Alternative splicing results in differential expression, activity, and localization of the two forms of arginyl-tRNA-protein transferase, a component of the N-end rule pathway.

The half-lives of intracellular proteins range from a few seconds to many days. The rates of processive proteolysis are a function of the cell’s physiological state and are controlled differentially for specific proteins. In particular, most of the damaged or otherwise abnormal proteins are metabolically unstable. Many other proteins, while long-lived as components of larger macromolecular structures such as ribosomes and oligomeric proteins, are metabolically unstable as free subunits. Regulatory proteins are often also short-lived in vivo, providing a way to generate their spatial gradients and to rapidly adjust their concentrations, or subunit compositions, through changes in the rate of their synthesis or degradation (20, 23, 28, 39, 44, 55). The posttranslational conjugation of arginine (Arg) to the N termini of eukaryotic proteins was described 35 years ago (26), but the function of this modification, and of the enzyme involved, Arg-tRNA-protein transferase (R-transferase) (47), remained unknown until the discovery that the identity of N-terminal residue in a protein influences its metabolic stability (4). The resulting relation was termed the N-end rule (54). Aspartate (Asp) and glutamate (Glu), the two N-terminal residues known to be arginylated by R-transferase (47), were shown to be destabilizing residues in the N-end rule (4). It was therefore proposed (4) that the function of R-transferase is to target proteins for degradation by conjugating Arg, one of the primary destabilizing residues, to secondary destabilizing N-terminal residues (Asp and Glu in fungi; Asp, Glu, and Cys in metazoans (18) (Fig. ​(Fig.1).1). It was also proposed (4) that the analogous prokaryotic enzyme Leu, Phe-tRNA-protein transferase (L, F-transferase) (47) mediates the activity of N-terminal Arg and Lys, which, in prokaryotes, would be the secondary destabilizing residues. These conjectures were confirmed (7, 17, 45, 53). FIG. 1 Comparison of enzymatic reactions that underlie the activity of the tertiary and secondary destabilizing residues among eukaryotes. (A) Mammals (reference 54 and this work); (B) the yeast S. cerevisiae (5). N-terminal residues are indicated by single-letter ... The similar but distinct degradation signals which together give rise to the N-end rule are called the N-degrons (54, 56). In eukaryotes, an N-degron comprises two determinants: a destabilizing N-terminal residue and an internal Lys residue of a substrate (5, 22). The Lys residue is the site of formation of a substrate-linked multiubiquitin chain (11). The N-end rule pathway is thus one pathway of the ubiquitin (Ub) system. Ub is a 76-residue protein whose covalent conjugation to other proteins plays a role in a multitude of processes, including cell growth, division, differentiation, and responses to stress (20, 23, 39, 55). In many of these processes, Ub acts through routes that involve the degradation of Ub-protein conjugates by the 26S proteasome, an ATP-dependent multisubunit protease (9, 13, 40, 43). (In the text that follows, names of mouse genes are in italics, with the first letter uppercase. Names of human and Saccharomyces cerevisiae genes are also in italics, all uppercase. If human and mouse genes are named in the same sentence, the mouse gene notation is used. Names of S. cerevisiae proteins are roman, with the first letter uppercase and an extra lowercase “p” at the end. Names of the corresponding mouse and human proteins are the same, except that all letters but the last “p” are uppercase. The latter usage is a modification of the existing convention (50), to facilitate simultaneous discussions of yeast, mouse, and human proteins. In some citations, the abbreviated name of a species precedes the gene’s name.) The N-end rule is organized hierarchically. In the yeast S. cerevisiae, Asn and Gln are tertiary destabilizing N-terminal residues in that they function through their conversion, by the NTA1-encoded N-terminal amidase (Nt-amidase) (6), to the secondary destabilizing N-terminal