From the Genetics and Microbiology Department,
Institute of Food Research, Norwich Research Park, Colney, Norwich
NR4 7UA, United Kingdom and the § Center for Enzyme
Research, Institute of Molecular Biology, University of Copenhagen,
83H Sølvgade DK-1307 Copenhagen K, Denmark
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ABSTRACT |
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The PRS gene family in
Saccharomyces cerevisiae consists of five genes each
capable of encoding a 5-phosphoribosyl-1( The enzyme 5-phosphoribosyl-1( PRS genes have been cloned and sequenced from a variety of
organisms; bacteria (5-9), mycoplasma (10), and protozoa (11) each
contain apparently one PRS gene. In nematodes (12, 13) and
the yeast Schizosaccharomyces pombe (14, 15) two
PRS genes have been found so far, whereas in Spinacia
oleracea four PRS cDNAs have been identified (16).
PRS genes have also been cloned in rat (17-20) and human
(21-23). Both rat and human have two ubiquitously expressed
PRS genes, with a third testis-specific transcript found in
humans (22).
The biochemistry of the rat liver enzyme has been widely studied and
led to the identification of the genes encoding it. PRSI and
PRSII encode the catalytic subunit, which consists of two highly homologous isoforms, PRSI and PRSII, each with a molecular mass
of 34 kDa (17, 24). The enzyme has two additional components of 39 and
41 kDa (24), which have been designated PRPP synthetase-associated proteins (PAPs). A negative regulatory role has been proposed for these
PAPs because their removal from the rat liver enzyme complex results in
an increased enzymatic activity (25). cDNAs corresponding to the
rat PAP39 and PAP41 have been obtained, the deduced amino acid
sequences of both are very similar to each other and to those of the
PRS catalytic subunits; therefore broadly speaking on the basis of
sequence similarity PAP39 and PAP41 can be considered to be members of
the PRS family (25, 26). In humans the situation appears to be
comparable with that in rat because human PAP39 and PAP41 cDNAs
have been obtained and found to be very similar to each other and to
rat PAP39 and PAP41 and human PRSI and PRSII (27, 28).
Our analysis of the PRS gene family in Saccharomyces
cerevisiae has identified five genes, each located on a different
chromosome (29, 30). The five predicted PRS polypeptides contain the characteristic motifs of PRS: a divalent cation-binding site and a
PRPP-binding site (5, 31). The predicted PRS2, PRS3, and PRS4
polypeptides are 318-320 amino acids long, whereas PRS1 and PRS5
polypeptides are longer and more divergent in their sequences because
they contain in-frame insertions bearing no similarity to any known
PRS and, in fact, to any known gene product. We have named
these regions nonhomologous regions (NHRs). PRS1 contains one NHR (NHR1-1) that is neither an intron nor processed by protein splicing (29, 32), and PRS5 contains two NHRs, NHR5-1 and NHR5-2, that are not introns (30). Our preliminary analyses of the
phenotypes associated with strains containing disruptions in each of
the PRS genes have shown that none of the five genes is
essential, although the contribution of each to the cell's metabolism
does not appear to be equal because Prs1p and Prs3p make a more
significant contribution than the other members of the family (30, 32).
However, the PRS5 null mutation is synthetically lethal in
combination with a disruption in either PRS1 or
PRS3, implying an important role for Prs5p in the production
of PRPP (30).
Because all PRS genes sequenced to date have a high degree
of similarity, studies on PRS in S. cerevisiae
could provide valuable information on the genetics and biochemistry of
eukaryotic PRS. In the present study we have constructed a collection
of strains bearing all the possible combinations of disruptions in the
five PRS genes and analyzed their phenotypes. The data
described here are consistent with the existence of two functionally
different entities, one consisting of Prs1p and Prs3p and the other
consisting of Prs2p, Prs4p, and Prs5p.
Standard DNA manipulations were carried out as described by
Sambrook et al. (33). Preparation of yeast genomic DNA,
total RNA, Northern blotting, and hybridization were performed as
described previously (29, 32). In Northern analysis
32P-labeled actin-encoding DNA was used as the loading
control. Unless otherwise stated yeast transformation was carried out
according to Elble (34). Quantification of radioactive signals were
performed using a Fujix BAS-1500 phosphorimager.
