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

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. In the yeast Saccharomyces cerevisiae, the UBR1-encoded ubiquitin ligase (E3) of the N-end rule pathway mediates the targeting of substrate proteins in part through binding to their destabilizing N-terminal residues. The functions of the yeast N-end rule pathway include fidelity of chromosome segregation and the regulation of peptide import. Our previous work described the cloning of cDNA and a gene encoding the 200-kDa mouse UBR1 (E3alpha). Here we show that mouse UBR1, in the presence of a cognate mouse ubiquitin-conjugating (E2) enzyme, can rescue the N-end rule pathway in ubr1Delta S. cerevisiae. We also constructed UBR1(-/-) mouse strains that lacked the UBR1 protein. UBR1(-/-) mice were viable and fertile but weighed significantly less than congenic +/+ mice. The decreased mass of UBR1(-/-) mice stemmed at least in part from smaller amounts of the skeletal muscle and adipose tissues. The skeletal muscle of UBR1(-/-) mice apparently lacked the N-end rule pathway and exhibited abnormal regulation of fatty acid synthase upon starvation. By contrast, and despite the absence of the UBR1 protein, UBR1(-/-) fibroblasts contained the N-end rule pathway. Thus, UBR1(-/-) mice are mosaics in regard to the activity of this pathway, owing to differential expression of proteins that can substitute for the ubiquitin ligase UBR1 (E3alpha). We consider these UBR1-like proteins and discuss the functions of the mammalian N-end rule pathway.     

A family of mammalian E3 ubiquitin ligases that contain the UBR box motif and recognize N-degrons

A subset of proteins targeted by the N-end rule pathway bear degradation signals called N-degrons, whose determinants include destabilizing N-terminal residues. Our previous work identified mouse UBR1 and UBR2 as E3 ubiquitin ligases that recognize N-degrons. Such E3s are called N-recognins. We report here that while double-mutant UBR1(-/-) UBR2(-/-) mice die as early embryos, the rescued UBR1(-/-) UBR2(-/-) fibroblasts still retain the N-end rule pathway, albeit of lower activity than that of wild-type fibroblasts. An affinity assay for proteins that bind to destabilizing N-terminal residues has identified, in addition to UBR1 and UBR2, a huge (570 kDa) mouse protein, termed UBR4, and also the 300-kDa UBR5, a previously characterized mammalian E3 known as EDD/hHYD. UBR1, UBR2, UBR4, and UBR5 shared a approximately 70-amino-acid zinc finger-like domain termed the UBR box. The mammalian genome encodes at least seven UBR box-containing proteins, which we propose to call UBR1 to UBR7. UBR1(-/-) UBR2(-/-) fibroblasts that have been made deficient in UBR4 as well (through RNA interference) were significantly impaired in the degradation of N-end rule substrates such as the Sindbis virus RNA polymerase nsP4 (bearing N-terminal Tyr) and the human immunodeficiency virus type 1 integrase (bearing N-terminal Phe). Our results establish the UBR box family as a unique class of E3 proteins that recognize N-degrons or structurally related determinants for ubiquitin-dependent proteolysis and perhaps other processes as well.     

Universality and structure of the N-end rule

Our previous work has shown that, in the yeast Saccharomyces cerevisiae, any of the eight stabilizing amino-terminal residues confers a long (greater than 20 h) half-life on a test protein beta-galactosidase (beta gal), whereas 12 destabilizing amino-terminal residues confer on beta gal half-lives from less than 3 min to 30 min. We now show that an analogous single-residue code (the N-end rule) operates in an in vitro system derived from mammalian reticulocytes. We also show that the N-end rule has a hierarchical structure. Specifically, amino-terminal Glu and Asp (and also Cys in reticulocytes) are secondary destabilizing residues in that they are destabilizing through their ability to be conjugated to primary destabilizing residues such as Arg. Amino-terminal Gln and Asn are tertiary destabilizing residues in that they are destabilizing through their ability to be converted, via selective deamidation, into secondary destabilizing residues Glu and Asp. Furthermore, in reticulocytes, distinct types of the N-end-recognizing activity are shown to be specific for three classes of primary destabilizing residues: basic (Arg, Lys, His), bulky hydrophobic (Phe, Leu, Trp, Tyr), and small uncharged (Ala, Ser, Thr). Features of the N-end rule in reticulocytes suggest that the exact form of the N-end rule may depend on the cell's physiological state, thereby providing a mechanism for selective destruction of preexisting proteins upon cell differentiation.     

