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INTRODUCTION |
It is generally thought that the
degree of conservation of genes and their respective proteins through
species evolution is a measure of their importance for the survival of
an organism. In fact, most highly conserved proteins, such as Hsp70,
are essential in most organisms (14). We recently identified
the Snz1 protein in Saccharomyces cerevisiae, which is the
most highly conserved protein present in all three phylogenetic
domains, exhibiting 60% identity with Snz proteins in archaea and
bacteria (4). Because of the high degree of conservation of
Snz1p, it was surprising to us that snz1 deletion mutants
exhibited no defects in viability or growth rate under a variety of
laboratory conditions.
Although Snz proteins are found in bacteria, archaea, and eucarya
including plants and fungi, very little is understood about the
specific function of these proteins. Snz proteins contain no distinct
functional motifs, although they exhibit very distant relationships
with proteins involved in amino acid, vitamin, and nucleic acid
biosynthesis, such as bacterial HisF, TrpC, and ThiG, and appear to
contain a phosphate-binding region (10). Nevertheless, Snz-related proteins do appear to have a role in stress responses. For
example, in the rubber tree Hevea brasiliensis, an
SNZ-related gene is induced in response to ethylene and
salicylic acid (31). In Bacillus subtilis, a
Snz homologue is guanylated during sporulation (23) and is induced in response to oxygen stress
(2). In the ascomycete Cercospora nicotianae,
Snz1-related protein Sor1 is required for resistance to singlet
oxygen-generating photosensitizers (7).
We initially identified Snz1p as a protein whose synthesis increases
dramatically as yeast cells enter stationary phase (9). There are three SNZ-related genes in yeast, SNZ1,
SNZ2, and SNZ3. Each of these genes is found
adjacent to another conserved gene family we have named SNO
(Snz-proximal open reading frame [ORF]), recently described as snzB
(10). Although Sno proteins, like Snz proteins, have an
unknown function, they show some sequence similarity to glutamine
amidotransferases (10).
The high degree of conservation of Snz and Sno proteins, the
conservation of their proximal chromosome location, and the unique pattern of expression of SNZ1 compelled us to investigate
the regulation and function of these gene families and their encoded proteins. We report here that adjacent SNZ and
SNO genes are coregulated during growth to stationary phase
and during nutrient starvation; that Snz1p and Au Sno1p interact, as
determined by a two-hybrid analysis; and that expression of
SNZ1 is repressed by expression of SNZ2 and
SNZ3 during growth to stationary phase. We have determined that snz1 and sno1 mutants are sensitive to
6-azauracil (6-AU), an inhibitor of purine and pyrimidine biosynthesis
(15), and methylene blue, a producer of singlet oxygen. Our
results support the hypotheses that SNZ- and
SNO-related genes have been linked through evolutionary time
and that they are involved in an ancient response to nutrient
limitation.
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MATERIALS AND METHODS |
Strains and media.
The following media were used to
cultivate yeast: YPD (1% yeast extract, 2% peptone, 2% glucose),
synthetic complete (SC) medium (0.67% Bacto-yeast nitrogen base
without amino acids [Difco], 2% glucose; supplemented with
auxotrophic requirements but lacking the amino acids for which one is
selecting, unless indicated otherwise) (27), and
nonsupplemented YNB (2% glucose, 0.17% Bacto-yeast nitrogen base
without amino acids and ammonium sulfate [Difco], 16.7% succinate
buffer) (18). When indicated (see Results), supplements were
added to the YNB media (supplemented YNB) to the following final
concentrations: adenine, 0.06 mg/ml; uracil, tryptophan, and histidine,
0.02 mg/ml; and leucine, 0.03 mg/ml. Solid media contained 2% agar.
For each liquid-culture experiment, yeast cells were shaken at 250 rpm
in 100 ml of medium at 30°C for the time indicated (see Results).
Yeast strains are listed in Table 1. Most
of the strains produced for this study were derived from the common
laboratory strains W303-1A (MW644) and W303-1B (MW647) (32).
All yeast transformations were performed by the lithium acetate
protocol or the quick-colony method (11). Transformants were
selected on SC media lacking only the auxotrophic requirement used in
the selection.
To obtain strains with single tryptophan (MW1128), histidine (MW1201),
uracil (MW1207), or adenine (MW1203) auxotrophies, PCR fragments
containing the appropriate wild-type genes were obtained from common
laboratory strain S288C (MW481) (25) and introduced by
linear transformation into a W303-1 strain (Table 1). To obtain strains
that are prototrophic for tryptophan, W303-1 strains were transformed
with the YCplac22 CEN plasmid (12). Escherichia coli XL2-Blue cells were used for propagation of
all plasmids and were cultured in Luria broth with ampicillin according to the manufacturer's recommendations (Stratagene).
Mutant construction.
The snz2-1 and
snz3-1 disruption alleles were constructed by inserting a
1.8-kb KpnI LEU2 fragment, generated by PCR from YCplac181 (12), into SNZ2 and a 1.4-kb
KpnI TRP1 fragment, generated by PCR from
YCplac22 (12), into the KpnI site of
SNZ3 (Fig. 1A). Chromosome
blot analysis and Southern blot analysis were used to confirm the
disruption of SNZ2 and SNZ3.
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FIG. 1.
SNZ and SNO deletion and
disruption mutations. (A) Disruption of SNZ2 and SNZ3.
SNZ2 was disrupted with a 1.8-kb LEU2 fragment cloned
into the KpnI site, and SNZ3 was disrupted with a
1.1-kb TRP1 fragment cloned into the KpnI site.