residues Asp and Glu. The destabilizing activity of N-terminal Asp and Glu requires their conjugation, by the S. cerevisiae ATE1-encoded Arg-tRNA-protein transferase (R-transferase), to Arg, one of the primary destabilizing residues (7) (Fig. ​(Fig.1B).1B). In mammals, the deamidation step is bifurcated, in that two distinct Nt-amidases specific, respectively, for N-terminal Asn and Gln, mediate the activity of tertiary destabilizing residues (19, 51) (Fig. ​(Fig.1A).1A). Mice lacking the Asn-specific Nt-amidase NTAN1p have recently been produced through targeted mutagenesis and found to be fertile, outwardly normal, but behaviorally distinct from their congenic wild-type counterparts (28a). In mammals, the set of secondary destabilizing residues contains not only Asp and Glu but also Cys, which is a stabilizing residue in yeast (18, 54) (Fig. ​(Fig.1).1). The primary destabilizing N-terminal residues are bound directly by the UBR1-encoded N-recognin (also called E3α), the recognition component of the N-end rule pathway (8). In S. cerevisiae, N-recognin is a 225-kDa protein that binds to potential N-end rule substrates through their primary destabilizing N-terminal residues, Phe, Leu, Trp, Tyr, Ile, Arg, Lys, and His (54). The Ubr1 genes encoding mouse and human N-recognins, also called Eα (21, 41), have been cloned (29), and mouse strains lacking Ubr1 have recently been constructed (29a). The known functions of the N-end rule pathway include the control of peptide import in S. cerevisiae, through the degradation of Cup9p, a transcriptional repressor of PTR2 which encodes the peptide transporter (2, 10); a role in regulating the Sln1p-dependent phosphorylation cascade that mediates osmoregulation in S. cerevisiae (38); the degradation of Gpa1p, a Gα protein of S. cerevisiae (34); and the conditional degradation of alphaviral RNA polymerase in virus-infected metazoan cells (16, 54). Physiological N-end rule substrates were also identified among the proteins secreted into the host cell’s cytosol by intracellular parasites such as the bacterium Listeria monocytogenes (46). Inhibition of the N-end rule pathway was reported to interfere with mammalian cell differentiation (24) and to delay limb regeneration in amphibians (52). Microarray-based comparisons of gene expression patterns in wild-type and congenic ubr1Δ strains of S. cerevisiae have shown that a number of yeast genes, of diverse functions, are significantly up- or down-regulated in the absence of the N-end rule pathway (39a). The mammalian counterpart of the yeast ATE1-encoded R-transferase was partially purified from rabbit reticulocytes and shown to cofractionate with Arg-tRNA synthetase (12). Recent studies of the Ub-dependent proteolysis of endogenous proteins in muscle extracts suggested that the N-end rule pathway plays a major role in catabolic states that result in muscle atrophy (48, 49). A significant fraction of the N-end rule pathway’s activity in muscle extracts was found to be tRNA dependent, indicating the involvement of R-transferase (48, 49). It was also reported that a crush injury to the rat sciatic nerve results in a ∼10-fold increase in the rate of arginine conjugation to the N termini of unidentified proteins in the nerve’s region upstream of the crush site (15, 57), suggesting an injury-induced increase in the concentration of R-transferase substrates and/or an enhanced activity of the N-end rule pathway. In this work, we began the functional analysis of mammalian R-transferase (ATE1p) by isolating mouse cDNA encoding this enzyme. Surprisingly, we found two Ate1 cDNA species, which were identical except for a 129-bp region that encoded similar but distinct sequences. One or the other, but not both, of the corresponding Ate1 exons is retained in the mature Ate1 mRNA, and the ratio of the resulting two species, Ate1-1 and Ate1-2, varies greatly among mouse tissues. We also show that ATE1-1p and ATE1-2p, while differing in activity, can arginylate N-terminal Asp and Glu in model substrates. However, neither of them can arginylate N-terminal Cys, the known secondary destabilizing residue (18), suggesting the existence of a distinct Cys-specific mammalian R-transferase.