Female lethality and apoptosis of spermatocytes in mice lacking the UBR2 ubiquitin ligase of the N-end rule pathway

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 E3alpha, 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.     

ClpS is the recognition component for Escherichia coli substrates of the N-end rule degradation pathway

The N-end rule degradation pathway states that the half-life of a protein is determined by the nature of its N-terminal residue. In Escherichia coli the adaptor protein ClpS directly interacts with destabilizing N-terminal residues and transfers them to the ClpA/ClpP proteolytic complex for degradation. The crucial role of ClpS in N-end rule degradation is currently under debate, since ClpA/ClpP was shown to process selected N-terminal degrons harbouring destabilizing residues in the absence of ClpS. Here, we investigated the contribution of ClpS to N-end rule degradation by two approaches. First, we performed a systematic mutagenesis of selected N-degron model substrates, demonstrating that ClpS but not ClpA specifically senses the nature of N-terminal residues. Second, we identified two natural N-end rule substrates of E. coli : Dps and PATase (YgjG). The in vivo degradation of both proteins strictly relied on ClpS, thereby establishing the function of ClpS as the essential discriminator of the E. coli N-end rule pathway.     

Altered activity, social behavior, and spatial memory in mice lacking the NTAN1p amidase and the asparagine branch of the N-end rule pathway

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. N-terminal asparagine and glutamine are tertiary destabilizing residues, in that they are enzymatically deamidated to yield secondary destabilizing residues aspartate and glutamate, which are conjugated to arginine, a primary destabilizing residue. N-terminal arginine of a substrate protein is bound by the Ubr1-encoded E3alpha, the E3 component of the ubiquitin-proteasome-dependent N-end rule pathway. We describe the construction and analysis of mouse strains lacking the asparagine-specific N-terminal amidase (Nt(N)-amidase), encoded by the Ntan1 gene. In wild-type embryos, Ntan1 was strongly expressed in the branchial arches and in the tail and limb buds. The Ntan1(-/-) mouse strains lacked the Nt(N)-amidase activity but retained glutamine-specific Nt(Q)-amidase, indicating that the two enzymes are encoded by different genes. Among the normally short-lived N-end rule substrates, only those bearing N-terminal asparagine became long-lived in Ntan1(-/-) fibroblasts. The Ntan1(-/-) mice were fertile and outwardly normal but differed from their congenic wild-type counterparts in spontaneous activity, spatial memory, and a socially conditioned exploratory phenotype that has not been previously described with other mouse strains.     

The N-end rule pathway controls the import of peptides through degradation of a transcriptional repressor.

Ubiquitin-dependent proteolytic systems underlie many processes, including the cell cycle, cell differentiation and responses to stress. One such system is the N-end rule pathway, which targets proteins bearing destabilizing N-terminal residues. Here we report that Ubr1p, the main recognition component of this pathway, regulates peptide import in the yeast Saccharomyces cerevisiae through degradation of Cup9p, a 35 kDa homeodomain protein. Cup9p was identified using a screen for mutants that bypass the previously observed requirement for Ubr1p in peptide import. We show that Cup9p is a short-lived protein (t1/2 approximately 5 min) whose degradation requires Ubr1p. Cup9p acts as a repressor of PTR2, a gene encoding the transmembrane peptide transporter. In contrast to engineered N-end rule substrates, which are recognized by Ubr1p through their destabilizing N-terminal residues, Cup9p is targeted by Ubr1p through an internal degradation signal. The Ubr1p-Cup9p-Ptr2p circuit is the first example of a physiological process controlled by the N-end rule pathway. An earlier study identified Cup9p as a protein required for an aspect of resistance to copper toxicity in S.cerevisiae. Thus, one physiological substrate of the N-end rule pathway functions as both a repressor of peptide import and a regulator of copper homeostasis.     