The resulting alleles are snz2-1 and snz3-1. (B)
SNZ2 and -3 and SNO2 and -3
deletion by insertion of a 1.8-kbp LEU2 fragment. The
resulting alleles are snz2 3sno23 and
snz33sno33. (C) SNZ1 deletion by insertion
of a 1.1-kbp URA3 fragment. The resulting allele is
snz12. (D) SNZ1 and SNO1 deletion
and insertion of a 1.1-kbp URA3 fragment. The resulting
allele is snz13sno13. (E) SNO1 disrupted
with the URA3 fragment carried on transposon mTn-4x. The
resulting allele is sno1-1.
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The sno23snz23 sno33snz33 strain was constructed
by inserting a 1.8-kb SalI LEU2 fragment
generated by PCR from YCplac181 (12) into the
SalI sites in SNO3 and SNZ3,
respectively. This resulted in the deletion of the SNZ2 and
-3 and SNO2 and -3 promoter region as
well as 178 bp of the SNO2 and -3 coding region
and 575 bp of the SNZ2 and -3 coding region (Fig.
1B). The deletions were confirmed by Southern analysis. The
snz13 sno13 strain was produced by outward-directed
PCR of the snz11 construct from the pWFY12 plasmid
(4). This modification resulted in the deletion of 751 bp of
the SNZ1 coding region and 159 bp of the SNO1
coding region as well as the 450 bp between the two genes (Fig. 1D). These strains were mated with sno23snz23
sno33snz33 strain, and haploid segregants from tetrads were
isolated to obtain the snz, sno sextuple mutant (MW980). The
construction of the snz12 strain (Fig. 1C) has been
previously described (4).
The heterozygous diploid sno1-1/SNO1 strain, carrying
sno1 disrupted by URA3 at amino acid 139 (of 224)
(Fig. 1E), and a control ssa4/SSA4 strain (MW1434) were
obtained from Mike Snyder (28). Conventional dissection
techniques were used to obtain the haploid mutant strains.
Chromosome analysis.
Yeast chromosomes were isolated and
separated according to protocols for the Bio-Rad CHEF-DR II gel
apparatus. Gels were blotted to GeneScreen membranes (NEN) and probed
with either an SNZ1 or an SNZ3 probe, labeled
with 32P by using random primers (Pharmacia).
Analysis of mRNA accumulation.
Total RNA was prepared with
the Purescript RNA isolation kit (Gentra Systems Inc.), except that
glass beads were used to lyse the cells. Briefly, the cells were
vortexed with the glass beads for 30 s and put on ice for an
additional 30 s, a cycle which was repeated three times. This
produced a better yield of total RNA, especially for stationary-phase
cells. Electrophoresis, hybridizations, and digitizing of
autoradiograms were performed as previously described (4).
Two-hybrid analysis.
Two-hybrid analysis was carried out by
using genomic GAL4 activation domain libraries YL2H-C1, -C2,
and -C3 and yeast host strain PJ69-4A (17). This experiment
was performed once, so it was not an exhaustive search. Amplification
of the libraries, resulting in greater than 20 million transformants,
was achieved with electrocompetent cells (Gibco BRL) and the Gibco BRL
electroporator. The transformants were pooled and inoculated into 3 liters of Terrific Broth medium and grown at 30°C to an optical
density (OD) of 1.1. (17). Plasmid DNA was isolated with
Maxiprep columns (Qiagen). Plasmids (100 ng) from the three activation
domain libraries were separately transformed (17) into
PJ69-4A harboring the GAL4-binding domain plasmid, pGBT9
(Clontech), into which the SNZ1 ORF had been subcloned in
frame.
Transformants were selected on SC medium lacking leucine and tryptophan
(to maintain plasmids) and containing histidine-2 mM 3-aminotriazole
(to test the two-hybrid interaction). In PJ69-4A, under these
conditions, the auxotrophic requirements can be met only when the
Gal4-Snz1 fusion proteins interact and activate transcription of the
GAL1-HIS3 reporter gene (17). Two additional reporter genes in PJ69-4A, GAL2-ADE2 and
GAL4-lacZ, were used to confirm two-hybrid interacting
candidates by screening for growth on medium lacking adenine and by
determining 
-galactosidase activity. -Galactosidase assays were
performed as described by Miller (22). Plasmids were
isolated from cells that showed positive two-hybrid interactions and
were retransformed into E. coli DH10B cells. The DNA of
isolated plasmids was sequenced (Amersham; Thermosequenase) and further
analyzed by using DNASIS 2.0 software (Hitachi) and the National Center
for Biotechnology Information Web site
(http://www.ncbi.n.lm.nih.gov/BLAST/). Positive controls,
i.e., proteins known to interact, p53 and simian virus 40 large T
antigen, were obtained from Clontech.
Analysis of the Snz1p/Sno1p complex by nondenaturing gel
electrophoresis.
Cell pellets (20 OD at 600 nm
[OD600] units) were collected from 8-day stationary-phase
cultures and resuspended in radioimmunoprecipitation assay lysis buffer
(0.1% sodium dodecyl sulfate, 1% Nonidet P-40, 0.5% deoxycholate,
150 mM NaCl, 50 mM TRIS, pH 8) containing complete protease inhibitors
(Boehringer Mannheim). Acid-washed glass beads were added to the cells,
and the suspension was vortexed for 10 min at 4°C. After
centrifugation in a microcentrifuge for 10 min at 22,000 × g, the supernatant was collected and assayed with bicinchoninic acid (Pierce) to determine protein concentration. Approximately 20 µg of protein was loaded per lane and separated (100 V, 20 h) on a 4-to-20% nondenaturing polyacrylamide gel
electrophoresis precast gel (FMC), along with native
high-molecular-weight standards (HMW calibration kit proteins;
Pharmacia), to determine the size of the complex. After
electrophoresis, proteins were blotted onto an Immobilon-P membrane
(Millipore) and probed with anti-Snz1,2,3p antibody (rabbit polyclonal
antibody; generated to the common N-terminal peptide sequence
ALESIPADMRKSGKVC) (QCB). The blot was dried and probed according to
Millipore's rapid chemiluminescence protocol. A peroxidase-conjugated
anti-rabbit secondary antibody (Amersham) was used to detect the
anti-Snz antibody bound to the blotted protein. The blot was then
developed with BLAZE SuperSignal chemiluminescent detection reagent
(Pierce). After detection, the blot was stained for total proteins with
GelCode Blue (Pierce).
