N-end Rule Pathway Research Articles

The N-End Rule Pathway inXenopusEgg Extracts

Ubiquitin-dependent degradation of intracellular proteins underlies a multitude of biological processes, including the cell cycle, cell differentiation, and responses to stress. One ubiquitin-dependent proteolytic system is the N-end rule pathway, whose targets include proteins that bear destabilizing N-terminal residues. This pathway, which has been characterized only in somatic cells, is shown here to be present also in germ line cells such as the eggs of the amphibianXenopus laevis.We demonstrate that the set of destabilizing residues in the N-end rule pathway ofXenopuseggs is similar, if not identical, to that of somatic cells such as mammalian reticulocytes and fibroblasts. It is also shown that the degradation of engineered N-end rule substrates in egg extracts can be strongly and selectively inhibited by dipeptides bearing destabilizing N-terminal residues. This result allowed us to ask whether selective inhibition of the N-end rule pathway in egg extracts influences the apoptosis-like changes that are observed in these extracts. A dipeptide bearing a bulky hydrophobic (type 2) destabilizing N-terminal residue was found to delay the apoptotic changes in egg extracts, whereas dipeptides bearing basic (type 1) destabilizing N-terminal residues had no effect. High activity of the N-end rule pathway in egg extracts provides an alternative to reticulocyte extracts for thein vitroanalyses of this pathway.

The N-end Rule Pathway Catalyzes a Major Fraction of the Protein Degradation in Skeletal Muscle

In skeletal muscle, overall protein degradation involves the ubiquitin-proteasome system. One property of a protein that leads to rapid ubiquitin-dependent degradation is the presence of a basic, acidic, or bulky hydrophobic residue at its N terminus. However, in normal cells, substrates for this N-end rule pathway, which involves ubiquitin carrier protein (E2) E214k and ubiquitin-protein ligase (E3) E3alpha, have remained unclear. Surprisingly, in soluble extracts of rabbit muscle, we found that competitive inhibitors of E3alpha markedly inhibited the 125I-ubiquitin conjugation and ATP-dependent degradation of endogenous proteins. These inhibitors appear to selectively inhibit E3alpha, since they blocked degradation of 125I-lysozyme, a model N-end rule substrate, but did not affect the degradation of proteins whose ubiquitination involved other E3s. The addition of several E2s or E3alpha to the muscle extracts stimulated overall proteolysis and ubiquitination, but only the stimulation by E3alpha or E214k was sensitive to these inhibitors. A similar general inhibition of ubiquitin conjugation to endogenous proteins was observed with a dominant negative inhibitor of E214k. Certain substrates of the N-end rule pathway are degraded after their tRNA-dependent arginylation. We found that adding RNase A to muscle extracts reduced the ATP-dependent proteolysis of endogenous proteins, and supplying tRNA partially restored this process. Finally, although in muscle extracts the N-end rule pathway catalyzes most ubiquitin conjugation, it makes only a minor contribution to overall protein ubiquitination in HeLa cell extracts.

The mouse and human genes encoding the recognition component 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. The N-end rule pathway is one proteolytic pathway of the ubiquitin system. The recognition component of this pathway, called N-recognin or E3, binds to a destabilizing N-terminal residue of a substrate protein and participates in the formation of a substrate-linked multiubiquitin chain. We report the cloning of the mouse and human Ubr1 cDNAs and genes that encode a mammalian N-recognin called E3alpha. Mouse UBR1p (E3alpha) is a 1,757-residue (200-kDa) protein that contains regions of sequence similarity to the 225-kDa Ubr1p of the yeast Saccharomyces cerevisiae. Mouse and human UBR1p have apparent homologs in other eukaryotes as well, thus defining a distinct family of proteins, the UBR family. The residues essential for substrate recognition by the yeast Ubr1p are conserved in the mouse UBR1p. The regions of similarity among the UBR family members include a putative zinc finger and RING-H2 finger, another zinc-binding domain. Ubr1 is located in the middle of mouse chromosome 2 and in the syntenic 15q15-q21.1 region of human chromosome 15. Mouse Ubr1 spans approximately 120 kilobases of genomic DNA and contains approximately 50 exons. Ubr1 is ubiquitously expressed in adults, with skeletal muscle and heart being the sites of highest expression. In mouse embryos, the Ubr1 expression is highest in the branchial arches and in the tail and limb buds. The cloning of Ubr1 makes possible the construction of Ubr1-lacking mouse strains, a prerequisite for the functional understanding of the mammalian N-end rule pathway.

