Altered Activity, Social Behavior, and Spatial Memory in Mice Lacking the NTAN1p Amidase and the Asparagine Branch of the N-End Rule Pathway

A multitude of regulatory circuits involve conditionally or constitutively short-lived proteins (26, 27, 44, 48, 49, 64). Features of proteins that confer metabolic instability are called degradation signals, or degrons (37, 63). The essential component of one degradation signal, termed the N-degron, is a destabilizing N-terminal residue of a protein (3). A set of N-degrons containing different N-terminal residues which are destabilizing in a given cell yields a rule, termed the N-end rule, which relates the in vivo half-life of a protein to the identity of its N-terminal residue. An N-end rule pathway is present in all organisms examined, from mammals and plants to fungi and prokaryotes (63). In eukaryotes, an N-degron comprises two determinants: a destabilizing N-terminal residue and an internal lysine of a substrate protein (4, 32, 60). The Lys residue is the site of formation of a substrate-linked multiubiquitin chain (15, 49). The N-end rule pathway is thus one pathway of the ubiquitin (Ub) system (25–27). Ub is a 76-residue eukaryotic protein that exists in cells either free or covalently conjugated to many other proteins. The Ub system plays a role in a vast range of processes, including cell growth, division, differentiation, and responses to stress. In most of these processes, Ub acts through routes that involve the degradation of Ub-protein conjugates by the 26S proteasome, an ATP-dependent multisubunit protease (10, 17, 20, 51). (Throughout the text, the names of mouse genes are in italics, with the first letter uppercase. The 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. The names of S. cerevisiae proteins are roman, with the first letter uppercase and an extra lowercase “p” at the end. The names of 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 [58], 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 has a hierarchic structure. 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 amidohydrolase (Nt-amidase), into the secondary destabilizing N-terminal residues Asp and Glu (6). Destabilizing activity of N-terminal Asp and Glu requires their conjugation, by the S. cerevisiae ATE1-encoded Arg-tRNA protein transferase (R-transferase) (8, 41), to Arg, one of the primary destabilizing residues (Fig. ​(Fig.1A).1A). In mammals, the deamidation step is mediated by two Nt-amidases, NtN-amidase and NtQ-amidase, which are specific, respectively, for N-terminal Asn and Gln (Fig. ​(Fig.1A)1A) (24, 59). The mammalian counterpart of the yeast R-transferase Ate1p exists as two distinct species, ATE1-1p and ATE1p-2, which are produced through alternative splicing of Ate1 pre-mRNA (34). In vertebrates, the set of secondary destabilizing residues contains not only Asp and Glu but also Cys, which is a stabilizing residue in yeast (Fig. ​(Fig.1A)1A) (18, 23). The primary destabilizing N-terminal residues are bound directly by the UBR1-encoded N-recognin, the targeting (E3) component of the N-end rule pathway. In S. cerevisiae, Ubr1p is a 225-kDa protein which recognizes 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 terminal residues Phe, Leu, Trp, Tyr, and Ile (35, 63). Ubr1p contains yet another substrate-binding site that targets proteins such as Cup9p and Gpa1p, which bear internal (non-N-terminal) degrons (12, 54). The Ubr1 genes encoding mouse and human N-recognins, also called E3α, have been cloned (36), and mouse strains lacking Ubr1 have recently been constructed (Y. T. Kwon and A. Varshavsky, unpublished data). FIG. 1 Deletion-disruption of the mouse Ntan1 gene. (A) Comparison of enzymatic reactions that underlie the activity of tertiary and secondary destabilizing residues in the yeast S. cerevisiae and the mouse. N-terminal residues are indicated by single-letter ... 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 (1, 12); a mechanistically undefined role in regulating the Sln1p-dependent phosphorylation cascade that mediates osmoregulation in S. cerevisiae (47); the degradation of alphaviral RNA polymerases and other viral proteins in infected metazoan cells (19, 38); and the degradation of Gpa1p, a Gα protein of S. cerevisiae (43, 54). Physiological N-end rule substrates were also identified among the proteins secreted into the mammalian cell's cytosol by intracellular parasites such as the bacterium Listeria monocytogenes. Short half-lives of these bacterial proteins are required for the efficient presentation of their peptides to the immune system (56). Inhibition of the N-end rule pathway was reported to interfere with mammalian cell differentiation (28) and to delay limb regeneration in amphibians (61). Studies of the Ub-dependent proteolysis of endogenous proteins in muscle extracts suggested that the N-end rule pathway plays a role in catabolic states that result in muscle atrophy (39, 57). A crush injury to the rat sciatic nerve was reported to result in a ∼10-fold increase in the rate of arginine conjugation to the N termini of proteins in the nerve's region upstream of the crush site, suggesting an injury-induced increase in the concentration of R-transferase substrates and/or an enhanced activity of the N-end rule pathway (65). Physiological substrates of either yeast or metazoan Nt-amidases and R-transferases are unknown. Engineered N-end rule substrates, including substrates of Nt-amidases and R-transferases, can be produced in vivo through the Ub fusion technique, in which a Ub-X reporter fusion is cleaved by deubiquitylating enzymes (DUBs) (66) after the last residue of Ub, yielding a reporter bearing the desired N-terminal residue X (3, 63). The mouse Asn-specific NtN-amidase is encoded by the 17-kb Ntan1 gene. The 1.6-kb Ntan1 mRNA specifies the 310-residue NtN-amidase (24). In the present work, we characterized the expression and intracellular localization of NtN-amidase. We also constructed mouse strains bearing a homozygous deletion-disruption of Ntan1 and showed that these mice lacked both NtN-amidase and the Asn-specific branch of the N-end rule pathway. The Ntan1−/− mice were fertile and outwardly normal but were found to differ from their congenic wild-type counterparts in spontaneous activity and spatial memory. Among these differences was a socially conditioned exploratory phenotype of Ntan1−/− mice that has not been previously described, to our knowledge, with other mouse strains.