©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Yeast N-terminal Amidase
A NEW ENZYME AND COMPONENT OF THE N-END RULE PATHWAY (*)

Rohan T. Baker (§) , Alexander Varshavsky (¶)

From the (1) Division of Biology, California Institute of Technology, Pasadena, California 91125

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

The in vivo half-lives of damaged or otherwise abnormal proteins are often shorter than half-lives of their normal counterparts. Many regulatory proteins are also metabolically unstable. Short lifetimes of regulatory proteins allow for rapid adjustments of their concentrations through changes in the rates of their synthesis or degradation. Features of proteins that confer metabolic instability are called degradation signals, or degrons (Varshavsky, 1992). The essential component of one degradation signal, termed the N-degron, is a destabilizing N-terminal residue of a protein (Bachmair et al., 1986). A set of N-degrons containing different destabilizing residues is referred to as the N-end rule, which relates the in vivo half-life of a protein to the identity of its N-terminal residue (for review, see Varshavsky(1992)). The N-degron comprises two determinants: a destabilizing N-terminal residue and an internal lysine (or lysines) of a substrate (Bachmair and Varshavsky, 1989; Johnson et al., 1990; Hill et al., 1993; Dohmen et al., 1994). The Lys residue is the site of formation of a multiubiquitin chain (Chau et al., 1989; Arnason and Ellison, 1994; Spence et al., 1995). Ubiquitin (Ub)() is a protein whose covalent conjugation to other proteins (often in the form of a multi-Ub chain) plays a role in a number of processes, primarily through routes that involve protein degradation (for review, see Hershko and Ciechanover (1992), Jentsch(1992), Varshavsky(1992), Hochstrasser(1992), and Ciechanover(1994)). Two classes of physiological N-end rule substrates were identified thus far. One is RNA polymerase of the Sindbis virus (and also, by inference, RNA polymerases of other alphaviruses) (deGroot et al., 1991). The other is G subunit of heterotrimeric G proteins, a major class of signal transducers in eukaryotes (Madura and Varshavsky, 1994).

The N-end rule is organized hierarchically. In eukaryotes such as the yeast Saccharomyces cerevisiae, Asn and Gln are tertiary destabilizing N-terminal residues (denoted as N-d) in that they function through their conversion, by enzymatic deamidation, into the secondary destabilizing N-terminal residues Asp and Glu (denoted as N-d), whose activity requires their conjugation, by Arg-tRNA-protein transferase (R-transferase), to Arg, one of the primary destabilizing N-terminal residues (denoted as N-d) (Gonda et al., 1989; Balzi et al., 1990). The N-d residues are bound directly by N-recognin (also called E3), the recognition component of the N-end rule pathway (Varshavsky, 1992). In S. cerevisiae, N-recognin is a 225-kDa protein (encoded by UBR1) that selects potential N-end rule substrates by binding to their N-d residues Phe, Leu, Trp, Tyr, Ile, Arg, Lys, or His (Bartel et al., 1990; Baker and Varshavsky, 1991; Madura et al., 1993).

An apparently enzymatic conversion of N-terminal Asn and Gln in cytosolic proteins into Asp and Glu was demonstrated in both yeast and mammalian cells (Gonda et al., 1989), but the postulated amidase(s) responsible for this conversion remained unknown. Stewart et al.(1994, 1995) described purification, cDNA isolation, and analysis of porcine N-terminal amidase (Nt-amidase) that deamidates N-terminal Asn but not Gln. We report a S. cerevisiae gene NTA1 that encodes an enzymatically distinct Nt-amidase; it deamidates either N-terminal Asn or Gln and is essential for the in vivo degradation of proteins bearing N-terminal Asn or Gln residues. (Nta1p has previously been referred to as Dea1p (Varshavsky, 1992).) In addition, we examine functional interactions between targeting components of the N-end rule pathway and consider a multienzyme targeting complex whose components include Nt-amidase (Nta1p), R-transferase (Ate1p), N-recognin (Ubr1p), and Ubc2p, one of at least 11 Ub-conjugating enzymes in S. cerevisiae.


EXPERIMENTAL PROCEDURES

Strains, Genetic Techniques, and -Galactosidase Assay

S. cerevisiae strains were BWG1 7a (MAT a his4 ura3 ade1 leu2) (Guarente et al., 1982), BWG9a-1 (MAT his4 ura3 ade6) (Bartel et al., 1990), RBY56 (MAT a nta1-1 his4 ura3 ade1 leu2); RBY561 (MAT nta1-1 his4 ura3 ade6 leu2), and YBY1 (MAT a nta1-1::URA3 his4 ura3 ade1 leu2) (this work). Cells were grown in rich (YPD) or synthetic media (Sherman et al., 1986), with the latter containing either 2% glucose (SD medium) or 2% galactose (SG medium). X-gal plates also contained 0.1 M KHPO-KHPO (pH 7.0) and 5-bromo-4-chloro-3-indolyl -D-galactoside (X-gal) at 40 µg/ml (Guthrie and Fink, 1991). Escherichia coli strains MC1061, JM101, and DH5F` (Ausubel et al., 1992) were grown in Luria Broth (LB) or in M9 minimal medium containing auxotrophic nutrients and antibiotics as required. Transformation of S. cerevisiae was carried out using the lithium acetate method (Ito et al., 1983; Baker, 1991). Yeast mating, sporulation, and tetrad analyses were performed as described previously (Guthrie and Fink, 1991). S. cerevisiae were cured of URA3-expressing plasmids using 5-fluoroorotic acid (Boeke et al., 1984). Activity of gal in yeast extracts was measured using o-nitrophenyl -D-galactoside (Baker and Varshavsky, 1991).