Strains, Plasmids, and Growth Conditions--
The S. cerevisiae strains used in this study are listed in Table I. The
strains were grown at 30 °C in yeast extract peptone dextrose medium
(YEPD) or in YEPD containing 200 mg/liter of the aminoglycoside
antibiotic geneticin G418 (Roche Molecular Biochemicals) (35) or
synthetic complete medium (36). Tetrads were dissected using a Singer®
micromanipulator, model MSM (Singer Instruments). 5-Fluoro-orotic acid
(5-FOA; Sigma-Aldrich) was used to select against uracil prototrophy
(37).
S. cerevisiae strains containing disruptions in two or three
PRS genes were obtained by one of the following methods. (i) a PCR-based method using the loxP-KanMX-loxP module as a
marker (38). DNA fragments containing the disruption cassette together with 40-45-nucleotide extensions at their ends that are homologous to
the regions immediately upstream and downstream of the start and stop
codons of each PRS gene were obtained by PCR (30). These DNA
fragments were used to transform (39) S. cerevisiae strains
already disrupted in a PRS gene (30), and the resulting transformants were selected on YEPD containing G418. The correct integration of the KanMXr cassette was verified
by PCR and Southern blotting. Derivatives from the strain HF7c (40)
containing disruptions in each PRS gene were obtained by the
same procedure. The KanMX marker was excised from the
deletants by a recombination event between the two flanking
loxP sites after transforming the corresponding strains with
plasmid pSH47, which contains the Cre recombinase under the GAL1 promoter. Plasmid pSH47 (URA+)
was removed from these strains by streaking the cells onto plates containing 5-FOA, which counterselects URA3+
plasmids (37). (ii) Strains YN94-22 and YN95-100 were obtained by
appropriate crosses of available strains (32). The diploids obtained
were sporulated, the resulting tetrads were dissected, and the spores
were selected for the presence of the markers used for the disruptions.
In all cases the disruptions were checked by PCR and Southern blotting.
Plasmids were amplified in Escherichia coli DH5 Determination of PRS Activity--
Crude cell extracts were
prepared from mid-log phase cultures. Activity of PRS was assayed by
thin layer chromatography (44) as modified by Carter et al.
(32). The relative amounts of radioactive material corresponding to ATP
and PRPP were determined with a Packard Instant Imager 2024. Specific
activity is expressed as nmol PRPP min Extraction and Quantification of Nucleotides--
Total
nucleotides were extracted from wild type and
Yeast Two-hybrid Analysis--
PRS genes were cloned
in the two-hybrid system (48) vectors pGAD424 and pGBT9
(CLONTECH, Palo Alto, CA), which contain the Gal4p
activation domain or DNA-binding domain, respectively. For this purpose
each PRS gene was amplified by PCR using primers that placed
an EcoRI restriction endonuclease site immediately upstream
of the initiation codon and a PstI restriction endonuclease site immediately downstream of the stop codon (PRS1-4) or
placed BamHI restriction endonuclease sites at both ends
(PRS5). The DNA fragments obtained were cloned into
appropriately restricted pGAD424 and pGBT9, and the constructs were
sequenced to confirm the correct insertion and to check for PCR-induced errors.
The yeast reporter strain HF7c (40) was transformed with every possible
pairwise combination of Gal4pAD-Prsp and Gal4pDBD-Prsp plasmids to
tryptophan and leucine prototrophy. Crude cell extracts were prepared
from mid-log phase cultures. Two-hybrid analyses were performed by
assaying for Construction of PRS Deletion Strains
A collection of S. cerevisiae strains containing all
possible combinations of disruptions in the five PRS genes
has been created by targeted gene disruption. The
loxP-KanMX-loxP cassette together with the Cre recombinase
expression system allows for repeated use of the
KanMXr marker and is therefore ideal for the
functional analysis of gene families. The strains created by this
system have the advantage that the only foreign DNA contained therein
is the loxP site remaining after the Cre recombinase-induced
event and all the deletants will be congenic (38). Using this procedure
the viable double deletants listed in Table
I were obtained. The viable double deletant strains )-pyrophosphate synthetase
polypeptide. To gain insight into the functional organization of this
gene family we have constructed a collection of strains containing all
possible combinations of disruptions in the five PRS genes.