Ubiquitin ligases of the N-end rule pathway: assessment of mutations in UBR1 that cause the Johanson-Blizzard syndrome

Background

Johanson-Blizzard syndrome (JBS; OMIM 243800) is an autosomal recessive disorder that includes congenital exocrine pancreatic insufficiency, facial dysmorphism with the characteristic nasal wing hypoplasia, multiple malformations, and frequent mental retardation. Our previous work has shown that JBS is caused by mutations in human UBR1, which encodes one of the E3 ubiquitin ligases of the N-end rule pathway. The N-end rule relates the regulation of the in vivo half-life of a protein to the identity of its N-terminal residue. One class of degradation signals (degrons) recognized by UBR1 are destabilizing N-terminal residues of protein substrates.

Methodology/Principal Findings

Most JBS-causing alterations of UBR1 are nonsense, frameshift or splice-site mutations that abolish UBR1 activity. We report here missense mutations of human UBR1 in patients with milder variants of JBS. These single-residue changes, including a previously reported missense mutation, involve positions in the RING-H2 and UBR domains of UBR1 that are conserved among eukaryotes. Taking advantage of this conservation, we constructed alleles of the yeast Saccharomyces cerevisiae UBR1 that were counterparts of missense JBS-UBR1 alleles. Among these yeast Ubr1 mutants, one of them (H160R) was inactive in yeast-based activity assays, the other one (Q1224E) had a detectable but weak activity, and the third one (V146L) exhibited a decreased but significant activity, in agreement with manifestations of JBS in the corresponding JBS patients.

Conclusions/Significance

These results, made possible by modeling defects of a human ubiquitin ligase in its yeast counterpart, verified and confirmed the relevance of specific missense UBR1 alleles to JBS, and suggested that a residual activity of a missense allele is causally associated with milder variants of JBS.     

The N-end rule pathway: from recognition by N-recognins, to destruction by AAA+proteases

Intracellular proteolysis is a tightly regulated process responsible for the targeted removal of unwanted or damaged proteins. The non-lysosomal removal of these proteins is performed by processive enzymes, which belong to the AAA+superfamily, such as the 26S proteasome and Clp proteases. One important protein degradation pathway, that is common to both prokaryotes and eukaryotes, is the N-end rule. In this pathway, proteins bearing a destabilizing amino acid residue at their N-terminus are degraded either by the ClpAP protease in bacteria, such as Escherichia coli or by the ubiquitin proteasome system in the eukaryotic cytoplasm. A suite of enzymes and other molecular components are also required for the successful generation, recognition and delivery of N-end rule substrates to their cognate proteases. In this review we examine the similarities and differences in the N-end rule pathway of bacterial and eukaryotic systems, focusing on the molecular determinants of this pathway.     

Bivalent inhibitor of the N-end rule pathway.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Ubr1p, the recognition (E3) component of the Saccharomyces cerevisiae N-end rule pathway, contains at least two substrate-binding sites. The type 1 site is specific for N-terminal basic residues Arg, Lys, and His. The type 2 site is specific for N-terminal bulky hydrophobic residues Phe, Leu, Trp, Tyr, and Ile. Previous work has shown that dipeptides bearing either type 1 or type 2 N-terminal residues act as weak but specific inhibitors of the N-end rule pathway. We took advantage of the two-site architecture of Ubr1p to explore the feasibility of bivalent N-end rule inhibitors, whose expected higher efficacy would result from higher affinity of the cooperative (bivalent) binding to Ubr1p. The inhibitor comprised mixed tetramers of beta-galactosidase that bore both N-terminal Arg (type 1 residue) and N-terminal Leu (type 2 residue) but that were resistant to proteolysis in vivo. Expression of these constructs in S. cerevisiae inhibited the N-end rule pathway much more strongly than the expression of otherwise identical beta-galactosidase tetramers whose N-terminal residues were exclusively Arg or exclusively Leu. In addition to demonstrating spatial proximity between the type 1 and type 2 substrate-binding sites of Ubr1p, these results provide a route to high affinity inhibitors of the N-end rule pathway.     