6-AU, methylene blue, and MPA sensitivities.
6-AU
sensitivity was evaluated on SC medium lacking uracil and supplemented
with 6-AU to a final concentration of 30.0 µg/ml (15).
Mycophenolic acid (MPA) sensitivity was scored on SC medium lacking
adenine, guanine, and uracil and supplemented with MPA at a final
concentration of 30.0 µg/ml (15). When indicated (see
Results) uracil was added to the SC medium to a final concentration of
30.0 µg/ml. Strains were incubated at 30°C for 2 days.
To evaluate methylene blue sensitivity, yeast strains were grown
overnight in YPD and the growth medium was diluted to a final OD600 of 0.25/ml and then diluted by 1/5 for a series of
five dilutions. Then, 6 µl from each of these dilutions was plated onto YPD medium, supplemented with 37.0 µg of methylene blue per ml,
and exposed to a light source, when indicated (see Results), for 3 days
at 25°C (7).
Sequence analysis.
Sequences were retrieved from databases
by using the National Center for Biotechnology Information Web site
(http://www.ncbi.nlm.nih.gov/BLAST/). Identification of
SNZ and SNO sequences was carried out with BLAST and BLAST2 software (1a). Genome sequences examined were
those for S. cerevisiae Sno1p (Swiss-Prot Q03144), Sno2p
(Swiss-Prot P53823), and Sno3p (Swiss-Prot P43544); B. subtilis SnzP (Swiss-Prot P37527) and SnoP (Swiss-Prot P37528); Haemophilus influenzae SnzP (Swiss-Prot P45293) and SnoP (Swiss-Prot P45294); Mycobacterium tuberculosis SnzP
(Swiss-Prot O06208) and SnoP (Swiss-Prot e1299817); Methanococcus
jannaschii SnzP (Swiss-Prot Q58090) and SnoP (Swiss-Prot Q59055); Pyrococcus horikoshii SnzP (DDBJ d1028463) and SnoP (DDBJ
d1028462); Mycobacterium leprae SnzP (Swiss-Prot O07145) and
SnoP (Swiss-Prot S72721); Methanobacterium
thermoautotrophicum SnzP (Swiss-Prot O26762) and SnoP (GenBank
2621878); and Archaeoglobus fulgidus SnzP (Swiss-Prot
O29742) and SnoP (GenBank 2650108). Multiple sequence alignments were
made with CLUSTAL W (32a). Percent identity values
correspond to the percentages of identical amino acids in multiple
sequence alignments, calculated with the ProtST program (1).
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RESULTS |
The SNZ family in yeast.
SNZ1 was previously
identified, based on Southern hybridization and sequence analysis, as a
member of a multigene family in yeast (4). The presence of
two additional SNZ1 homologues, SNZ2 and
SNZ3, was confirmed after publication of the entire yeast genome (13). SNZ1 is situated proximal to the
centromere on the right arm of chromosome XIII. SNZ2 and
SNZ3 are located in the telomeric regions on the left arms
of chromosomes XIV and VI, respectively, within a 7-kb region that is
nearly identical between these two chromosomes. This duplicated region
is not observed in the telomeric regions of other yeast chromosomes
(13). The Snz2p sequence is approximately 99% identical to
that of Snz3p and approximately 80% identical to that of Snz1p.
Duplicated genes are often present in variable numbers even in closely
related yeast strains (5, 6, 24). To determine whether this
was also true for SNZ genes, we assessed the numbers of
SNZ genes in several laboratory yeast strains by Southern
hybridization of chromosome blots. All of the strains we examined
carried a single copy of a gene closely related to SNZ1 but
carried variable numbers of SNZ2 and -3 genes.
S288C (25), W303 (32), and YPH (30)
contain a single SNZ1 homologue and two genes more closely related to SNZ2 and -3 (data not shown). At least
one strain, 
1278, which grows pseudohyphally (19),
contains a single copy of SNZ1 and does not contain genes
related to SNZ2 or SNZ3. Finally, DS10, which is
derived from S288C (34), contains a fourth gene related to
SNZ2 and -3 on chromosome II. Although we do not
know the chromosomal position of this fourth SNZ gene, its
similarity to SNZ2 and SNZ3 suggests that it may
have arisen from gene duplication in the telomeric region.
SNZ genes are adjacent to members of a second highly
conserved gene family, the SNO genes.
An analysis of
the sequences adjacent to the yeast SNZ genes revealed an
additional conserved, duplicated gene upstream of each SNZ
gene, which we called SNO (for SNZ-proximal ORF).
The presence of SNO2 and SNO3 genes adjacent to
SNZ2 and SNZ3 was expected because of the
chromosomal duplication that included the entire region. The presence
of the SNO1 gene adjacent to the more centromeric
SNZ1 was surprising and suggested that the original duplication of SNZ1 also included the entire SNO1
gene. Like SNZ2 and SNZ3 genes, SNO2
and SNO3 encode proteins that are almost 100% identical to
each other and that are approximately 72% identical to Sno1p. The Sno
proteins are predicted to have a molecular mass of 21.5 kDa.