Isolation of nta1 Mutants

BWG1-7a was transformed with pUB23-N, a high copy (2 µm-based), URA3-containing plasmid expressing Ub-Asn-gal from the galactose-inducible P hybrid promoter (Bachmair et al., 1986). The cells were mutagenized with ethyl methanesulfonate (Sherman et al., 1986) to 20% survival and plated on SD medium lacking uracil. After 3 days of growth at 23 °C, colonies were replica plated onto X-gal-SG plates and incubated for 2 days at 23 or 36 °C. About 100 colonies (out of 3 10 colonies screened) that turned blue at 23 °C and 30 colonies that turned blue only at 36 °C were picked and retested. Extracts from 43 colonies (23 °C) and 15 colonies (36 °C) that passed the second X-gal test were examined for gal activity using the o-nitrophenyl -D-galactoside assay. Thirty putative 23 °C mutants and eight putative temperature-sensitive (ts) mutants with high gal activity were cured of pUB23-N using 5-fluoroorotic acid and were retransformed with the same (unmutagenized) plasmid. Seven apparently plasmid-linked mutants were eliminated at this stage. The rest were transformed with pUB23-X plasmids expressing Ub-Met-gal, Ub-Pro-gal, Ub-Asn-gal, Ub-Asp-gal, or Ub-Arg-gal (Bartel et al., 1990). This analysis segregated each mutant into one of three classes: (i) those with increased levels of Asn-gal, Asp-gal, and Arg-gal (23 mutants); (ii) those with increased levels of Asn-gal and Asp-gal (two mutants); and (iii) those with increased levels of Asn-gal but not Asp-gal or Arg-gal (six mutants). All mutants had wt levels of Met-gal and Ub-Pro-gal. Mutants of the third class were examined by complementation and found to harbor recessive mutations in a single complementation group. One mutant, RBY56, was crossed to the parental strain BWG9a-1; sporulation and tetrad analysis of the resulting diploid showed a 2:2 segregation of high Asn-gal levels. Two back-crosses of RBY56 to BWG9a-1 and BWG1-7a yielded the haploid RBY561 carrying the original mutation. Curing RBY561 of pUB23-N, transforming it with pUB23-X plasmids expressing different Ub-X-gals (Bachmair and Varshavsky, 1989; X = Asn, Gln, Asp, Glu, Leu, and Arg), and measuring gal activity in the transformants (Fig. 1B) showed that among the normally short-lived gals, only those bearing N-d residues (Asn and Gln) accumulated to high levels in RBY561. Pulse-chase analyses of X-gals (Fig. 2) confirmed these results. The gene a mutation in which produced the phenotype of RBY561 was termed NTA1.


Figure 1: Three classes of mutants that stabilize short-lived Asn-gal. A, levels of gal activity (plotted as percentages of Met-gal activity) in extracts from the parental (wt) S. cerevisiae strain BWG1-7a expressing different X-gal (Ub-X-gal) test proteins. B, C, and D, representative mutants RBY56 (nta1-1), RBY42 (ate1), and RBY45 (ubr1), respectively. These mutants were isolated through their inability to degrade the normally short-lived Asn-gal. Each of the strains was transformed with plasmids expressing X-gals whose N termini bore either tertiary (N, Asn; Q, Gln) or secondary (D, Asp; E, Glu) destabilizing residues and also a primary destabilizing (R, Arg) or a stabilizing (M, Met) residue. The striped upper portions of the bars indicate differences between the activities of a given X-gal in the wt strain (A) and in its mutant derivatives (B-D). Values shown are the means from duplicate measurements, which yielded results within 10% of each other.




Figure 2: Metabolic stabilization of Gln-gal in the nta1-1 mutant. A, lanesa-c, BWG1-7a (wt) cells that expressed Gln-gal (Ub-Gln-gal) were labeled with TranS-label for 5 min at 30 °C, followed by a chase for 0, 10, and 30 min, respectively; extraction and immunoprecipitation of test proteins with an antibody to gal; and SDS-PAGE and fluorography (see ``Experimental Procedures''). Lanesd-f, same as lanesa-c but with Arg-gal; lanesg-i, same as lanesa-c but with Met-gal. B, same as the corresponding lanes in A but with RBY561 (nta1-1) cells. The X-gal bands are indicated. An asterisk denotes a 90 kDa, long-lived gal cleavage product characteristic of short-lived gal-based proteins (Bachmair et al., 1986; Baker and Varshavsky, 1991). Note the absence of this species from nta1-1 cells expressing Gln-gal. Note also the much weaker labeling of Arg-gal (in comparison with longer-lived X-gals) even at the beginning of chase; this ``zero-point effect'' (Baker and Varshavsky, 1991) is caused by degradation of Arg-gal during the labeling.



Isolation of NTA1

The nta1-1 strain RBY561, carrying pNL, a derivative of pUB23-N (expressing Ub-Asn-gal) in which the URA3 marker had been replaced by LEU2, was transformed with S. cerevisiae genomic DNA library carried in the URA3, CEN4-based vector YCp50 (Rose et al., 1987). About 4 10 transformants were screened for white colonies (low levels of gal) on SG-X-gal plates that lacked uracil and leucine. Two of the six initially selected transformants remained white upon retesting on SG-X-gal plates; these results were confirmed using the o-nitrophenyl -D-galactoside assay for gal. When the two transformants were cured of their library-derived plasmids on 5-fluoroorotic acid plates and retested on SG-X-gal plates, only one isolate yielded blue colonies (high levels of gal). Plasmid DNA (Hoffman and Winston, 1987) from these cells was used to transform E. coli MC1061 to ampicillin resistance. Transformants carrying YCp50 library-derived plasmids were distinguished from those carrying pNL by picking white E. coli colonies on LB/ampicillin/X-gal plates. A 4.3-kb subclone of the 14.3-kb insert in the plasmid thus obtained (termed pRB8) complemented the defect in degradation of Asn-gal in RBY561 and the other five nta1 mutants. This subclone was sequenced, revealing two complete open reading frames (ORFs) and a portion of a third (Fig. 3A). Comparisons of the amino acid sequences encoded by these ORFs with sequences in GenBank showed that one of the two complete ORFs had similarities to known amidotransferases. A low copy plasmid expressing only the putative NTA1 portion of the 4.3-kb insert (Fig. 3A) was constructed and found to complement all six nta1 mutants. Conversely, the low copy plasmid pRB8E2.5 that expressed only the smaller complete ORF was unable to complement nta1 mutants.


Figure 3: The NTA1 locus. A, ORFs are shown as arrow-shapedboxes indicating the direction of transcription. The incompletely sequenced ORFa is shown as a jagged-endbox. Subcloned regions are also indicated, with (+) or (-) denoting their ability to complement the nta1-1 phenotype. Dashedlines delineate the region of NTA1 that has been replaced with URA3 in the nta1-1 allele. Restriction sites: E, EcoRI; H, HindIII; K, KpnI; P, PstI; S, SalI; Sc, ScaI; Spe, SpeI; X, XbaI. The scale (in kb) is above the map, with zero denoting the end of yeast genomic DNA insert in pRB8. Nucleotide sequence encompassing NTA1 (a 3747-bp region from the XhoI to the SalI site) has been submitted to GenBank (accession number L35564). An ORF located 346 bp upstream of NTA1 and oriented in the opposite direction has been identified as the RRN4 gene encoding the 125-residue A12.2 subunit of S. cerevisiae RNA polymerase I (Nogi et al., 1993). A partially sequenced ORFa 156 bp downstream of NTA1 is oriented in the opposite direction and encodes a protein of at least 740 residues. ORFa is transcriptionally active (data not shown) and encodes a protein highly similar to the product of a putative S. cerevisiae ORF on chromosome XI (GenBank accession numbers Z28200 and Z28201). The functions of either of these ORFs are unknown. B, deduced amino acid sequence of the Nta1p. Amino acid residues are numbered on the right. The sequence Ile-Gly-Ile-Cys-Met that matches a portion of the consensus active-site region of several amidotransferases and contains an essential Cys residue is doublyunderlined. A blackrectangle indicates the position of the 12-residue ha epitope tag in the Nta1p-ha derivative of Nta1. C, alignment of the Nta1p sequence with the 11-residue consensus sequence encompassing the putative active-site Cys residue of glutamine amidotransferases (Nyunoya and Lusty, 1984). Alternate amino acids in the consensus among these enzymes are shown above the consensus sequence. The region of identity between Nta1p and the consensus sequence is boxed. The essential Cys-187 of Nta1p is doublyunderlined. D, nucleotide sequence at the 5` region of NTA1. Positions of the major and minor 5` ends of NTA1 mRNAs are indicated by closed and opencircles, respectively. ATG codons are boxed. A start codon at position +1 was inferred in part from the 5` mapping data. The in-frame ATG codon at position +31 is likely to be used as an in vivo translation initiation site as well (see text). The ATG codon at position -51 is indicated by an asterisk. This in-frame ATG is absent from the presently detectable NTA1 mRNAs; it is followed by two in-frame (underlined) stop codons. Motifs that are present in the promoter regions of genes encoding components of the N-end rule pathway (Fig. 6) are doublyunderlined.