PINK1 is degraded through the N-end rule pathway

PINK1, a mitochondrial serine/threonine kinase, is the product of a gene mutated in an autosomal recessive form of Parkinson disease. PINK1 is constitutively degraded by an unknown mechanism and stabilized selectively on damaged mitochondria where it can recruit the E3 ligase PARK2/PARKIN to induce mitophagy. Here, we show that, under steady-state conditions, endogenous PINK1 is constitutively and rapidly degraded by E3 ubiquitin ligases UBR1, UBR2 and UBR4 through the N-end rule pathway. Following precursor import into mitochondria, PINK1 is cleaved in the transmembrane segment by a mitochondrial intramembrane protease PARL generating an N-terminal destabilizing amino acid and then retrotranslocates from mitochondria to the cytosol for N-end recognition and proteasomal degradation. Thus, sequential actions of mitochondrial import, PARL-processing, retrotranslocation and recognition by N-end rule E3 enzymes for the ubiquitin proteosomal degradation defines the rapid PINK1 turnover. PINK1 steady-state elimination by the N-end rule identifies a novel organelle to cytoplasm turnover pathway that yields a mechanism to flag damaged mitochondria for autophagic elimination.     

PRT6/At5g02310 encodes an Arabidopsis ubiquitin ligase of the N-end rule pathway with arginine specificity and is not the CER3 locus

The eukaryotic N-end rule pathway mediates ubiquitin- and proteasome-dependent turnover of proteins with a bulky amino-terminal residue. Arabidopsis locus At5g02310 shows significant similarity to the yeast N-end rule ligase Ubr1. We demonstrate that At5g02310 is a ubiquitin ligase and mediates degradation of proteins with amino-terminal Arg residue. Unlike Ubr1, the Arabidopsis protein does not participate in degradation of proteins with amino-terminal Phe or Leu. This modified target specificity coincides with characteristic differences in domain structure. In contrast to previous publications, our data indicate that At5g02310 is not identical to CER3, a gene involved in establishment of a protective surface wax layer. At5g02310 has therefore been re-designated PROTEOLYSIS 6 (PRT6), in accordance with its ubiquitin ligase function.     

UBR1 promotes protein kinase quality control and sensitizes cells to Hsp90 inhibition

UBR1 and UBR2 are N-recognin ubiquitin ligases that function in the N-end rule degradation pathway. In yeast, the UBR1 homologue also functions by N-end rule independent means to promote degradation of misfolded proteins generated by treatment of cells with geldanamycin, a small molecule inhibitor of Hsp90. Based on these studies we examined the role of mammalian UBR1 and UBR2 in the degradation of protein kinase clients upon Hsp90 inhibition. Our findings show that protein kinase clients Akt and Cdk4 are still degraded in mouse Ubr1(-)/(-) cells treated with geldanamycin, but that their levels recover much more rapidly than is found in wild type cells. These findings correlate with increased induction of Hsp90 expression in the Ubr1(-)/(-) cells compared with wild type cells. We also observed a reduction of UBR1 protein levels in geldanamycin-treated mouse embryonic fibroblasts and human breast cancer cells, suggesting that UBR1 is an Hsp90 client. Further studies revealed a functional overlap between UBR1 and the quality control ubiquitin ligase, CHIP. Our findings show that UBR1 function is conserved in controlling the levels of Hsp90-dependent protein kinases upon geldanamycin treatment, and suggest that it plays a role in determining the sensitivity of cancer cells to the chemotherapeutic effects of Hsp90 inhibitors.     