It was recently reported that the Sno proteins are conserved in all
three phylogenetic domains (10). Our comparison of the predicted yeast Sno1 protein sequence to the protein sequences of Sno1p
homologues extends this observation (Table
2). Yeast Sno1p is 40% identical to the
B. subtilis homologue (SnzB) and 37% identical to the
M. jannaschii homologue. The M. jannaschii and
B. subtilis (SnzB) proteins are 42% identical. Overall, the Sno proteins are less conserved than other highly conserved proteins such as the Hsp70 and Snz proteins (Table 2), suggesting that there are
fewer constraints on the structure and sequence of Sno proteins.
On the basis of the microbial genomes that have been entirely
sequenced, all species that contain an SNZ gene also contain a SNO gene. In organisms for which the complete genome
sequences are available, the proximal location of the SNZ
and SNO genes is relatively conserved but the orientations
of the two genes differ between eukaryotes and prokaryotes. In yeast,
SNZ and SNO genes are adjacent and divergently
transcribed (this work and reference 10), whereas in
the bacteria B. subtilis, H. influenzae, and
M. leprae the SNZ and SNO homologues
are in apparent operons (10), i.e., they are adjacent and
transcribed in the same direction. In the bacterium M. tuberculosis, SNZ and SNO are separated by a
single gene, whose product has some homology to thioesterases. In the
archaea P. horikoshii and A. fulgidus SNZ and
SNO are also in an apparent operon. These observations
suggest that there has been strong selective pressure for the retention
of the proximal location of SNZ and SNO genes
through evolution. M. jannaschii (10) and
M. thermoautotrophicum are the two exceptions; for them the
chromosomal location is not conserved between SNZ and SNO genes.
SNZ and SNO gene expression during growth
to stationary phase.
We previously reported that SNZ1
was induced at low levels during the postdiauxic phase and induced 8- to 10-fold in stationary phase (4). Similar patterns of
SNZ1 mRNA accumulation in other laboratory strains have been
observed, indicating that the unique regulation of SNZ1 is
not strain dependent (data not shown).
Northern analysis of total RNA was used to assay SNZ2 and
SNZ3 mRNA accumulation during growth to stationary phase
(Fig. 2A). We refer to these mRNAs as
SNZ2/3 because the close identity between SNZ2
and SNZ3 does not allow distinction between their mRNAs. The
SNZ2/3 pattern of expression differs noticeably from that of
SNZ1 in cells grown to stationary phase (Fig. 2A).
SNZ2/3 mRNAs accumulate slightly before the diauxic shift,
decrease in abundance at the diauxic shift, and increase for a short
period after the diauxic shift. Unlike SNZ1 mRNA,
SNZ2/3 mRNAs are not detectable in Northern analysis of
total RNA from stationary-phase cells. rRNAs are used as a control for
loading because most mRNAs, e.g., that for actin, decrease in abundance
in stationary phase and we currently have no RNA that remains constant
in both exponential and stationary phases and that therefore could be
used as an internal reference. For these analyses, we have used
SNZ1 and BCY1 as controls in some blots to allow
us to demonstrate the intactness of mRNAs in cells grown to stationary
phase (Fig. 2), because the mRNA levels of both SNZ1 and
BCY1 in cells grown to stationary phase are known
(4).
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FIG. 2.
Northern analysis of SNZ and SNO
gene expression in yeast cells grown to stationary phase. (A) Northern
blot probed with SNZ3, SNO3, and SNZ1
to show the relative timing of SNZ2 and -3 and
SNO2 and -3 expression relative to that of
SNZ1. Ethidium bromide-stained rRNAs are presented to show
relative loading. Lanes: 1, early exponential phase (OD600 = 0.95); 2, late exponential phase (OD600 = 5.0); 3, diauxic shift (OD600 = 7.1; determined by glucose
exhaustion); 4, 24 h after the diauxic shift (OD600 = 10.5); 5, stationary phase (5 days after inoculation); 6, stationary
phase (8 days after inoculation). (B) Northern blot probed with
SNO1, SNZ1, and BCY1 to show the
timing of SNO1 expression relative to that of
SNZ1. Lanes: 1, early exponential phase (OD600 = 1.4); 2, late exponential phase (OD600 = 6.6); 3, diauxic
shift (OD600 = 7.1); 4, 22 h after the diauxic shift
(OD600 = 10.5); 5, 48 h after the diauxic shift
(OD600 = 19.6); 6, stationary phase (5 days after
inoculation); 7, stationary phase (8 days after inoculation).
BCY1 was used as a control. Autoradiographs were exposed for
3 days (SNZ2 and -3, SNO2 and
-3, BCY1, and SNZ1) or 5 days
(SNO1).
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The pattern of SNZ1 expression during growth to stationary
phase is altered dramatically in snz2,3 disruption mutants
(Fig. 3). In snz2,3 mutants,
SNZ1 mRNA levels increase during late exponential phase,
decrease at the diauxic shift and shortly thereafter, increase again
24 h after the diauxic shift, and remain high in stationary phase.
This novel pattern of SNZ1 mRNA accumulation prior to the diauxic shift in snz2,3 mutants is similar to that of
SNZ2/3 mRNA accumulation in control strains. These results
suggest that SNZ2 and -3 directly or indirectly
regulate SNZ1 expression. SNZ2/3 mRNA
accumulation is not altered in an snz12 mutant grown to stationary phase (data not shown), which indicates that the converse regulation of SNZ2 and -3 by SNZ1 does
not occur.
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FIG. 3.