Phenotypically these deletant strains can be classified into three
groups: (i) a lethal phenotype that corresponds to strains containing a
double disruption in PRS2 and PRS4 in
combination with a disruption in either PRS1 or
PRS3; simultaneous deletion of PRS1 and
PRS5 or PRS3 and PRS5 are also
lethal combinations; (ii) a second phenotype that is encountered in
strains containing disruptions in PRS1 and PRS3
together or in combination with any of the other PRS genes
manifests itself as a reduction in growth rate, enzyme activity, and
nucleotide content; (iii) a third phenotype that corresponds to strains
that, although affected in their phosphoribosyl pyrophosphate-synthesizing ability, are unimpaired for growth and have
nucleotide profiles virtually the same as the wild type. Deletions of
PRS2, PRS4, and PRS5 or
combinations thereof cause this phenotype. These results suggest that
the polypeptides encoded by the members of the PRS gene
family may be organized into two functional entities. Evidence that
these polypeptides interact with each other in vivo was
obtained using the yeast two-hybrid system. Specifically PRS1 and PRS3
polypeptides interact strongly with each other, and there are
significant interactions between the PRS5 polypeptide and either the
PRS2 or PRS4 polypeptides. These data suggest that yeast phosphoribosyl
pyrophosphate synthetase exists in vivo as multimeric complex(es).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
)-pyrophosphate synthetase
(ATP:D-ribose-5-phosphate pyrophosphotransferase; EC
2.7.6.1) (PRS)1 catalyzes the
reaction at a key junction in intermediary metabolism. PRS transfers
the pyrophosphate moiety released from ATP to ribose-5-phosphate, thus
giving rise to phosphoribosyl-pyrophosphate (PRPP) (1), and the enzyme
therefore directs ribose-5-phosphate from energy generated by the
pentose phosphate pathway to the important biosynthetic intermediate
PRPP. PRPP is a precursor for the production of purine, pyrimidine, and
pyridine nucleotides and the amino acids histidine and tryptophan (2).
PRPP is required for both the de novo and the
salvage pathways of nucleotide metabolism (3). It has been shown that in Mycobacterium spp. PRPP is also required for
the biosynthesis of polyprenylphosphate pentoses that contribute to the
arabinosyl residues of the cell wall (4).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(supE44
lacU169 (
80 lacZ
M15)
hsdR17 recA1 endA1 gyrA96
thi-1 relA1; Ref. 41). Plasmid pRS21 consists of
the 2562-bp HindIII fragment containing the PRS2
coding sequence plus 1470 and 135 bp upstream and downstream,
respectively (29), inserted in plasmid pRS416 (42). pRS41 is plasmid
pRS416 containing the 2950-bp SalI/BglII fragment
that corresponds to the PRS4 coding sequence plus 509 and
1429 bp upstream and downstream thereof (29). Plasmid pVT1 consists of
the 1346-bp PstI/SspI fragment containing the
PRS1 coding sequence plus 42 and 21 bp upstream and
downstream, respectively (29), inserted into the appropriately
restricted plasmid pVT100-U (43). pVT3 is plasmid pVT100-U containing
the 1113-bp SnaBI fragment that corresponds to the
PRS3 coding sequence plus 73 and 77 bp upstream and
downstream thereof (29).
1 mg
1
protein. Protein content was determined according to Bradford (45)
using bovine serum albumin as the standard.
prs::loxP strains after growth in complete
medium to approximately mid-log phase (46). The extracts were
resuspended in 150 µl of 7 mM KH2PO4, pH 4.0. Nucleotide pools were
determined by high pressure liquid chromatography using 50 µl of the
extract (47). The elutant was monitored with a diode array detector,
and peaks were quantified by comparison of peak areas at 260 nm with
those of known amounts of 98% pure standards (Sigma-Aldrich).
