine, or glutamine produced mutant enzymes with apparent affinities for 3-P-glycerate 10-160-fold lower than that of the wild-type enzyme. The glutamic acid mutant enzyme was inhibited by 3-P-glycerate. These mutations had lesser impact on the kinetic constants for the substrates and inhibitor, P(i), and on the thermal stability. These results indicate that both the charge and size of the residue at position 382 influence the binding of 3-P-glycerate. Site-directed mutagenesis was also performed to obtain a K382R-K419R double mutant. The apparent affinity for 3-P-glycerate of this double-mutant enzyme was 104-fold lower than that of the wild-type enzyme, and the specificity for activator of this mutant enzyme was altered. The K382R-K419R enzyme could not be phosphopyridoxylated, suggesting that other lysine residues are not involved in the binding of 3-P-glycerate.