Northern analysis of SNZ1 expression in an
snz2 snz3 mutant during growth to stationary phase. The
Northern blot was probed with SNZ1 to show the relative
timing of SNZ1 expression in an snz2-1 snz3-1
strain. Ethidium bromide-stained rRNAs are presented to show relative
loading. Lanes: 1, early exponential phase (OD600 = 3.7);
2, late exponential phase (OD600 = 5.7); 3, diauxic shift
(determined by glucose exhaustion) (OD600 = 6.2); 4, 2 h after the diauxic shift (OD600 = 7.0); 5, 24 h after
the diauxic shift; 6, 48 h after the diauxic shift; 7, stationary
phase (5 days after inoculation); 8, stationary phase (8 days after
inoculation). BCY1 was used as a control. Autoradiographs
were exposed for 4 days (BCY1) or 2 days
(SNZ1).
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All three SNZ-SNO gene pairs in yeast are divergently
transcribed and are separated by 400 to 450 bp. Because the positions of the SNZ and SNO genes are conserved during
evolution and because their relative orientations in yeast suggested
that they share common promoter elements, we wanted to determine
whether they were coregulated. Northern analysis of total RNAs revealed
that SNO1 and SNZ1 mRNAs do exhibit the same
pattern of expression in cells grown to stationary phase (Fig. 2B).
Likewise, SNO2/3 mRNAs, which are also indistinguishable
from each other by Northern analysis, accumulate at the same time as
SNZ2/3 mRNAs (Fig. 2A). We concluded from this that the
SNZ-SNO gene pairs are coregulated and that the ability to
coregulate these genes might have been an important factor in
maintaining their proximity and orientations during evolution.
Snz and Sno proteins interact.
In order to determine if Snz
proteins interact as a higher-order complex, we analyzed extracts of
wild-type and various snz mutants via nondenaturing gradient
polyacrylamide gel electrophoresis. This method involves running
samples on a 4-to-20% acrylamide gel for 20 h through a matrix of
decreasing pore size until proteins stop at an impassable acrylamide
percentage. Because of the long run, proteins that migrate slowly due
to low charge still run to the limiting pore size of the gel. A
protein's migration on these gels is based solely on shape and size,
allowing specific size comparisons, as opposed to that of proteins
analyzed by continuous nondenaturing electrophoresis, where protein
migration is based on size, charge, and shape (21). The
analysis revealed that Snz proteins are part of a complex, in
stationary-phase cells, with an apparent molecular mass of
approximately 230 kDa (Fig. 4). The
antibody to the common N-terminal peptide that was produced is capable
of recognizing all three Snz proteins in yeast (data not shown).
However, the complex is only present when Snz1p is present (Fig. 4).
Thus, we conclude that Snz1p is part of a protein complex in
stationary-phase cells.
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FIG. 4.
Western analysis of Snz proteins separated by
nondenaturing polyacrylamide gel electrophoresis. Proteins were
isolated, seperated, and blotted as described in Materials and Methods.
Snz proteins were visualized by using a rabbit polyclonal antibody to
the N termini of Snz proteins and a secondary anti-rabbit horseradish
peroxidase-conjugated antibody in combination with a chemiluminescence
assay (Pierce) as described in Materials and Methods. The arrow
indicates the Snz-reactive band. Lanes (from left to right): early
exponential phase (OD600 = 2.7; wild-type strain
[MW1072]); late exponential phase (OD600 = 7.2; wild-type
strain [MW1072]); stationary phase, wild-type strain (MW1072);
stationary phase, sextuple mutant (MW983); stationary phase,
snz2/33 sno2/33 mutant (MW 976); stationary phase,
snz13 sno13 mutant (MW908).
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To learn more about the Snz1p complex, we used two-hybrid analysis to
identify proteins that might interact with Snz1p (8). We
first examined whether Snz1p could interact with itself by cloning the
SNZ1 ORF in frame with the GAL4-binding domain
(GAL4-bd) and GAL4 activation domain
(GAL4-ad). By themselves, the Gal4-Snz1 activation or
binding domain fusion proteins did not activate GAL7-lacZ
(Table 3) or allow growth on media
lacking histidine or adenine. When cotransformed into yeast cells, the
activation and binding domain fusion proteins activated the
transcription of a GAL7-lacZ reporter gene (Table 3) and
allowed strain PJ69-4A, which is conditionally auxotrophic for
histidine and adenine, to grow in the absence of these supplements.
These results suggest that Snz1p could function in the cell as a dimer
or higher-order oligomer.
To identify other proteins that interact with Snz1p, yeast cells were
transformed with the GAL4-bd-SNZ1 vector and a yeast GAL4-ad library (17). Plasmids from cells that
grew on medium lacking histidine were isolated for further study. The
results of this screen yielded two plasmids with activating genes
SNZ2 and SNO1 (Table 3). The plasmid containing
SNO1 includes all of the ORF except for the first 40 nucleotides. The plasmid containing SNZ2 includes more than
three-fourths of the coding region. Based on -galactosidase assays,
the interaction between Snz1p and Snz2p is not as strong as the
interaction between Snz1p and Snz1p. However, the interactions between
Snz1p and Sno1p and between Snz1p and Snz1p are equally strong. These
results suggest that Snz1p and Sno1p may function in the cell as an
oligomeric complex. The apparent physical interaction between Snz1p and
Sno1p, the coregulation of their genes, and their conserved proximity
on the chromosome provide strong support for the hypothesis that these
two proteins are involved in the same pathway.
SNZ1 is induced in response to starvation for specific
nutrients.
