Construction and Analysis of Mouse Strains Lacking the Ubiquitin Ligase UBR1 (E3α) of the N-End Rule Pathway

Many biological processes are regulated by circuits that involve conditionally or constitutively short-lived proteins. Features of proteins that confer metabolic instability are called degradation signals, or degrons (16, 36, 72). The essential component of one degradation signal, termed the N-degron, is a destabilizing N-terminal residue of a protein (3, 71). A set of amino acid residues that are destabilizing in a given cell 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. Variants of the underlying proteolytic system, called the N-end rule pathway, are present in all organisms examined, from mammals and plants to fungi and prokaryotes (51, 71). In eukaryotes, an N-degron consists of two determinants: a destabilizing N-terminal residue and an internal lysine of a substrate protein (4, 29, 66). The recognition of N-degron by the targeting machinery involves stochastic selection of second-determinant Lys residues from among the substrate's sterically suitable lysines (4, 29, 66). This Lys residue is the site of formation of a substrate-linked multiubiquitin (multi-Ub) chain (10, 48, 78). The N-end rule pathway is, thus, one pathway of the Ub system (15, 20, 24–26, 57). Ub is a 76-residue protein whose covalent conjugation to other proteins plays a role in a vast range of biological processes, including cell growth, division, differentiation, and responses to stress (24, 26, 49, 74). In most of these processes, Ub acts through routes that involve processive degradation of Ub-protein conjugates by the 26S proteasome, an ATP-dependent protease (14, 52, 75). The N-end rule has a hierarchic structure. In the yeast Saccharomyces cerevisiae, Asn and Gln are tertiary destabilizing N-terminal residues in that they function through their deamidation, by the NTA1-encoded N-terminal amidase (Nt-amidase), to yield the secondary destabilizing N-terminal residues Asp and Glu (6, 63). The destabilizing activity of N-terminal Asp and Glu requires their conjugation, by the ATE1-encoded Arg-tRNA-protein transferase (R-transferase), to Arg, one of the primary destabilizing residues (7, 40, 71) (Fig. ​(Fig.1).1). These latter residues are bound directly by UBR1 (N-recognin), the E3 (recognition) component of the N-end rule pathway. S. cerevisiae UBR1 is a 225-kDa E3 which binds to potential N-end rule substrates through its type 1 and type 2 substrate-binding sites. The type 1 site binds the basic N-terminal residues Arg, Lys, and His. The type 2 site binds the bulky hydrophobic N-terminal residues Phe, Leu, Trp, Tyr, and Ile (34, 71) (Fig. ​(Fig.1).1). S. cerevisiae UBR1 also contains a third substrate-binding site which targets proteins such as CUP9 and GPA1 through their internal (non-N-terminal) degrons (9, 56, 68). The UBR1-encoded E3, in a complex with the RAD6-encoded E2 (Ub-conjugating) enzyme, catalyzes the synthesis of a substrate-linked multi-Ub chain (17, 71) and may also mediate the delivery of substrates to the 26S proteasome (80). UBR1 contains a functionally essential RING-H2 domain (79), a feature of many otherwise distinct E3s (28, 70, 78). FIG. 1 (A) The N-end rule pathway in mammals (12, 32, 33). N-terminal residues are indicated by single-letter abbreviations for amino acids. The ovals denote the rest of a protein substrate. The Asn-specific N-terminal amidase (NtN-amidase) NTAN1 converts N-terminal ... The term Ub ligase denotes either an E2-E3 complex or its E3 component. The numerous proteolytic pathways of the Ub system have in common their dependence on Ub conjugation and the proteasome and differ largely through their utilization of distinct E2-E3 complexes. The RAD6-UBR1 (E2-E3) Ub ligase of the N-end rule pathway is one example of such a complex. Specific E3s recognize (bind to) specific degrons of their protein substrates. The diversity of E3s underlies the enormous range of substrates that are recognized and destroyed by the Ub system in ways that are regulated both temporally and spatially. There are dozens of E3s in S. cerevisiae and possibly hundreds of distinct E3s in mammals (78). In contrast to yeast, where N-terminal Asn and Gln are deamidated by a single Nt-amidase, in mammals there are two enzymes, NtN-amidase and NtQ-amidase, which are specific for N-terminal Asn and Gln, respectively (19, 32, 64) (Fig. ​(Fig.1).1). In vertebrates, the set of secondary destabilizing residues contains not only Asp and Glu but also Cys, the latter being a stabilizing residue in the yeast N-end rule (11, 18). The two known species of mammalian R-transferase, ATE1-1 and ATE1-2, are produced through alternative splicing of ATE1 pre-mRNA (33). The substrate specificities of ATE1-1 and ATE1-2 are similar to those of the ATE1-encoded R-transferase of S. cerevisiae in that they can arginylate N-terminal Asp and Glu but cannot arginylate N-terminal Cys (33). However, recent work revealed that mouse ATE1−/− cells are incapable of arginylating any of the three secondary destabilizing N-terminal residues—Asp, Glu, and Cys (Y. T. Kwon, A. Kashina, I. Davydov, and A. Varshavsky, unpublished data). The known functions of the N-end rule pathway include the control of peptide import in S. cerevisiae through the conditional degradation of CUP9, a transcriptional repressor of the peptide transporter PTR2 (1, 9, 68). It remains to be determined whether the N-end rule pathway has similar import-regulating functions in prokaryotes and multicellular eukaryotes. The S. cerevisiae N-end rule pathway is also essential for chromosome stability, through degradation, at the metaphase-anaphase transition, of a fragment of cohesin complexes that hold together sister chromatids (51). Given the evolutionary conservation of separase and cohesin (76), this function of the yeast N-end rule pathway may be relevant to other eukaryotes as well. Besides CUP9 and SCC1, several other proteins were also found to be substrates of the N-end rule pathway. These proteins include Sindbis virus RNA polymerase (and homologous polymerases of other alphaviruses) (13), HIV integrase (45), a bacterial protein, p60, which is secreted by Listeria monocytogenes into the cytosol of infected mammalian cells (59), the mammalian GTPase-accelerating (GAP) proteins RGS4 and RGS16 (12), the S. cerevisiae GPA1-encoded Gα protein (43, 56), and the encephalomyocarditis (EMC) virus 3C protease (37). Physiological functions, if any, of the degradation of these proteins by the N-end rule pathway are either unknown or have not been established definitively. In yeast only one E3, encoded by UBR1, mediates the recognition of substrates by the N-end rule pathway (8, 71). Studies of the Ub-dependent proteolysis in rabbit reticulocyte extracts suggested that the same may be true in mammalian cells, since only one E3 of the N-end rule pathway, called E3α, was apparent in these extracts (23). However, the cloning of cDNA and genes encoding mouse UBR1 (E3α) indicated the existence of at least two UBR1 homologs in the mouse (and human) genome, termed UBR2 and UBR3 (35). The sequences of UBR2 and UBR3 cDNAs and genes (Y. T. Kwon, T. Tasaki, and A. Varshavsky, unpublished data) suggested that at least mouse UBR2 may functionally overlap with UBR1. To address this and related questions, we initiated genetic and biochemical dissection of the UBR protein family in the mouse. In the present study, the first in a projected series, we constructed and analyzed mouse strains lacking UBR1.