The nta1-1 Allele

To construct nta1-1, the 3.7-kb XhoI-SalI fragment of pRB8S13 (Fig. 3A) that contained NTA1 was subcloned into SalI-cut pUC9, yielding pUC3.7. The yeast URA3 gene, isolated as a 1.1-kb XbaI-KpnI fragment of pRBU1 (Tobias and Varshavsky, 1991), was ligated between the SpeI site at the 5` end of the NTA1 ORF and the KpnI site near the 3` end of the NTA1 ORF in pUC3.7, yielding p3.7::URA3, which lacked 82% of the NTA1 ORF (Fig. 3A). The 3.5-kb HindIII fragment of p3.7::URA3 containing nta1-1::URA3 was used to replace NTA1 in ura3 strains of S. cerevisiae by homologous recombination (Rothstein, 1991). Southern hybridization analysis of Ura transformants, using restriction endonuclease cuts diagnostic of the transplacement, was used to confirm the predicted structure of the integrated nta1-1 allele (data not shown).

To verify that the isolated NTA1 gene was allelic to the gene of the original nta1-1 mutation, a diploid strain YRB200 was constructed by crossing RBY561 (nta1-1) to YRB1 (nta1-1::URA3). This strain was unable to degrade N-end rule substrates bearing N-terminal Asn or Gln, but it retained the ability to degrade N-end rule substrates of other classes (data not shown). Since both the nta1-1 and nta1-1::URA3 mutations were recessive, this result indicated that the cloned NTA1 gene was allelic to nta1-1.

Other DNA Constructs

DNA fragments were isolated from agarose gels using Geneclean (Bio 101). To verify that expression of the NTA1 ORF was sufficient for complementation of the nta1 phenotype, a low copy plasmid was constructed whose only yeast ORF was NTA1. The 944-bp ScaI-EcoRI fragment of pRB8S13 that contained the 5` region of NTA1 was ligated into SmaI/EcoRI-cut pRS316 (a CEN4, URA3-based plasmid; Sikorski and Hieter(1989)), yielding p316ESc. The 998-bp EcoRI fragment of pRB8S13 that contained the rest of NTA1 (Fig. 3A) was then ligated into the EcoRI site of p316ESc, yielding pNTA1, which contained the reassembled NTA1 ORF, in addition to 335 bp of yeast DNA 5` to the (inferred) start codon of NTA1 and 236 bp of yeast DNA 3` to NTA1 stop codon. An NTA1-overexpressing plasmid was constructed by ligating the 2-kb BamHI-SalI fragment of pNTA1 into BamHI/SalI-cut YEplac195 (a 2 µm, URA3-based vector; Gietz and Sugino(1988)), yielding p195NTA1. To construct the NTA1-ha allele, the 2-kb BamHI-SalI fragment of pNTA1 was ligated into BamHI/SalI-cut RF DNA of the phage M13mp18 (Ausubel et al., 1992). A synthetic oligodeoxynucleotide (5`-GTATAGCTTCTGTTCATCATCCAAGCTAGCGTAATCTGGAACATCGTATGGGTAATCATCTTCAGGATATCC-3`) was used for insertional mutagenesis with the MutaGene kit (Bio-Rad), yielding an ORF that encoded a modified Nta1p (Nta1p-ha) containing the sequence Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala-Ser-Leu-Asp between residues 112 and 113 of Nta1p. This sequence contained the ha epitope recognizable by the monoclonal antibody 12CA5 (Johnson et al., 1992). The insertion (containing a diagnostic NheI site) was verified by NheI mapping and DNA sequencing, and the 1-kb BamHI-SalI fragment of the resulting plasmid was ligated into BamHI/SalI-cut YEplac195, yielding p195NTA1-ha. To mutate the Cys-187 residue of Nta1p, two oligonucleotides (5`-CTTAAGTCCATAGATATCCCTATTGATGC 3` and 5`-GGACTTAAGTCCATCGCGATACCTATTGATGC-3`) were used with the M13mp18 subclone described above to produce ORFs encoding Nta1p-C187S and Nta1p-C187A, in which Cys-187 was converted, respectively, into Ser and Ala. The oligos contained diagnostic restriction sites (EcoRV and NruI, respectively); both mutations were confirmed by sequencing as well. The 2-kb BamHI-SalI fragments of the resulting plasmids were ligated into BamHI/SalI-cut YEplac195, yielding p195NTA1-CS and p195NTA1-CA, which expressed, respectively, Nta1p-C187S and Nta1p-C187A from the P promoter. The ha tag was linked to Nta1p-C187S and Nta1p-C187A by ligating the 472-bp SpeI-HindIII fragment of p195NTA1-ha, together with either the 897-bp HindIII-XbaI fragment of p195NTA1-CS or an analogous fragment of p195NTA1-CA, into SpeI/XbaI-cut p195NTA1-ha, yielding, respectively, p195NTA1-CS-ha and p195NTA1-CA-ha.

Plasmids expressing S. cerevisiae Ate1p either alone or together with Nta1p were constructed as follows. The 4-kb HindIII fragment of pA4-H4 (Balzi et al., 1990) that contained ATE1 was subcloned into the HindIII-cut high copy plasmid YEplac195, yielding p195ATE1. The same fragment was also subcloned into the low copy plasmid YCplac33 (Gietz and Sugino, 1988), yielding p33ATE1. pNTA1/ATE1, a high copy plasmid that overexpressed both Nta1p and Ate1p, was constructed by subcloning the above HindIII fragment into p195NTA1 that had been partially digested with HindIII, and screening for products in which the insert was located at the polylinker HindIII site.