PRT1 of Arabidopsis thaliana encodes a component of the plant N-end rule pathway.

Mutants in the PRT1 gene of Arabidopsis thaliana are impaired in the degradation of a normally short-lived intracellular protein that contains a destabilizing N-terminal residue. Proteins bearing such residues are the substrates of an ubiquitin-dependent proteolytic system called the N-end rule pathway. The chromosomal position of PRT1 was determined, and the PRT1 gene was isolated by map-based cloning. The 45-kDa PRT1 protein contains two RING finger domains and one ZZ domain. No other proteins in databases match these characteristics of PRT1. There is, however, a weak similarity to Rad18p of Saccharomyces cerevisiae. The RING finger domains have been found in a number of other proteins that are involved in ubiquitin conjugation, consistent with the proposed role of PRT1 in the plant N-end rule pathway.

Sequence elements that contribute to the degradation of yeast G alpha.

Gpa1 is the alpha subunit of the yeast G-protein that regulates signal transduction during mating. The stability of Galpha/Gpa1 is influenced by the ubiquitin-dependent N-end rule pathway, suggesting that the regulation of G alpha levels may be important for effective mating response and recovery. The G alpha sequences that confer sensitivity to degradation by the N-end rule pathway were identified. The insertion of this degradation signal (G1-Deg) into the ordinarily stable Gpa2 protein conferred proteolytic targeting. We examined G alpha degradation under different conditions and found that it was efficiently degraded in haploid and diploid cells, but was stable if it was synthesized prior to expression of the N-end rule pathway. Interestingly, a specific mutation in G alpha that is believed to promote the GTP-bound form (N388K) caused accelerated degradation. A region encompassing a putative effector-binding domain (G1-Deg) is required for G alpha degradation via the N-end rule pathway. Our studies have shown that G alpha is susceptible to proteolysis soon after synthesis. These results are in agreement with the idea that G alpha is more unstable in the GTP-bound form, which is the predominant state of monomeric/free G alpha soon after synthesis. It is likely that the signal transduced by Gbetagamma can be regulated by adjusting the levels of G alpha through proteolysis.

Requirement for an aromatic amino acid or histidine at the N terminus of Sindbis virus RNA polymerase.

The N terminal amino acid of nonstructural protein nsP4, the viral RNA polymerase, is a tyrosine in all sequenced alphaviruses; this is a destabilizing amino acid for the N-end rule pathway and results in rapid degradation of nsP4 produced in infected cells or in reticulocyte lysates. We have constructed 11 mutants of Sindbis virus bearing Phe, Ala, Thr, Cys, Leu, Met, Asn, Gln, Glu, Arg, or Pro at the N terminus of nsP4. Translation of RNAs in reticulocyte lysates showed that cleavage at the nsP3/nsP4 site occurred efficiently for all mutants except for Glu-nsP4, which was cleaved inefficiently, and Pro-nsP4, which was not detectably cleaved, and that Tyr, Cys, Leu, Arg, and Phe destabilized nsP4 but Ala, Met, Thr, Asn, Gln, and Glu stabilized nsP4 to various extents. The viability of the mutants was examined by transfection of chicken cells at 30 or 40 degrees C. The Phe-nsP4 mutant formed large plaques at both temperatures. The Met-nsP4 mutant was also viable but formed small plaques at 30 degrees C and minute plaques at 40 degrees C. The remaining mutants did not form plaques at either temperature. However, after prolonged incubation at 30 degrees C, all the mutants except Glu-nsP4 and Pro-nsP4 produced viable viruses. In the case of Cys-, Leu-, Asn-, Gln-, or Arg-nsP4, revertants that were indistinguishable in plaque phenotype from the wild-type virus arose by same-site reversion to Tyr, Trp, Phe, or His by a single nucleotide substitution in the original mutant codon. Viable viruses also arose from the Ala-, Leu-, Cys-, Thr-, Asn-, Gln-, and Arg-nsP4 mutants that retained the original mutations at the N terminus of nsP4, but these viruses formed smaller plaques than the wild-type virus and many were temperature sensitive. Our results indicate that only nsP4s bearing N-terminal Tyr, Phe, Trp, or His have wild-type or near-wild-type activity for RNA replication and that rapid degradation of nsP4 is not a prerequisite for its function. nsP4s bearing other N-terminal residues, with the exception of Met-nsP4, have only very low or negligible activity, so that no detectable infectious virus can be produced. However, suppressor mutations can arise that enable most such nsP4s to regain significant but still suboptimal activity.