The Substrate Recognition Domains of the N-end Rule Pathway

The N-end rule pathway is a ubiquitin-dependent system where E3 ligases called N-recognins, including UBR1 and UBR2, recognize type-1 (basic) and type-2 (bulky hydrophobic) N-terminal residues as part of N-degrons. We have recently reported an E3 family (termed UBR1 through UBR7) characterized by the 70-residue UBR box, among which UBR1, UBR2, UBR4, and UBR5 were captured during affinity-based proteomics with synthetic degrons. Here we characterized substrate binding specificity and recognition domains of UBR proteins. Pull-down assays with recombinant UBR proteins suggest that 570-kDa UBR4 and 300-kDa UBR5 bind N-degron, whereas UBR3, UBR6, and UBR7 do not. Binding assays with 24 UBR1 deletion mutants and 31 site-directed UBR1 mutations narrow down the degron-binding activity to a 72-residue UBR box-only fragment that recognizes type-1 but not type-2 residues. A surface plasmon resonance assay shows that the UBR box binds to the type-1 substrate Arg-peptide with K_d of ∼3.4 μm. Downstream from the UBR box, we identify a second substrate recognition domain, termed the N-domain, required for type-2 substrate recognition. The ∼80-residue N-domain shows structural and functional similarity to 106-residue Escherichia coli ClpS, a bacterial N-recognin. We propose a model where the 70-residue UBR box functions as a common structural element essential for binding to all known destabilizing N-terminal residues, whereas specific residues localized in the UBR box (for type 1) or the N-domain (for type 2) provide substrate selectivity through interaction with the side group of an N-terminal amino acid. Our work provides new insights into substrate recognition in the N-end rule pathway.The N-end rule pathway is a ubiquitin (Ub)²-dependent proteolytic system in which N-terminal residues of short-lived proteins function as an essential component of degradation signals (degrons) called N-degrons (Fig. 1A) (1-15). An N-degron can be created from a pre-N-degron through specific N-terminal modifications (12). Specifically, in mammals, N-terminal Asn and Gln are tertiary destabilizing residues that function through their deamidation by N-terminal amidohydrolases into the secondary destabilizing N-terminal residues Asp and Glu, respectively (6, 16) (Fig. 1A). N-terminal Asp and Glu are secondary destabilizing residues that function through their arginylation by ATE1 R-transferase, which creates the primary destabilizing residue Arg at the N terminus (4, 8) (Fig. 1A). N-terminal Cys can also function as a tertiary destabilizing residue through its oxidation in a manner depending on nitric oxide and oxygen (O₂); the oxidized Cys residue is subsequently arginylated by ATE1 (8, 13, 17).Open in a separate windowFIGURE 1.A, the mammalian N-end rule pathway. N-terminal residues are indicated by single-letter abbreviations for amino acids. Yellow ovals denote the rest of a protein substrate. C^* denotes oxidized N-terminal Cys, either Cys-sulfinic acid [CysO₂(H)] or Cys-sulfonic acid [CysO₃(H)]. The Cys oxidation requires nitric oxide and oxygen (O₂) or its derivatives. The oxidized Cys is arginylated by ATE1 Arg-tRNA-protein transferase (R-transferase). N-recognins also recognize internal (non-N-terminal) degrons in other substrates of the N-end rule pathway. B, the X-peptide pull-down assay. Left, a 12-mer peptide bearing N-terminal Arg (type 1), Phe (type 2), Trp (type 2), or Gly (stabilizing control) residue was cross-linked through its C-terminal Cys residue to Ultralink Iodoacetyl beads. Right, the otherwise identical 12-mer peptide, bearing C-terminal biotinylated Lys instead of Cys, was conjugated, via biotin, to the streptavidin-Sepharose beads. C, the X-peptide pull-down assay of endogenous UBR proteins using testes extracts. Extracts from mouse testes were mixed with bead-conjugated X-peptides bearing N-terminal Phe (F), Gly (G), or Arg (R). After centrifugation, captured proteins were separated and subjected to anti-UBR immunoblotting. Mo, a pull-down reaction with mock beads. D, the X-peptide pull-down assays using rat testis extracts were performed in the presence of varying concentrations of NaCl. After incubation and washing, bound proteins were eluted by 10 mm Tyr-Ala for Phe-peptide, 10 mm Arg-Ala for Arg-peptide, and 5 mm Tyr-Ala and 5 mm Arg-Ala for Val-peptide. Eluted proteins were subjected to immunoblotting for UBR1 and UBR5. E, cytoplasmic fractions of wild-type (+/+), Ubr1^-/-, Ubr2^-/-, Ubr1^-/-Ubr2^-/-, and Ubr1^-/-Ubr2^-/-Ubr4^RNAi MEFs were subjected to X-peptide pull-down assay. Precipitated proteins were separated and