The different patterns of SNZ1 and
SNZ2/3 mRNA accumulation during growth to stationary phase
are likely to reflect a response to the changing nutritional
environment. To determine whether limitation for specific nutrients
affects the regulation of SNZ genes, auxotrophic cells
(MW644) that have all wild-type SNZ and SNO genes
were transferred from rich, glucose-based medium to nonsupplemented,
nitrogen-limiting medium (YNB medium). Northern analysis revealed that
SNZ1 and SNO1 mRNAs accumulate in cells transferred to nonsupplemented YNB medium whereas SNZ2/3 and
SNO2/3 mRNAs did not (Fig. 5).
These results indicate that under specific starvation conditions as
well as in cells grown to stationary phase, SNZ and
SNO genes are coregulated and that SNZ1 and
SNZ2 and -3 are controlled by distinct regulatory
mechanisms.
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FIG. 5.
Northern analysis of SNZ and SNO
mRNA accumulation in nitrogen-starved cells. Total RNA was isolated
from yeast cells (MW644) grown overnight in YPD medium to an
OD600 of 2.06 (YPD lanes). YNB-labeled lanes show RNA from
yeast cells transferred from YPD to YNB medium for 90 min. Ethidium
bromide-stained rRNAs are shown to indicate relative loading. (A)
Northern blot probed with SNZ3 and SNZ1. (B)
Northern blot probed with SNO3 and SNO1. Northern
blots were probed with ACT1 (actin gene) as a control.
Autoradiographs were exposed for 1 day.
|
|
To determine whether the accumulation of SNZ1 mRNA in cells
incubated in YNB was due to starvation for specific nutrients or a
general nitrogen starvation signal, we examined SNZ1
expression in auxotrophic, W303-derived cells (MW644) transferred from
rich, glucose-based medium to YNB medium supplemented with auxotrophic requirements (Fig. 6). Under these
conditions, SNZ1 mRNA does not accumulate, suggesting that
SNZ1 induction is not due to general nitrogen limitation but
to the absence of one or more auxotrophic requirements.
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FIG. 6.
Northern analysis of SNZ1 expression in
auxotrophic and prototrophic strains in the presence or absence of
auxotrophic requirements. A Northern blot of total RNA isolated from
cells grown overnight in YPD medium to an OD600 of 2.0 to
3.0 and from cells transferred from YPD to YNB medium for 90 min is
shown. The blot was probed with SNZ1. Auxotrophic strains
were incubated with (+) or without ( ) their specific auxotrophic
requirements. Genotypes of the strains are as follows: leu2-3,112
trp1-1 ura3-1 ade2-1 his3-11,15 can1-100 (MW644);
trp1-1 (MW1128); his3-11,15 (MW1201);
ade2-1 (MW1203); ura3-1 (MW1207); and
can1-100 (MW1199). Ethidium bromide-stained rRNAs are shown
to indicate loading. The Northern blot was probed with ACT1
as a control. The autoradiograph was exposed for 1 day.
|
|
To identify the nutrient limitation responsible for the dramatic
increase in SNZ1 expression, we evaluated SNZ1
mRNA accumulation in W303-derived strains that are auxotrophic for a
single nutrient (Table 1 and Fig. 6). SNZ1 mRNA accumulated
in the trp1-1 (MW1128), ade2-1 (MW1203), and
ura3-1 (MW1207) mutants in YNB medium but did not accumulate
in the his3-11 (MW1201) or the prototrophic (MW1199) strains
incubated in YNB medium (Fig. 6). SNZ1 mRNA did not
accumulate when tryptophan was added to YNB medium in which the
trp1-1 mutant (MW1128) was incubated. SNZ1 mRNA
accumulation in leu2-3,112 trp1-1 ura3-1 ade2-1 his3-11,15
can1-100 (MW644) cells incubated in YNB medium with supplements
was suppressed with the addition of uracil and adenine. Surprisingly,
the SNZ1 mRNA accumulation in ade2-1 (MW1203) and
ura3-1 (MW1207) mutants incubated in YNB medium with
supplements was not suppressed (Fig. 6). These results indicate that an
imbalance between nucleotide levels could have an effect on
SNZ1 mRNA accumulation. The difference in SNZ1
mRNA accumulation in the leu2-3,112 trp1-1 ura3-1 ade2-1 his3-11,15 can1-100 (MW644) cells versus the ura3-1
(MW1207) or ade2-1 (MW1203) cells incubated in YNB medium
with supplements cannot be directly determined because of the
additional auxotrophies in the MW644 cells which could affect the
SNZ1 mRNA levels.
Strains derived from another common laboratory strain, S288C, were
examined to determine whether SNZ1 induction in YNB medium was a strain-specific phenomenon. As in W303-derived strains, SNZ1 is induced when S288C strains with multiple
auxotrophies, e.g., MW751 (his3200 leu21 lys2202
trp163 ura3-52), are incubated in YNB medium but is not induced
in a prototrophic strain (MW481) (data not shown). We conclude from
these results that SNZ1 induction in response to limitation
of specific nutrients is a function of the auxotrophies of a given
strain and is not a function of strain background.
snz and sno mutant sensitivity to
6-AU.
To further investigate Snz and Sno protein function, we
evaluated the phenotypes of snz and sno mutants.
Yeast strains carrying snz1, sno1, snz2,3
sno2,3, and snz1,2,3 sno1,2,3 mutations are viable and
grow at rates indistinguishable from those of wild-type cells on rich,
glucose-based medium (data not shown). To determine if Snz and Sno
proteins were essential under other conditions, we tested these mutants
for their ability to survive and grow under a variety of different
medium and temperature regimes. None of the snz sno mutants
exhibited changes in growth rates or viability during growth to
stationary phase on glucose medium, acetate-based medium, or
nitrogen-limiting medium (YNB medium) at 30 and 37°C, compared with
control cells.