Modulation of the intracellular stability and toxicity of diphtheria toxin through degradation by the N-end rule pathway

The enzymatically active A-fragment of diphtheria toxin enters the cytosol of sensitive cells where it inhibits protein synthesis by inactivating elongation factor 2 (EF-2). We have constructed a number of diphtheria toxin mutants that are degraded by the N-end rule pathway in Vero cells, and that display a wide range of intracellular stabilities. The degradation could be inhibited by the proteasome inhibitor lactacystin, indicating that the proteasome is responsible for N-end rule-mediated degradation in mammalian cells. Previously, the N-end rule has been investigated by studying the co-translational degradation of intracellularly expressed beta-galactosidase. Our work shows that a mature protein entering the cytosol from the exterior can also be degraded by the N-end rule pathway with a similar, but not identical specificity to that previously found. We found a correlation between the intracellular stability of the mutants and their toxic effect on cells, thus demonstrating a novel manner of modulating the toxicity of a protein toxin. The data also indicate that the inactivation of EF-2 is the rate-limiting step in the intoxication process.

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.

NF-κB p105 Processing via the Ubiquitin-Proteasome Pathway

The p50 subunit of NF-kappa B is generated by proteolytic processing of a 105-kDa precursor (p105) in yeast and mammalian cells. Here we show that yeast mutants in the ubiquitin-proteasome pathway inhibit or abolish p105 processing. Specifically, p105 processing is inhibited by a mutation in a 20 S proteasome subunit (pre1-1), by mutations in the ATPases located in the 19 S regulatory complexes of the proteasome (yta1, yta2/sug1, yta5, cim5), and by a mutation in a proteasome-associated isopeptidase (doa4). A ubiquitinated intermediate of the p105 processing reaction accumulates in some of these mutants, strongly suggesting that ubiquitination is required for processing. However, none of the ubiquitin conjugating enzyme mutants tested (ubc1, -2, -3, -4/5, -6/7, -8, -9, -10, -11) had an effect on p105 processing, suggesting that more than one of these enzymes is sufficient for p105 processing. Interestingly, a mutant "N-end rule" ligase does not adversely affect p105 processing, showing that the N-end rule pathway is not involved in degrading the C-terminal region of p105. Unexpectedly, we found that a glycine-rich region of p105 that is required for p105 processing in mammalian cells is not required for processing in yeast. Thus, p105 processing in both yeast and mammalian cells requires the ubiquitin-proteasome pathway, but the mechanisms of processing, while similar, are not identical.

The N-end rule pathway of protein degradation.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Similar but distinct versions of the N-end rule operate in all organisms examined, from mammals to fungi and bacteria. In eukaryotes, the N-end rule pathway is a part of the ubiquitin system. Ubiquitin is a 76-residue protein whose covalent conjugation to other proteins plays a role in many biological processes, including cell growth and differentiation. I discuss the current understanding of the N-end rule pathway.

The N-end rule: functions, mysteries, uses.

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Similar but distinct versions of the N-end rule operate in all organisms examined, from mammals to fungi and bacteria. In eukaryotes, the N-end rule pathway is a part of the ubiquitin system. I discuss the mechanisms and functions of this pathway, and consider its applications.

Cdc48p interacts with Ufd3p, a WD repeat protein required for ubiquitin-mediated proteolysis in Saccharomyces cerevisiae.

A library of random 10 residue peptides fused to the N-terminus of a reporter protein was screened in the yeast Saccharomyces cerevisiae for sequences that can target the reporter for degradation by the N-end rule pathway, a ubiquitin (Ub)-dependent proteolytic system that recognizes potential substrates through binding to their destabilizing N-terminal residues. One of the N-terminal sequences identified by this screen was used in a second screen for mutants incapable of degrading the corresponding reporter fusion. A mutant thus identified had an abnormally low content of free Ub. This mutant was found to be allelic to a previously isolated mutant in a Ub-dependent proteolytic system distinct from the N-end rule pathway. We isolated the gene involved, termed UFD3, which encodes an 80 kDa protein containing tandem repeats of a motif that is present in many eukaryotic proteins and called the WD repeat. Both co-immunoprecipitation and two-hybrid assays demonstrated that Ufd3p is an in vivo ligand of Cdc48p, an essential ATPase required for the cell cycle progression and the fusion of endoplasmic reticulum membranes. Further, we showed that, similarly to Ufd3p, Cdc48p is also required for the Ub-dependent proteolysis of test substrates. The discovery of the Ufd3p--Cdc48p complex and the finding that this complex is a part of the Ub system open up a new direction for studies of the function of Ub in the cell cycle and membrane dynamics.