analyzed by immunoblotting for UBR1 and UBR4.N-terminal Arg together with other primary destabilizing N-terminal residues are directly bound by specific E3 Ub ligases called N-recognins (3, 7, 9). Destabilizing N-terminal residues can be created through the removal of N-terminal Met or the endoproteolytic cleavage of a protein, which exposes a new amino acid at the N terminus (12, 13). N-terminal degradation signals can be divided into type-1 (basic; Arg, Lys, and His) and type-2 (bulky hydrophobic; Phe, Leu, Trp, Tyr, and Ile) destabilizing residues (2, 12). In addition to a destabilizing N-terminal residue, a functional N-degron requires at least one internal Lys residue (the site of a poly-Ub chain formation) and a conformational feature required for optimal ubiquitylation (1, 2, 18). UBR1 and UBR2 are functionally overlapping N-recognins (3, 7, 9). Our proteomic approach using synthetic peptides bearing destabilizing N-terminal residues captured a set of proteins (200-kDa UBR1, 200-kDa UBR2, 570-kDa UBR4, and 300-kDa UBR5/EDD) characterized by a 70-residue zinc finger-like domain termed the UBR box (10-12). UBR5 is a HECT E3 ligase known as EDD (E3 identified by differential display) (19) and a homolog of Drosophila hyperplastic discs (20). The mammalian genome encodes at least seven UBR box-containing proteins, termed UBR1 through UBR7 (10). UBR box proteins are generally heterogeneous in size and sequence but contain, with the exception of UBR4, specific signatures unique to E3s or a substrate recognition subunit of the E3 complex: the RING domain in UBR1, UBR2, and UBR3; the HECT domain in UBR5; the F-box in UBR6 and the plant homeodomain domain in UBR7 (Fig. 2B). The biochemical properties of more recently identified UBR box proteins, such as UBR3 through UBR7, are largely unknown.Open in a separate windowFIGURE 2.The binding properties of the UBR box family members to type-1 and type-2 destabilizing N-terminal residues. A, the X-peptide pull-down assay with overexpressed, full-length UBR proteins: UBR2, UBR3 (in S. cerevisiae cells), UBR4, UBR5 (in COS7 cells), and UBR6 and UBR7 (in the wheat germ lysates). Precipitates were analyzed by immunoblotting (for UBR2, UBR3, UBR4, and UBR5) with tag-specific antibodies as indicated in B or autoradiography (for UBR6 and UBR7). B, the structures of UBR box proteins. Shown are locations of the UBR box, the N-domain, and other E3-related domains. UBR, UBR box; RING, RING finger; UAIN, UBR-specific autoinhibitory domain; CRD, cysteine-rich domain; PHD, plant homeodomain; HECT, HECT domain.Studies using knock-out mice implicated the N-end rule pathway in cardiac development and signaling, angiogenesis (8, 15), meiosis (9), DNA repair (21), neurogenesis (15), pancreatic functions (22), learning and memory (23, 24), female development (9), muscle atrophy (25), and olfaction (11). Mutations in human UBR1 is a cause of Johanson-Blizzard syndrome (22), an autosomal recessive disorder with multiple developmental abnormalities (26). Other functions of the pathway include: (i) a nitric oxide and oxygen (O₂) sensor controlling the proteolysis of RGS4, RGS5, and RGS16 (8, 13, 17), (ii) a heme sensor through hemin-dependent inhibition of ATE1 function (27), (iii) the regulation of short peptide import through the peptide-modulated degradation of the repressor of the import (28, 29), (iv) the control of chromosome segregation through the degradation of a separate produced cohesin fragment (30), (v) the regulation of apoptosis through the degradation of a caspase-processed inhibitor of apoptosis (31, 32), (vi) the control of the human immunodeficiency virus replication cycle through the degradation of human immunodeficiency virus integrase (10, 33), and (vii) the regulation of leaf senescence in plants (34).In the present study we characterized substrate binding specificities and recognition domains of UBR proteins. In our binding assays, UBR1, UBR2, UBR4, and UBR5 were captured by N-terminal degradation determinants, whereas UBR3, UBR6, and UBR7 were not. We also report that in contrast to other E3 systems that usually recognize substrates through protein-protein interface, UBR1 and UBR2 have a general substrate recognition domain termed the UBR box. Remarkably, a 72-residue UBR box-only fragment fully retains its structural integrity and thereby the ability to recognize type-1 N-end rule substrates. We also report that the N-domain, structurally and functionally related with bacterial N-recognins, is required for recognizing type-2 N-end rule substrates. We discuss the evolutionary relationship between eukaryotic and prokaryotic N-recognins.     