Since SNZ1 mRNA accumulates in ura3 and
ade2 mutants starved for uracil and adenine, we tested
snz1, sno1, snz2,3 sno2,3, and
snz1,2,3 sno1,2,3 mutants for sensitivity to 6-AU, an
inhibitor of both pyrimidine and purine biosynthesis. 6-AU inhibits IMP dehydrogenase, encoded by PUR5, and OMP decarboxylase,
encoded by URA3 (15). Strains carrying any of the
snz1 mutant alleles (Fig. 1), including snz12
and snz13 sno13, and strains carrying sno1
mutations are extremely sensitive to 6-AU (Fig.
7A). The growth inhibition is specific to
strains carrying snz1 or sno1 mutations, and is
not observed with SNZ1 snz2,3 sno2,3 mutants or wild-type
controls (Fig. 7A). The 6-AU sensitivity cosegregates with the
snz1 mutation through numerous crosses in both S288C and
W303 strain backgrounds.
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FIG. 7.
Phenotype analysis of snz and sno
mutants. (A) Growth of snz and sno mutants and
control strains on minimal medium without uracil, with or without 6-AU.
A control is shown for each mutant strain; the control has the same
auxotrophic markers as the mutant strain. Strains are as follows:
control A, MW740; snz12 strain, MW926;
snz13sno13 strain, MW908; snz12 YCplac19
strain, MW1435; control B MW1071; sextuple snz1,2,33
sno1,2,33 mutant strain, MW980; control C, MW1283;
snz2,33 sno2,33 strain, MW1286; control D, MW1434;
heterozygous sno1-1/SNO1 strain, MW1359; sno1-1
strain, MW1427. (B) Growth of snz and sno mutants
and control strains on YPD medium supplemented with methylene blue, in
the presence or absence of light. Strains are as follows: control
strain, MW740; snz12 strain, MW926; sno1-1
strain, MW1427; snz13 sno13 strain, MW908;
heterozygous diploid sno1-1/SNO1 strain, MW1359.
|
|
The addition of uracil to the medium or the introduction of
URA3 on a 2µm plasmid suppresses the 6-AU growth
inhibition of snz1 strains (data not shown). However, the
introduction of the centromeric plasmid YCplac19 (URA3 TRP1)
to the snz12 mutant does not suppress the 6-AU
sensitivity (Fig. 7A). Thus, one copy or a few copies of plasmid-borne
URA3 are not sufficient to suppress the 6-AU sensitivity of
snz1 mutant strains. However, snz12 mutants were not complemented by mating to a SNZ1 ura3 strain or by
transformation with a CEN plasmid carrying the
SNZ1 structural gene and 953 bp of the region upstream of
the start codon. This data suggests that the snz12 mutant
allele may cause a dominant-negative effect or an imbalance between
Snz1p and Sno1p resulting in a mutant phenotype under these conditions
or that the snz12 mutant allele results in an alteration
of SNO1 expression.
We examined complementation by comparison of sno1-1/SNO1
heterozygous diploid and sno1-1 haploid strains which both
contain a single gene disrupted by transposon mutagenesis (Fig. 1). To ensure that the 6-AU sensitivity was not due to the URA3
gene used to disrupt SNO1, an
ssa4::URA3 mutant was used as a
control. The 6-AU sensitivity of the sno1 mutant is
complemented in diploids that are heterozygous for SNO1,
heterozygous for URA3 at the SNO1 locus, and
homozygous at the ura3-52 locus, i.e., they contain only the
URA3 gene used to disrupt SNO1. Additionally,
sno1 mutants have a slower growth rate on SC-uracil media
than the ssa4::URA3 mutant control and
the sno1-1 heterozygous diploid (Fig. 7A). We conclude from
these results that loss of either Snz1 or Sno1 protein function is
responsible for the 6-AU sensitivity observed in these mutants.
To determine whether the 6-AU sensitivity of snz1 mutants
was due to inhibition of IMP dehydrogenase or OMP decarboxylase or
both, we examined the growth of snz1 mutants in SC medium
containing MPA, a specific inhibitor of IMP dehydrogenase
(15). The growth of snz1 mutants was not affected
by MPA (data not shown), suggesting that the sensitivity occurs through
an effect of Snz1p on pyrimidine biosynthesis.
Sensitivity of snz and sno mutants to
methylene blue.
A recent report indicated that a mutant of the
SNZ1 orthologue in the ascomycete C. nicotianae
was sensitive to singlet oxygen generators, including methylene
(7). To determine whether S. cerevisiae snz and
sno mutants were also sensitive to methylene blue, various
mutants and parental controls were grown on methylene blue plates in
the light, which stimulates the production of singlet oxygen within the
cell. Our results indicated that methylene blue sensitivity is specific
to cells carrying snz1 or sno1 mutations (Fig.
7B). Surprisingly, the snz1,2,33 sno1,2,33, the
snz2,33 sno2,33 (data not shown), and the
snz13sno13 alleles are not sensitive to growth on
methylene blue (Fig. 7B). Thus, sensitivity to methylene blue is only
exhibited when Snz1p or Sno1p is absent. These results indicate that an
imbalance between Snz1p and Sno1p, i.e., the presence of either Snz1p
or Sno1p and the absence of the other protein, results in the
sensitivity to methylene blue. The sno1-1 mutant sensitivity
to methylene blue is complemented in a sno1-1/SNO1
heterozygous mutant (Fig. 7B).
| |
DISCUSSION |
We have identified two gene families, named SNZ and
SNO, which are highly conserved. Adjacent SNZ and
SNO genes are coregulated, and different SNZ-SNO
gene pairs are induced at alternate times during growth to stationary
phase. The induction of SNZ1 mRNA accumulation in a
snz2/3 mutant during late-exponential and postdiauxic phases
is reminiscent of the cross-regulation observed in the HSP70
genes in yeast (34). SNZ1 is also induced in
auxotrophic mutants in response to the limitation of adenine, uracil,
and tryptophan. The observation that Snz1 and Sno1 proteins interact, based on the two-hybrid assay, and the result that both snz1
and sno1 mutants are sensitive to 6-AU and methylene blue,
support the hypothesis that these two genes function in the same
pathway and that they play a role in nucleic acid metabolism.