Southern Hybridization and DNA Sequencing

DNA was isolated from S. cerevisiae as described by Hoffman and Winston (1987), digested with restriction endonucleases, and processed for electrophoresis and hybridization as described by Bartel et al.(1990). Restriction fragments of the NTA1-containing a 4.3-kb fragment of pRB8S13 (Fig. 3A) were subcloned into M13mp18 and M13mp19 (Ausubel et al., 1992) and were sequenced using the Sequenase kit (U. S. Biochemical Corp.). The entire 4.3-kb fragment was sequenced on both strands. The nucleotide sequence of NTA1 (GenBank accession L35564) and the predicted amino acid sequence of Nta1p (Fig. 3B) were compared with sequences in the GenBank/EMBL data base using the GCG program (Deveraux et al., 1984) (version 7.2, Genetics Computer Group, Madison, WI).

5` Mapping of NTA1 mRNAs

For mapping by primer extension, RNA was isolated from an exponential culture of BWG1-7a in YPD, using the phenol/chloroform/glass bead procedure of Sprague et al. (1983). Poly(A) RNA was isolated from total RNA by chromatography on oligo(dT)-agarose (Pharmacia Biotech Inc.). Primer extension analysis was carried out as described by Teem and Rosbash (1983), using 3 µg of poly(A) RNA/sample. The oligodeoxynucleotide primer was complementary to the coding strand of NTA1 between nucleotide positions +34 and +67. For mapping by S1 nuclease, the procedure described by Nyunoya and Lusty(1984) was used, with 50 µg of total RNA or 2 µg of poly(A) RNA/sample. The probe was a single-stranded DNA from an M13 subclone containing the 400-bp ScaI-SpeI fragment of pRB8S13 (NTA1 nucleotide positions -338 to +63) uniformly labeled with P (Baker et al., 1992).

Phenotypic Characterization of the nta1-1 Mutant

Assays measuring sensitivity of yeast cells to chronic heat stress (at 39 °C), sensitivity to canavanine, and survival in stationary phase were carried out as described by Finley et al.(1987). Sensitivity to acute heat stress was determined by exposing cells (which have been growing exponentially in YPD at 30 °C) to YPD at 50 °C for 0-15 min prior to plating on YPD to assay colony formation at 30 °C. Ability to use glycerol as a carbon source was assayed on YPD plates lacking glucose and containing 3% (v/v) glycerol. Ability to utilize Asn or Gln as a nitrogen source was assayed on synthetic media plates containing 0.17% yeast nitrogen base (without amino acids and ammonium sulfate; Difco), 2% glucose, auxotrophic nutrients at standard concentrations (Sherman et al., 1986), and 0.1% (w/v) of either Asn or Gln as a major nitrogen source. Control plates lacked Asn and Gln, and in addition either contained or lacked 0.1% (w/v) ammonium sulfate.

We did not detect a significant difference between the S. cerevisiae nta1-1 mutant and a congenic wt strain in their sensitivity to acute or chronic heat stress; in their survival at stationary phase after growth in rich or poor media, or upon starvation for either carbon or nitrogen; in their sensitivity to canavanine (a toxic arginine analog); in their ability to grow on glycerol as a carbon source; and in their ability to utilize either Asn or Gln as a source of nitrogen. No short-lived yeast proteins detectable by a pulse-chase and two-dimensional electrophoresis were significantly stabilized in the nta1-1 mutant (data not shown). ubr1 mutants (in which normally short-lived N-end rule substrates are metabolically stable) grow slightly (3%) slower than wt cells and have a defect in sporulation, increased fraction of asci with fewer than four spores (Bartel et al., 1990). The growth rate phenotype was not observed with the nta1-1 mutant (data not shown), while the sporulation of an nta1/nta1 strain has yet to be investigated.

Protein Labeling, Pulse-Chase Analysis, and Immunoblotting

Pulse labeling with TranS-label (ICN), a chase in the presence of cycloheximide, preparation of cell extracts, immunoprecipitation with a polyclonal antibody to gal (Sigma), and electrophoretic analysis of X-gals by SDS-PAGE in 6% gels were carried out as described by Bachmair et al. (1986), with slight modifications (Baker and Varshavsky, 1991). Immunoblotting of extracts from the nta1-1 strain RBY561 that has been transformed with either p195NTA1, p195NTA1-ha, p195NTA1-ha-CS, or p195NTA1-ha-CA was carried out after SDS-PAGE in a 10% gel, using a monoclonal anti-ha antibody (Bartel et al., 1990; Madura et al., 1993), a phosphatase-linked second antibody, a chromogenic phosphatase substrate, and procedures described by Tobias and Varshavsky(1991) and Baker et al. (1992).

RESULTS AND DISCUSSION

Isolation of nta1 Mutants and Cloning of the NTA1 Gene

To screen for S. cerevisiae mutants defective in the N-end rule pathway, we used a strain carrying a plasmid that expressed Ub-Asn-gal. Ub fusions are rapidly cleaved in vivo after the last residue of Ub, making possible the production of otherwise identical proteins bearing different N-terminal residues (Bachmair et al., 1986; Baker et al., 1992). Since Asn-gal is short-lived in wt cells (t of 3 min at 30 °C; Bachmair and Varshavsky(1989)), its steady-state level is low, and the corresponding yeast colonies are white on plates containing the chromogenic gal substrate X-gal (Bartel et al., 1990). By contrast, cells that express long-lived X-gals such as Met-gal (t > 30 h) have high gal activity and form blue colonies on X-gal plates. Cells expressing Asn-gal were mutagenized, plated on X-gal plates, and screened for blue colonies. These were tested further, and the putative nta1 mutants among them were identified as described under ``Experimental Procedures.''

The NTA1 gene was cloned by complementation (see ``Experimental Procedures''). The position of the start (ATG) codon of the NTA1 ORF was inferred so as to yield the largest ORF (Fig. 3). The 1,371-bp NTA1 encodes an acidic (calculated pI of 4.9), 457-residue (51.8 kDa) protein. The codon adaptation index of NTA1 (calculated according to Sharp and Li(1987)) is 0.125, characteristic of weakly expressed yeast genes.

An Essential Cysteine in Nta1p

Weak sequence similarities were detected between Nta1p and an aliphatic amidase from Pseudomonas aeruginosa (Ambler et al., 1987) as well as several other amidotransferases. The substrates of aliphatic amidase (acetamide and propionamide) are the side chains of Asn and Gln, which are the substrates of Nta1p when these residues are present at the proteins' N termini. Nyunoya and Lusty(1986) identified an 11-residue region conserved among 7 glutamine amidotransferases from five species, including E. coli, S. cerevisiae, and Neurospora crassa (Fig. 3C). The conserved region contains a Cys residue that could be labeled with reactive glutamine analogs in two of these enzymes, suggesting that this cysteine is a part of the active center (Nyunoya and Lusty, 1986). Nta1p contains a 5-residue sequence that is identical to the sequence in the middle of the 11-residue consensus stretch and includes the conserved cysteine (Cys-187) (Fig. 3C).

