Arginyltransferase, Its Specificity, Putative Substrates, Bidirectional Promoter, and Splicing-derived Isoforms

Abstract

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...

The N-end rule pathway is a sensor of heme

“…Hemin at 5 and 10 M inhibited either the mouse or yeast R-transferases by more than 90% and 95%, respectively, as measured by the 14 C-Arg-based in vitro arginylation assay (2,3) (Fig. 2).…”

“…6 E and G). [''ATE1'' is used interchangeably with ''R-transferase'' (2).] Gel filtration-based spectroscopic assays (16)(17)(18) revealed the characteristic Soret band at 372 nm, with a shoulder at 423 nm, indicating a complex of hemin and R-transferase, as distinguished from free hemin (Fig.…”

Cytoplasmic protein quality control degradation mediated by parallel actions of the E3 ubiquitin ligases Ubr1 and San1

“…N-end rule-dependent degradation can also occur when a "fast" N terminus is added to a stable N terminus by the action Ate1 arginyl transferase (25). Accordingly, we tested Ubr1-dependent degradation in ate1Δ and ate1Δsan1Δ nulls (Fig.…”

Mouse Dfa Is a Repressor of TATA-box Promoters and Interacts with the Abt1 Activator of Basal Transcription

“…Other destabilizing N-terminal residues, called secondary or tertiary destabilizing residues, must be modified through deamidation and/or arginylation (6,7,14) or, alternatively, through N-terminal acetylation (13) before the corresponding proteins can be targeted by cognate N-recognins. In particular, the destabilizing activity of N-terminal Asp and Glu requires their conjugation, by the Ate1 R-transferase (6, 7, 12, 14 -16), to Arg, one of the primary destabilizing residues.…”