The Leucyl/Phenylalanyl-tRNA-Protein Transferase: OVEREXPRESSION AND CHARACTERIZATION OF SUBSTRATE RECOGNITION, DOMAIN STRUCTURE, AND SECONDARY STRUCTURE

Previous work has shown that, in the bacterium Escherichia coli, the aat gene is essential for the degradation of proteins bearing amino-terminal Arg and Lys residues via the N-end rule pathway of protein degradation. We now show that the aat gene encodes directly the leucyl/phenylalanyl-tRNA-protein transferase (L/F-transferase). This enzyme catalyzes the transfer of Leu, Phe, and, less efficiently, Met and Trp, from aminoacyl-tRNAs, to the amino terminus of acceptor proteins. We have used the cloned aat gene to overexpress and purify an affinity tagged L/F-transferase. The recombinant L/F-transferase is as active as the previously purified wild type enzyme and contains no detectable RNA component. We have used the recombinant enzyme to demonstrate that both the solubility and substrate specificity, for aminoacyl-tRNA substrates, of the L/F-transferase are dependent on ionic strength conditions and that the modified nucleotides found in natural tRNAs are not essential for recognition by the enzyme. Limited digestion of the L/F-transferase with trypsin removes the proline rich NH2 terminus of the enzyme identifying a globular core, and circular dichroism demonstrates that the L/F-transferase is predominantly alpha-helical. Finally, a region of sequence conservation between the L/F-transferase and the NH2-terminal protein acetylases has been identified.

A Proteolytic Pathway That Recognizes Ubiquitin as a Degradation Signal

Previous work has shown that a fusion protein bearing a "nonremovable" N-terminal ubiquitin (Ub) moiety is short-lived in vivo, the fusion's Ub functioning as a degradation signal. The proteolytic system involved, termed the UFD pathway (Ub fusion degradation), was dissected in the yeast Saccharomyces cerevisiae by analyzing mutations that perturb the pathway. Two of the five genes thus identified, UFD1 and UFD5, function at post-ubiquitination steps in the UFD pathway. UFD3 plays a role in controlling the concentration of Ub in a cell: ufd3 mutants have greatly reduced levels of free Ub, and the degradation of Ub fusions in these mutants can be restored by overexpressing Ub. UFD2 and UFD4 appear to influence the formation and topology of a multi-Ub chain linked to the fusion's Ub moiety. UFD1, UFD2, and UFD4 encode previously undescribed proteins of 40, 110, and 170 kDa, respectively. The sequence of the last approximately 280 residues of Ufd4p is similar to that of E6AP, a human protein that binds to both the E6 protein of oncogenic papilloma viruses and the tumor suppressor protein p53, whose Ub-dependent degradation involves E6AP. UFD5 is identical to the previously identified SON1, isolated as an extragenic suppressor of sec63 alleles that impair the transport of proteins into the nucleus. UFD5 is essential for activity of both the UFD and N-end rule pathways (the latter system degrades proteins that bear certain N-terminal residues). We also show that a Lys --> Arg conversion at either position 29 or position 48 in the fusion's Ub moiety greatly reduces ubiquitination and degradation of Ub fusions to beta-galactosidase. By contrast, the ubiquitination and degradation of Ub fusions to dihydrofolate reductase are inhibited by the UbR29 but not by the UbR48 moiety. ufd4 mutants are unable to ubiquitinate the fusion's Ub moiety at Lys29, whereas ufd2 mutants are impaired in the ubiquitination at Lys48. These and related findings suggest that Ub-Ub isopeptide bonds in substrate-linked multi-Ub chains involve not only the previously identified Lys48 but also Lys29 of Ub, and that structurally different multi-Ub chains have distinct functions in Ub-dependent protein degradation.

Yeast N-terminal Amidase: A NEW ENZYME AND COMPONENT 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. Tertiary destabilizing N-terminal residues asparagine and glutamine function through their conversion, by enzymatic deamidation, into the secondary destabilizing residues aspartate and glutamate, whose activity requires their enzymatic conjugation to arginine, one of the primary destabilizing residues. We isolated a Saccharomyces cerevisiae gene, termed NTA1, that encodes an amidase (Nt-amidase) specific for N-terminal asparagine and glutamine. Alterations at the putative active-site cysteine of the 52-kDa Nt-amidase inactivate the enzyme. Null nta1 mutants are viable but unable to degrade N-end rule substrates that bear N-terminal asparagine or glutamine. The effects of overexpressing Nt-amidase and other components of the N-end rule pathway suggest interactions between these components and the existence of a multienzyme targeting complex.