We converted Cys-187 of Nta1p into Ser and Ala. The resulting Nta1p-C187S and Nta1p-C187A (expressed from either low or high copy plasmids) lacked Nt-amidase activity, as inferred from their inability to restore the degradation of Asn-gal in the nta1-1 mutant ( Fig. 4 and data not shown). Nta1p was then tagged with a 12-residue sequence containing the ha epitope, making it possible to immunoprecipitate Nta1p-ha with an anti-ha antibody (Field et. al., 1988; Johnson et al., 1992). The ha tag was positioned within a putative loop between residues 112 and 113 of Nta1p (Fig. 3B). Nta1p-ha and the unmodified Nta1p were equally effective in restoring the degradation of Asn-gal in the nta1-1 mutant, whereas no complementation was observed with Nta1p-ha-C187S or Nta1p-ha-C187A (Fig. 4). Internal tagging of Nta1p was necessitated by inactivity of the C-terminally tagged Nta1p (data not shown) and by an a priori drawback of N-terminal tagging, given uncertainties about the location of a start codon in NTA1 (see below). The levels of Nta1p-ha in cells, measured by immunoblotting of cell extracts after SDS-PAGE, were the same irrespective of whether Nta1p-ha contained or lacked a substitution at Cys-187 (Fig. 5). Thus, Cys-187 is required for the activity of Nta1p, in agreement with the observation that a thiol-blocking reagent N-ethylmaleimide inhibits the conversion of N-terminal Gln into Glu in reticulocyte extract (Gonda et al., 1989).


Figure 4: Nt-amidase contains an essential cysteine residue. Substitution of Cys-187 in Nta1p with either Ser or Ala results in metabolic stabilization of Asn-gal. Congenic S. cerevisiae strains BWG1-7a (NTA1) and RBY561 (nta1-1) carrying a plasmid that expressed Asn-gal were transformed with either a high copy vector YEplac195 (a control) or an otherwise identical plasmid expressing either the wt Nta1p, the ha epitope-tagged Nta1p (Nta1p-ha), or Nta1p-ha in which Cys-187 was converted into either Ser (Nta1p-ha-C187S) or Ala (Nta1p-ha-C187A). Extracts from cultures in exponential growth were assayed for gal activity. Values shown are the means of at least three independent measurements. Standard deviations are indicated above the bars. Note a discontinuity in the ordinate scale.




Figure 5: The Nta1p protein. Immunoblotting of extracts from cells that expressed Nta1p-ha containing either the wt Cys-187 (Nta1p-ha), Ser-187 (Nta1p-ha-CS), or Ala-187 (Nta1p-ha-CA). Equal amounts of total protein in extracts from the nta1-1 strain RBY561 that has been transformed with plasmids expressing Nta1p-ha-CA (lanea), Nta1p-ha-CS (laneb), Nta1p-ha (lanec), or the untagged (control) Nta1p (laned) were fractionated by SDS-PAGE and analyzed by immunoblotting with anti-ha antibody (see ``Experimental Procedures''). Note the presence of two Nta-ha species (53 kDa and 52 kDa; see text).



Location of Start Codons in NTA1

There is an ATG codon 51 bp upstream of (and in frame with) the inferred NTA1 start codon but with two stop codons in between (Fig. 3D). If NTA1 mRNA were to contain the -51 ATG codon, initiation of translation at this codon would result in the synthesis of an 8-residue peptide and might also interfere with initiation at the downstream (+1) ATG codon. A short translated ORF is present in the 5` leader region of the yeast GCN4 mRNA, which encodes transcriptional activator of the regulon for amino acid biosynthesis (Hinnebusch and Liebman, 1991). A potentially more relevant example is CPA1, which encodes glutamine amidotransferase, a subunit of carbamoyl-phosphate synthetase and component of the arginine biosynthetic pathway. In vivo translation of the upstream ORF in CPA1 mRNA yields a 25-residue peptide that down-regulates translation of the major CPA1 ORF in the presence of arginine (Werner et al., 1987).

To determine whether NTA1 mRNA contains the -51 start codon, we used primer extension and S1 nuclease mapping (see ``Experimental Procedures''). Both tests identified major 5` ends of NTA1 mRNA at positions -14 and -15 relative to the inferred (+1) start codon, with less abundant 5` ends at -15 and -13 (Fig. 3D). No transcripts extending beyond -19 were detected, indicating that most of NTA1 mRNAs lack the(-51) start codon. Thus, NTA1 appears not to be regulated in a way observed with CPA1. The 5` mapping also showed that in a minor but significant fraction of NTA1 mRNAs, their 5` ends are located immediately upstream, or even downstream of the inferred (+1) NTA1 start codon. Specifically, S1 mapping detected minor 5` ends largely at positions -4, -3, and +1, while primer extension detected sites at +2, +3, +4, +5, and also at +13 relative to the inferred (+1) start codon (the 5` end at +13 was not detected by S1 mapping) (Fig. 3D and data not shown). Thus, there exist NTA1 mRNAs that either lack the inferred (+1) start codon or contain it too close to the 5` end of the message for efficient initiation of translation at that position. An in-frame ATG is present 30 bp downstream of the inferred (+1) NTA1 start codon (Fig. 3D). Initiation of translation at this (+31) ATG should yield a 50.8-kDa protein lacking the first 10 residues of the inferred Nta1p (51.8 kDa). The (+31) ATG lies within a relatively favorable context for translation initiation (Kozak, 1992), with As in positions -3 and +4 (AAGATGA), whereas the (+1) ATG is located in a less favorable context, with pyrimidines at -3 and +4 (TGAATGC).

Immunoblot Analysis of Nta1p-ha

Two nearly comigrating, Nta1p-specific, ha-containing species of 52 and 53 kDa, were observed upon immunoblot analysis of Nta1-ha, the smaller protein being less abundant and partially obscured by the band of the 53-kDa Nta1p-ha ( Fig. 5and data not shown). The 1-kDa difference between these species of Nta1p is consistent with the possibility that the translation start site of the larger (53 kDa) Nta1p-ha is at the inferred (+1) ATG codon of NTA1 (predicted Nta1p-ha of 53.2 kDa, including the ha tag), whereas the smaller (52 kDa) Nta1p-ha is initiated at the (+31) ATG codon (predicted Nta1p-ha of 52.2 kDa, including the ha tag) (Fig. 3D). The results of mRNA mapping are consistent with this interpretation, inasmuch as the set of NTA1 mRNAs contains both the species whose 5` ends encompass the (+1) ATG and the species whose 5` ends are located between the (+1) and the (+31) ATG (Fig. 3D). The 29-residue region between Asp-4 and Asp-34 in the larger Nta1p (Fig. 3, B and D) resembles mitochondrial translocation signals (von Heijne, 1986). However, more extensive testing will be required to verify the conjecture that the larger Nta1p species might be a mitochondrial protein.