Behavioral characterization of mice lacking the ubiquitin ligase UBR1 of the N-end rule pathway

The N-end rule pathway, a subset of the ubiquitin pathway, relates the in vivo half-life of a protein to the identity of its N-terminal residue. Mice lacking NTAN1, a component of the N-end rule pathway, showed altered learning and memory, and socially conditioned behavioral alteration ( Balogh, Kwon, & Denenberg 1999, 2000 ; Kwon, Balogh et al . 2000 ; Balogh et al . 2001 ). Mice lacking UBR1 (E3α), one of at least three recognition components of the N-end rule pathway, are viable and outwardly normal. Here we describe behavioral characterizations of UBR1 knockout (UBR1^–/–) mice. Compared to congenic littermates, UBR1^–/– mice exhibited less spontaneous activity in an open field and took longer to locate the hidden platform during eight-week Morris water maze retention. In contrast, they performed better in horizontal–vertical discrimination and Lashley III maze testing. No statistically significant differences in inhibitory learning were observed. With the exception of enhanced Lashley III maze performance, these data parallel findings with NTAN1^–/– mice lacking the upstream component of UBR1. These results suggest that, like NTAN1, UBR1 is involved in learning and memory.     

Cloning and functional analysis of the arginyl-tRNA-protein transferase gene ATE1 of Saccharomyces cerevisiae

Aminoacyl-tRNA-protein transferases (Arg-transferases) catalyze post-translational conjugation of specific amino acids to the amino termini of acceptor proteins. A function of these enzymes in eukaryotes has been shown to involve the conjugation of destabilizing amino acids to the amino termini of short-lived proteins, these reactions being a part of the N-end rule pathway of protein degradation (Gonda, D. K., Bachmair, A., Wünning, I., Tobias, J. W., Lane, W. S., and Varshavsky, A. (1989) J. Biol. Chem. 264, 16700-16712). We have cloned the ATE1 gene of the yeast Saccharomyces cerevisiae which encodes arginyl-tRNA-protein transferase. ATE1 gives rise to a approximately 1.6-kilobase mRNA and codes for a 503-residue protein. Expression of the yeast ATE1 gene in Escherichia coli, which lacks Arg-transferases, was used to show that the ATE1 protein possesses the Arg-transferase activity. Null ate1 mutants are viable but lack the Arg-transferase activity and are unable to degrade those substrates of the N-end rule pathway that start with residues recognized by the Arg-transferase.     

Biochemical and genetic studies of UBR3, a ubiquitin ligase with a function in olfactory and other sensory systems

Our previous work identified E3 ubiquitin ligases, termed UBR1-UBR7, that contain the approximately 70-residue UBR box, a motif important for the targeting of N-end rule substrates. In this pathway, specific N-terminal residues of substrates are recognized as degradation signals by UBR box-containing E3s that include UBR1, UBR2, UBR4, and UBR5. The other E3s of this set, UBR3, UBR6, and UBR7, remained uncharacterized. Here we describe the cloning and analyses of mouse UBR3. The similarities of UBR3 to the UBR1 and UBR2 E3s of the N-end rule pathway include the RING and UBR domains. We show that HR6A and HR6B, the E2 enzymes that bind to UBR1 and UBR2, also interact with UBR3. However, in contrast to UBR1 and UBR2, UBR3 does not recognize N-end rule substrates. We also constructed UBR3-lacking mouse strains. In the 129SvImJ background, UBR3-/- mice died during embryogenesis, whereas the C57BL/6 background UBR3-/- mice exhibited neonatal lethality and suckling impairment that could be partially rescued by litter size reduction. The adult UBR3-/- mice had female-specific behavioral anosmia. Cells of the olfactory pathway were found to express beta-galactosidase (LacZ) that marked the deletion/disruption UBR3- allele. The UBR3-specific LacZ expression was also prominent in cells of the touch, vision, hearing, and taste systems, suggesting a regulatory role of UBR3 in sensory pathways, including olfaction. By analogy with functions of the UBR domain in the N-end rule pathway, we propose that the UBR box of UBR3 may recognize small compounds that modulate the targeting, by this E3, of its currently unknown substrates.