Methotrexate Inhibits Proteolysis of Dihydrofolate Reductase by 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 eukaryotes, the N-end rule pathway is a ubiquitin-dependent, proteasome-based system that targets and processively degrades proteins bearing certain N-terminal residues. Arg-DHFR, a modified dihydrofolate reductase bearing an N-terminal arginine (destabilizing residue in the N-end rule), is short lived in ATP-supplemented reticulocyte extract. It is shown here that methotrexate, which is a folic acid analog and high affinity ligand of DHFR, inhibits the degradation but not ubiquitination of Arg-DHFR by the N-end rule pathway. The degradation of other N-end rule substrates is not affected by methotrexate. We discuss implications of these results for the mechanism of proteasome-mediated protein degradation.

The Sequence of Porcine Protein NH2-terminal Asparagine Amidohydrolase: A NEW COMPONENT OF THE N-END RULE PATHWAY

Co- and post-translational amino-terminal processing of proteins is one mechanism by which intracellular proteins can be either protected from or targeted to degradation by the N-end Rule pathway (Bachmair, A., Finley, D., and Varshavsky, A. (1986) Science 234, 179-186). A novel enzyme, protein NH2-terminal asparagine amidohydrolase, which can function in this pathway by potentially directing critical regulatory proteins possessing an amino-terminal asparagine residue formed from the removal of N-acetylmethionine, has recently been purified and characterized (Stewart, A.E., Arfin, S. M., and Bradshaw, R. A. (1994) J. Biol. Chem. 269, 23509-23517). Here, we report the isolation and characterization of a cDNA for porcine protein NH2-terminal asparagine amidohydrolase, which indicates that it is a new type of enzyme, not homologous to any previously identified protein. This provides strong evidence for the importance of regulated protein degradation in cellular functioning.

Degradation of G alpha by the N-end rule pathway

The N-end rule relates the in vivo half-life of a protein to the identity of its amino-terminal residue. Overexpression of targeting components of the N-end rule pathway in Saccharomyces cerevisiae inhibited the growth of haploid but not diploid cells. This ploidy-dependent toxicity was shown to result from enhanced degradation of Gpa1, the alpha subunit (G alpha) of a heterotrimeric guanine nucleotide-binding protein (G protein) that regulates cell differentiation in response to mating pheromones. Sst2, a protein whose absence renders cells hypersensitive to pheromone, was essential for degradation of G alpha but not other N-end rule substrates, suggesting the involvement of an indirect, or trans-, targeting mechanism. G alpha degradation by the N-end rule pathway adds another regulatory dimension to the multitude of signaling functions mediated by G proteins.

Heat-Inducible Degron: a Method for Constructing Temperature-Sensitive Mutants

A temperature-sensitive (ts) mutant retains the function of a gene at a low (permissive) temperature but not at a high (nonpermissive) temperature. Arg-DHFR, a dihydrofolate reductase bearing an amino-terminal (N-terminal) arginine, is long-lived in the yeast Saccharomyces cerevisiae, even though arginine is a destabilizing residue in the N-end rule of protein degradation. A ts derivative of Arg-DHFR was identified that is long-lived at 23 degrees C but rapidly degraded by the N-end rule pathway at 37 degrees C. Fusions of ts Arg-DHFR to either Ura3 or Cdc28 of S. cerevisiae confer ts phenotypes specific for these gene products. Thus, Arg-DHFRts is a heat-inducible degradation signal that can be used to produce ts mutants without a search for ts mutations.

A yeast protein similar to bacterial two-component regulators.

Many bacterial signaling pathways involve a two-component design. In these pathways, a sensor kinase, when activated by a signal, phosphorylates its own histidine, which then serves as a phosphoryl donor to an aspartate in a response regulator protein. The Sln1 protein of the yeast Saccharomyces cerevisiae has sequence similarities to both the histidine kinase and the response regulator proteins of bacteria. A missense mutation in SLN1 is lethal in the absence but not in the presence of the N-end rule pathway, a ubiquitin-dependent proteolytic system. The finding of SLN1 demonstrates that a mode of signal transduction similar to the bacterial two-component design operates in eukaryotes as well.