A Null nta1 Mutant and Biochemical Aspects of Nt-amidase

A deletion/disruption allele of NTA1 (Fig. 3A) was used to produce the nta1-1 mutant (see ``Experimental Procedures''). As expected from the phenotype of the original nta1 mutants (Fig. 1B and 2B), Asn-gal and Gln-gal but not the other normally short-lived X-gals were long-lived in the nta1-1 mutant (t > 10 h) (data not shown), whereas they were short-lived in the congenic NTA1 strain (t of 3 and 10 min, respectively (Varshavsky, 1992)). The normally long-lived X-gals (bearing stabilizing N-terminal residues) remained long-lived in the nta1-1 mutant. These results supported the conjecture that NTA1 encodes an amidase specific for N-terminal Asn and Gln. These data also indicated that Nta1p is the only such amidase in S. cerevisiae.

In a biochemical test, purified, S-labeled Asn-dihydrofolate reductase or Gln-dihydrofolate reductase (dihydrofolate reductase-based N-end rule substrates (Bachmair and Varshavsky, 1989)) were incubated with extracts prepared from E. coli that either expressed or lacked Nta1p, and then fractionated by isoelectric focusing in a polyacrylamide gel. Isoelectric points of both substrates became more acidic after incubation with the Nta1p-containing E. coli extract but not after incubation with the control (Nta1p-lacking) extract. Moreover, the isoelectric point of Met-dihydrofolate reductase, an otherwise identical protein bearing a non-amide N-terminal residue, was not altered by incubation with either of E. coli extracts. These findings() confirmed the inferred deamidating activity of Nta1p and the confinement of this activity to N-terminal Asn and Gln.

Stewart et al.(1994) have purified a distinct Nt-amidase from porcine liver. They also isolated a cDNA encoding this enzyme (Stewart et al., 1995). In contrast to the 52-kDa yeast Nta1p, which deamidates either N-terminal Asn or N-terminal Gln, the activity of the 33-kDa porcine Nt-amidase is confined to N-terminal Asn. This finding (Stewart et al., 1994) suggests the existence of yet another mammalian Nt-amidase (the one specific for N-terminal Gln) and hence a bifurkation at the deamidation step in the N-end rule pathway of mammals but not of yeast. The amino acid sequence of the Asn-specific mouse Nt-amidase, deduced from sequences of the corresponding cDNA and the gene,() is highly similar to the sequence of porcine Nt-amidase but lacks similarities to the sequence of yeast Nta1p.

No significant phenotypic differences (other than the metabolic stabilization of Asn-gal and Gln-gal) were observed between the nta1-1 and congenic wt strains (see ``Experimental Procedures''). Ubr1p (N-recognin) was recently found to be required for the peptide import in S. cerevisiae; ubr1 mutants do not express PTR2, which encodes a peptide transporter, and are unable to import peptides from the medium (Alagramam et al., 1995). It is unknown whether this function of Ubr1p is mediated by the N-end rule pathway or another Ubr1p-dependent mechanism. Unlike ubr1 mutants, the nta1-1 mutant is able to import peptides.()

Overexpression of R-Transferase and Nt-amidase

Bartel et al.(1990) found that overexpression of S. cerevisiaeN-recognin (Ubr1p) accelerated the degradation of N-end rule substrates. We asked whether R-transferase (Ate1p) and Nt-amidase (Nta1p) are also rate limiting for certain classes of these substrates. Overexpression of R-transferase from a high copy plasmid in cells expressing either Glu-gal or Gln-gal decreased the levels of gal activity by 2- and 3-fold, respectively (Fig. 6A). Thus, the arginylation of N-terminal Glu in Glu-gal by R-transferase appears to be rate-limiting for the degradation of Glu-gal and Gln-gal. Even a weaker overexpression of R-transferase (from a low copy plasmid) resulted in a small but significant decrease of Glu-gal (Fig. 6A). Previous work (Bartel et al., 1990; Balzi et al., 1990; Baker and Varshavsky, 1991; Dohmen et al., 1991; Madura et al., 1993) has shown that the concentration of an X-gal test protein in yeast cells is a sensitive indicator of its metabolic stability.


Figure 6: Effects of overexpressing Nt-amidase and R-transferase. A, levels of gal activity in extracts from cells overexpressing Ate1p (R-transferase) and/or Nta1p (Nt-amidase), and also expressing Glu-gal (Ub-Glu-gal) or Gln-gal (Ub-Gln-gal) (indicated by E and Q, single-letter abbreviations of Glu and Gln). The strain BWG1-7a expressing either Glu-gal or Gln-gal was transformed with either the high-copy vector YEplac195 (control) or an otherwise identical plasmid expressing either Ate1p or Nta1p from their natural promoters. Alternatively, BWG1-7a was transformed with a low copy (CEN-based) plasmid expressing either Ate1p or Nta1p from their natural promoters, as indicated. Values of gal activity shown are the means of at least three independent measurements. Standard deviations are indicated above the bars. B, same as in A but with cells expressing either Tyr-gal (Ub-Tyr-gal) or His-gal (Ub-His-gal) (indicated by Y and H, single-letter abbreviations of Tyr and His).



Surprisingly, overexpression of Nt-amidase in cells expressing Gln-gal increased the level of Gln-gal by 5-fold (Fig. 6A). In other words, overexpression of Nt-amidase inhibited the degradation of Gln-gal. This result is likely to be related to an earlier finding that Gln-gal, which bears an N-d residue and therefore requires two modifications (deamidation and arginylation) prior to its binding by N-recognin, has a shorter half-life (t of 10 min at 30 °C) than Glu-gal (t of 30 min), which bears an N-d residue and is therefore only one step (arginylation) away from its binding by N-recognin (Bachmair and Varshavsky, 1989; Gonda et al., 1989). No such ``inverse'' order of half-lives was observed with Asn-gal and Asp-gal (t of 3 min for both substrates) (op. cit.). The following assumptions are sufficient to account for these apparently paradoxical findings: (i) R-transferase arginylates Asp-gal significantly faster than Glu-gal; (ii) Nt-amidase is about equally effective in deamidating Asn-gal and Gln-gal; (iii) in wt cells, Nt-amidase exists largely as an Nt-amidaseR-transferase complex. Specifically, the R-transferase-mediated arginylation of Glu-gal that has been produced from Gln-gal by the Nt-amidaseR-transferase complex is presumed to occur kinetically in preference to the arginylation of Glu-gal that reaches this complex directly from the bulk solvent, a feature known as ``substrate channeling'' in other multistage enzymatic reactions (Srere, 1987; Ovádi, 1991; Knowles, 1991; Negrutskii and Deutscher, 1991; Knighton et al., 1994).