Other reports have provided additional clues to Snz and Sno protein
function. SNZ genes are induced in response to oxidative stress in B. subtilis (2) and are required for
resistance to singlet oxygen in the ascomycete C. nicotianae
(7). Results of sequence analysis suggest that Snz proteins
are closely related to metabolic enzymes such as ThiG, TrpC, and HisF
and that Sno proteins are related to glutamine amidotransferases such
as GuaA and HisH (10). Several glutamine amidotransferase
reactions are part of both purine and pyrimidine biosynthesis (26,
35); thus, it is possible that Snz1p and Sno1p have an enzymatic
function in these pathways.
Mutations in two other yeast genes, PPR1 and
PPR2, also result in 6-AU sensitivity (15).
PPR1 encodes a transcriptional regulator of pyrimidine
pathway genes URA1, URA3, and URA4
(29). ppr1 sensitivity to 6-AU is also suppressed
by the addition of uracil to the medium (29).
PPR2 encodes TFIIS, a general transcriptional elongation
factor, which is sensitive to decreased GTP pools in the cell
(3). ppr2 sensitivity to 6-AU is suppressed by
the addition of guanine to the medium. The suppression of the
snz1 6-AU sensitivity by the addition of uracil suggests
that Snz1p, like Ppr1p, may play a role in pyrimidine metabolism.
However, the two proteins are not likely to have the same function
because, unlike Ppr1p, Snz1p has no motifs that suggest it is a
DNA-binding protein.
It is not readily apparent why snz1 and sno1
mutants are sensitive to both methylene blue, a singlet oxygen
generator, and 6-AU, a drug which lowers nucleotide pools within the
cell. However, there may be some similarities at the subcellular level
as a result of these two drugs. Singlet oxygen generators, such as
methylene blue, can cause damage to lipids, DNA, and RNA. Singlet
oxygen results in modified guanine residues (16, 20), breaks
in RNA, cross-linking between strands of RNA, and cross-linking between RNA and proteins (33). It seems possible that methylene blue causes a turnover in RNA or an increase in DNA repair, which results in
an alteration of intracellular nucleotide concentrations, in turn
resulting in an induction of SNZ1 and SNO1. The
sensitivity of either snz1 or sno1 mutants, but
not the double mutant, to methylene blue, in addition to the physical
interaction of Snz1 and Sno1 proteins, provides evidence that the
balance and physical interaction of these proteins may be needed to
maintain metabolites that are targets for the drug. Based on our
results, we hypothesize that the Snz1p-Sno1p complex, which may have a
glutamine amidotransferase activity, plays a role in nucleotide
metabolism and is induced in response to stresses that cause an
imbalance in, the maintenance of, or a decrease in the concentrations
of nucleotides. This hypothesis may seem inconsistent with the fact
that SNZ1 and SNO1 are expressed during
stationary phase, a time of low concentrations of a carbon source.
However, it is conceivable that starvation for a carbon source, in
stationary-phase cells, may result in a depletion of other important
metabolites such as nucleotides. Thus, it is possible that Snz1p and
Sno1p participate in nucleotide metabolism, a process that is likely to
be important to starved cells.
Interestingly, although SNZ and SNO genes are
found in all three phylogenetic domains, these genes are not present in
all organisms. Examples of organisms lacking SNZ and
SNO orthologues include E. coli (10),
Borrelia burgdorferi, Synechocystis spp., Helicobacter pylori, Mycoplasma pneumoniae, and
Mycoplasma genitalium. The majority of the organisms that
lack SNZ1 and SNO1 orthologues reside in
nutrient-rich environments within the host, e.g., in the stomach or
intestine, or adjacent to damaged cells. Thus, one hypothesis that
could explain this gene distribution is that SNZ and
SNO are important for survival in nutrient-poor conditions and organisms that reside in relatively nutrient-rich conditions may
have no selective pressure to maintain these genes. The presence of
SNZ and SNO genes in plants seems inconsistent
with this hypothesis but in fact may be indicative of the frequency of
nutrient limitation experienced by plants due either to poor soils or
drought. Snz and Sno proteins have not been identified in animals,
which are organisms that are unable to maintain viability under
frequent severe starvation conditions. Understanding the specific
functions of Snz and Sno proteins allows us to learn more about the
life cycles of different organisms as well as the conserved mechanisms used by organisms to deal with strong selective pressures such as
starvation.
We thank Mary Anne Nelson and Sepp D. Kohlwein for carefully
reading the manuscript and participating in helpful discussions. We
thank Fred Winston, Mike Snyder, Phil James, and Rodney Rothstein for
generously providing strains and Robert H. White for participating in
helpful discussions. We thank Stephanie Atencio, JoAnna Bernacik, and
Amy Hahn for excellent technical assistance.
This work was supported by grant MCB9418149, RIMI grant HRD-9550649,
and PYI grant MCB-9057514 to M.W.-W. from the National Science
Foundation and by MBRS grant 5SO6GM52576-03 from the National Science
Foundation and a Patricia Roberts Harris Fellowship to P.A.P.
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Abstract
Introduction
