From the
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.
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)
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
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.
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-
Plasmids expressing S.
cerevisiae Ate1p either alone or together with Nta1p were
constructed as follows. The
We did not
detect a significant difference between the S. cerevisiae
nta1-
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.
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-
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).
In a biochemical test, purified,
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,
No significant phenotypic differences
(other than the metabolic stabilization of Asn-
This model accounts for the observed stabilization
of Gln-
These findings (Fig. 6B) are especially
illuminating in conjunction with the data suggesting the existence of
an Nt-amidase
This model
(Fig. 8) postulates an
Nt-amidase
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.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
We thank S. Grigoryev for his advice about the in
vitro deamidation assay and B. Bartel, M. Hochstrasser, and K.
Madura for helpful discussions.
(
)
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).
) 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).
Strains, Genetic Techniques, and
S. cerevisiae strains were BWG1 7a
(MAT
a his4 ura3 ade1 leu2) (Guarente et
al., 1982), BWG9a-1 (MAT-Galactosidase
Assay
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
K
HPO
-KH
PO
(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 DH5
F` (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
Tran
S-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-
To construct
nta1-1 Allele
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).
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.
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-
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.
1
Mutant
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.''
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).
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).
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.
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.
(
)
is highly
similar to the sequence of porcine Nt-amidase but lacks similarities to
the sequence of yeast Nta1p.
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-amidase
R-transferase complex. Specifically, the
R-transferase-mediated arginylation of Glu-
gal that has been
produced from Gln-
gal by the Nt-amidase
R-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).
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-amidase
R-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).
R-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.
R-transferase
N-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-amidase
R-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-amidase
R-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-amidase
R-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).
/EMBL Data Bank with accession number(s) L35564.
, 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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.