This model accounts for the observed stabilization of Gln-gal upon overexpression of Nt-amidase. Indeed, under these conditions, a greater fraction of Gln-gal is converted into Glu-gal by the free (overexpressed) Nt-amidase. As a result, a greater fraction of the deamidation-produced Glu-gal will have to reach the Nt-amidaseR-transferase complex directly from the bulk solvent, a kinetically inefficient route to arginylation. The resulting delay in formation of Arg-Glu-gal (which can be bound by N-recognin) would cause the observed stabilization of Gln-gal in cells that overexpress Nt-amidase (Fig. 6A). Note that overexpression of Nt-amidase raised the level of Gln-gal to that of Glu-gal (Fig. 6A). This result is also predicted by the model, because the bulk of Gln-gal in cells that overexpress Nt-amidase is deamidated by the free (overexpressed) Nt-amidase rather than by the less abundant form of Nt-amidase that exists in the complex with R-transferase. Another prediction of the model is that expression of R-transferase and Nt-amidase is likely to be coregulated in wt cells to maintain optimal ratios of these apparently interacting enzymes. This conjecture is consistent with the presence of common sequence motifs in the 5` regions of genes that encode components of the N-end rule pathway ( Fig. 7and below).


Figure 7: Common sequence motifs in promoters of genes encoding components of the N-end rule pathway. Alignment of the 5` regions of the S. cerevisiae genes UBR1 (Bartel et al., 1990), ATE1 (Balzi et al., 1990), and NTA1 (the present work; Fig. 3, A and D) revealed two distinct regions of similarity: an 11-mer motif 1 (consensus TTTCATTGCTA) and a 14-mer motif 2 (consensus CTTTAATTTCRCAT; R = purine). Mismatches to the consensus are in lowercase. Arrows show the direction of transcription. While no other S. cerevisiae gene in GenBank contains both motifs, several genes, including three that encode known components of the Ub system, contain one of these motifs. Motifs 1 or 2 in the genes of this class (UBC2, UBI1, and UBI2) (Dohmen et al., 1991; zkaynak et al., 1987) are also shown. The numbers indicate locations of the motifs relative to either the known (UBC2, UBI1, UBI2) or inferred start codons.



Overexpression of R-transferase Perturbs the Function of N-Recognin: Evidence for a Targeting Complex

Overexpression of R-transferase accelerated the degradation of Gln-gal and Glu-gal, which bear, respectively, an N-d and an N-d residue (Fig. 6A). However, the same overexpression inhibited the degradation of N-end rule substrates bearing a type 1 N-d residue (Fig. 6B). The yeast N-recognin and its mammalian counterparts contain a binding site for type 1 (basic) N-d residues Arg, Lys, and His, and another physically distinct binding site for type 2 (bulky hydrophobic) N-d residues Phe, Leu, Trp, Tyr, and Ile (Reiss et al., 1988; Gonda et al., 1989; Baker and Varshavsky, 1991; Varshavsky, 1992). Overexpression of R-transferase increased by 2-fold the steady state level of His-gal (bearing a type 1 N-d residue), and slightly but reproducibly decreased the level of Tyr-gal (bearing a type 2 N-d residue) (Fig. 6B). Overexpression of Nt-amidase resulted in a slight inhibition of His-gal degradation, whereas overexpression of both Nt-amidase and R-transferase caused a stronger inhibition of His-gal degradation equal to the one observed upon overexpression of R-transferase alone (Fig. 6B). Thus, overexpression of R-transferase interferes with the function of the type 1 binding site in N-recognin, but slightly stimulates its type 2 binding site. The latter effect is consistent with the data indicating that an occupation of the type 1 site in either yeast or mammalian N-recognins with dipeptides bearing a type 1 N-d residue stimulates the activity of the other (type 2) site in N-recognin (Gonda et al., 1989; Baker and Varshavsky, 1991).

These findings (Fig. 6B) are especially illuminating in conjunction with the data suggesting the existence of an Nt-amidaseR-transferase complex (Fig. 6A). Taken together, our results suggest that the 58-kDa R-transferase is physically associated with the 225-kDa N-recognin in proximity to its type 1 binding site. The ``proximity'' aspect of the postulated complex is invoked to account for the markedly different effects of overexpressed R-transferase on the functions of type 1 and type 2 binding sites in N-recognin (Fig. 6B). Specifically, a physical proximity of the bound R-transferase to the type 1 site in N-recognin is presumed to decrease the steric accessibility of this site to an N-end rule substrate bearing a type 1 N-d residue that approaches the type 1 site directly from the bulk solvent. Conversely, a substrate that acquired Arg (a type 1 N-d residue) through the arginylation by N-recognin-bound R-transferase would get access to the (nearby) type 1 binding site of N-recognin in kinetic preference to an otherwise identical substrate that has to reach the type 1 site directly from the bulk solvent.

This model (Fig. 8) postulates an Nt-amidaseR-transferaseN-recognin complex in which the access to the type 1 binding site of N-recognin directly from the bulk solvent may be partially obstructed by the bound Nt-amidaseR-transferase complex. Similarly, the access to the active site of R-transferase from the bulk solvent is presumed to be at least partially obstructed by the bound Nt-amidase. In this view, which accounts for the entire set of otherwise paradoxical interference data in Fig. 6, the spatially distinct type 2 binding site of N-recognin would not be inhibited by the presence of the Nt-amidaseR-transferase complex near the type 1 binding site of N-recognin, thereby explaining the observed dichotomy between the effects of overexpressed R-transferase on the reactions mediated by the type 1 and type 2 binding sites of N-recognin (Fig. 6B). The postulated targeting complex in Fig. 8 includes the Ubc2p Ub-conjugating enzyme, whose physical association with N-recognin was demonstrated directly (Dohmen et al., 1991; Madura et al., 1993).


Figure 8: Model of a targeting complex in the S. cerevisiae N-end rule pathway. The Ub-conjugating enzyme Ubc2p, which has been shown to be physically associated with Ubr1p (N-recognin) (Dohmen et al., 1991; Madura et al., 1993), is depicted carrying activated Ub linked to Cys-88 of Ubc2 through a thioester bond (Sung et al., 1990). In the diagram, Ate1p (R-transferase) reversibly binds to Ubr1p in proximity to the type 1 substrate-binding site of Ubr1p. Furthermore, Nta1p (Nt-amidase) reversibly binds to Ate1p (or possibly to both Ate1p and Ubr1p; not shown). While this model accounts for the apparently paradoxical effects of overexpressed Ate1p (R-transferase) and/or Nta1p (Nt-amidase) on the activity of the N-end rule pathway (see the main text), the postulated targeting complex remains to be demonstrated directly.



N-recognin may partition in vivo between R-transferase-bound and free states. In this view, the free N-recognin would bind substrates bearing either type 1 or type 2 N-d residues directly from the bulk solvent, whereas the R-transferase-bound form of N-recognin would be preferentially accessible to substrates bearing N-d, N-d, or type 2 N-d residues (in comparison to substrates bearing type 1 N-d residues). Substrates bearing N-d or N-d residues would be ``channeled'' to the type 1 binding site of N-recognin after their modification by the N-recognin-bound Nt-amidaseR-transferase complex (Fig. 8). The mechanics of channeling may involve diffusion of an N-end rule substrate in proximity to surfaces of the targeting complex, similar to the channeling mechanism described for the bifunctional enzyme dihydrofolate reductase-thymidylate synthetase, where the channeling of dihydrofolate results from its movement across the surface of the protein (Knighton et al., 1994). Overexpression of R-transferase would partition more of N-recognin into an R-transferase-bound form that is less active toward substrates bearing type 1 N-d residues, resulting in a slower degradation of these substrates, as observed (Fig. 6B).

Common Sequence Motifs in Promoters of Genes That Encode Components of the N-end Rule Pathway

We examined 5` regions of NTA1, ATE1, and UBR1 for common sequence elements and found two of them, 11- and 14-bp long, at different distances from the (inferred) start codons in each of these loci (Fig. 7). The 11-bp sequence TTTCATTGCTA (motif 1) is present in both UBR1 and ATE1; a single-mismatch variant of this sequence is also present in NTA1. Variants of the 14-bp consensus sequence CTTTAATTTCRCAT (motif 2) are also present in NTA1, ATE1, and UBR1 (Fig. 7). No other S. cerevisiae gene in data bases contains both of these motifs, suggesting that these sequences are recognized by transcriptional regulators whose combination is specific for genes encoding targeting components of the N-end rule pathway. The 5` regions of about 15 S. cerevisiae genes in data bases contain one or the other but not both of the two motifs. Three of these genes, UBI1, UBI2, and UBC2, encode components of the Ub system. UBI1 and UBI2 encode identical precursors of Ub (zkaynak et al., 1987). UBC2 (Fig. 7) encodes the Ubc2p Ub-conjugating (E2) enzyme that is physically associated with the UBR1-encoded N-recognin (Dohmen et al., 1991; Madura et al., 1993) (Fig. 8). However, unlike Ubr1p, Ate1p, and Nta1p, which appear to have no functions outside of the N-end rule pathway, Ubc2p has other functions as well, mediated by complexes of Ubc2p with recognins distinct from N-recognin (Sung et al., 1990; Sharon et al., 1991; Ellison et al., 1991).

Concluding Remarks

The NTA1-encoded N-terminal amidase (Nt-amidase) of the yeast S. cerevisiae mediates the conversion of tertiary (Asn or Gln) into secondary (Asp or Glu) destabilizing N-terminal residues in a substrate of the N-end rule pathway. The hierarchical organization of N-end rule, with its tertiary (N-d), secondary (N-d) and primary (N-d) destabilizing residues, is a feature that is more conserved in evolution than the Ub dependence of N-end rule pathways or the precise identity of enzymatic reactions that mediate the hierarchy of destabilizing amino acids in an N-end rule. For example, in bacteria such as E. coli, which lack Ub and Ub-specific enzymes, the N-end rule has both N-d and N-d residues (it lacks N-d residues) (Tobias et al., 1991). However, the identities of N-d residues in E. coli (Arg and Lys) are different from those in eukaryotes (Asp and Glu in S. cerevisiae, Asp, Glu, and Cys in rabbit reticulocytes) (Varshavsky, 1992). Bacterial and eukaryotic enzymes that implement the coupling between N-d and N-d residues are also different: Leu,Phe-tRNA-protein transferase in E. coli and R-transferase in eukaryotes (Shrader et al., 1993; Balzi et al., 1990; Ciechanover et al., 1988). Nonetheless, both bacterial Leu,Phe-tRNA-protein transferase and eukaryotic R-transferase catalyze reactions of the same type (conjugation of an amino acid to an N-terminal residue of a polypeptide) and use the same source of activated amino acid (aminoacyl-tRNA).

Hierarchical organization of the N-end rule ``distributes'' domains that recognize specific destabilizing N-terminal residues among several proteins such as Nt-amidase, R-transferase, and N-recognin. It is likely that cells can produce different N-recognins and can also regulate either synthesis or activity of Nt-amidase and R-transferase. The resulting changes of N-end rule may occur in response to physiologically relevant alterations in the state of a cell, for example, during cell differentiation. A change of the N-end rule may provide a way to destroy a set of previously long-lived proteins or to stabilize a set of previously short-lived proteins. A variety of indirect evidence supports this conjecture (Varshavsky, 1992) but a definitive test remains to be done. Physiological substrates of Nt-amidase and R-transferase remain to be identified as well.

Experiments in which Nt-amidase, R-transferase, or both of these enzymes were overexpressed in S. cerevisiae suggested a substrate channeling in the N-end rule pathway and a specific organization of its multienzyme targeting complex ( Fig. 6and Fig. 8 and above). These ideas are consistent with the presence of two distinct sequence motifs in the promoter regions of genes encoding Nt-amidase, R-transferase, and N-recognin (Fig. 7). No other S. cerevisiae gene in data bases contains both of these motifs, suggesting that they are recognized by transcriptional regulators whose combination is specific for genes encoding targeting components of the N-end rule pathway.


FOOTNOTES

*
This work was supported by Grants DK39520 and GM31530 from the National Institutes of Health (to A. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) L35564.

§
Supported by fellowships from the Fulbright Foundation and the Life Sciences Research Foundation. Present address: Molecular Genetics Group, John Curtin School of Medical Research, Australian National University, P. O. Box 334, Canberra, ACT 2601, Australia.

To whom correspondence should be addressed: Div. of Biology, 147-75, Caltech, Pasadena, CA 91125. Tel.: 818-395-3785; Fax: 818-440-9821; E-mail: varshavskya@starbase1. caltech.edu.

The abbreviations used are: Ub, ubiquitin; N-d, a tertiary destabilizing N-terminal residue; N-d, a secondary destabilizing N-terminal residue; N-d, a primary destabilizing N-terminal residue; Nt-amidase, amidase specific for N-terminal Asn and Gln; R-transferase, Arg-tRNA-protein transferase; gal, E. coli -galactosidase; X-gal, 5-bromo-4-chloro-3-indolyl -D-galactoside; wt, wild-type; kb, kilobase(s); ORF, open reading frame; bp, base pair(s); PAGE, polyacrylamide gel electrophoresis.

R. T. Baker, S. Grigoryev, and A. Varshavsky, unpublished data.

S. Grigoryev, A. Stewart, S. Arfin, R. Bradshaw, and A. Varshavsky, unpublished data.

C. Byrd and A. Varshavsky, unpublished data.


ACKNOWLEDGEMENTS

We thank S. Grigoryev for his advice about the in vitro deamidation assay and B. Bartel, M. Hochstrasser, and K. Madura for helpful discussions.


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