-galactosidase activity using a chemiluminescent
substrate (Galacto-Light PlusTM, Tropix Inc, MA) according
to the manufacturer's instructions. Chemiluminescence was measured
using a Lumat LB9501 luminometer (Berthold, Wildbad, Germany). Results
were recorded as relative light units/µg protein and expressed
relative to the values obtained with control strains containing empty
two-hybrid vectors.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
prs2::loxP
prs5::loxP (YN97-89) and
prs4::loxP
prs5::loxP (YN97-90)
have been described previously, and we have shown that the double
deletant strains
prs1::loxP
prs5::loxP and
prs3::loxP
prs5::loxP are inviable (30). The triple deletants
prs1::loxP
prs2::loxP
prs3::loxP (YN97-169),
prs1::loxP
prs3::loxP
prs4::loxP (YN97-170), and
prs2::loxP
prs4::loxP
prs5::loxP (YN97-91) (Table I) were obtained by
transformation of appropriate double deletant strains. Our success in
constructing these deletant strains shows that the combined loss of
these PRS genes is not a lethal event.
S. cerevisiae strains used in this study
Synthetic Lethal Combinations
Repeated attempts were made to obtain the triple disruptants
prs1
prs2
prs4 and
prs2
prs3
prs4, but none was successful. This observation suggested that
a double disruption of PRS2 and PRS4 might be
synthetically lethal in combination with a disruption in either
PRS1 or PRS3.
To analyze this possibility we performed a cross between the strain
YN97-3 (prs2::loxP-KanMX-loxP) containing a
URA3-based plasmid with an intact copy of either
PRS2 (pRS21), PRS4 (pRS41), or PRS1
(pVT1) with the double disruptant strain YN94-22
(
prs1::HIS3
prs4::LEU2) (a similar
experiment was performed for PRS3 using the strain
YN95100 (
prs3::TRP1
prs4::LEU2) containing one of the following
plasmids: pRS21, pRS41, or pVT3). The resulting diploids were
sporulated, the tetrads were dissected, and the spores were incubated
at 30 °C. Viable
prs1::HIS3
prs2::loxP-KanMX-loxP
prs4::LEU2 and
prs2::loxP-KanMX-loxP
prs3::TRP1
prs4::LEU2 isolates were now
recovered but always containing the corresponding plasmid (Table
II). These strains were sensitive to
medium containing 5-FOA, indicating that any cells losing the plasmid
were inviable. Wild type, single deletant, and double deletant strains
carrying the relevant plasmids gave rise to colonies in
5-FOA-containing medium indicating their survival after plasmid loss.
This observation was also confirmed by transformation of the double
deletant strain
prs2::loxP
prs4::loxP (YN97-13) containing plasmid pRS21 with a
DNA fragment containing either
prs1::loxP-KanMX-loxP or
prs3::loxP-KanMX-loxP. In both cases all the
transformants (5 and 7, respectively) with the correct integration of
the cassette failed to survive in medium containing 5-FOA, whereas the
recipient strain gave rise to colonies on this medium (data not shown).
These data confirm that a PRS2 PRS4 null mutant is
synthetically lethal in combination with a deletion in either
PRS1 or PRS3. Therefore although PRS2
and PRS4 are not essential genes, loss of Prs2p and Prs4p
together cannot be tolerated if either the Prs1p or Prs3p function is
compromised.
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Characterization of prs Strains
To characterize the phenotypes associated with the multiple PRS disruptants, we have determined their growth rates, ability to produce PRPP, and nucleotide content.
Doubling Times--
The analysis of growth rates showed that
strains containing disruptions affecting PRS1 and
PRS3 together or in combination with disruption of other
PRS genes are affected severely in their growth rates. The
double disruptants have doubling times between 3.5 and 4.0 h, and
the triple deletants prs1
prs2
prs3 (YN97-169) and
prs1
prs3
prs4 (YN97-170) have doubling times of
4.8 and 5.0 h, respectively. However, strains carrying disruptions
in PRS2, PRS4, or PRS5 grow at the
same rate as the wild type with a doubling time of 2 h (Table
III).
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PRPP Synthesizing Ability--
We have examined PRS activity in
five single, nine double, and three triple congenic deletant strains.
As shown in Table III each of the deletants is impaired in its PRS
activity. The single, double, and triple deletants containing
disruptions eliminating PRS2, PRS4, or
PRS5 have an enzyme activity varying between 11 and 17% of
the wild type activity (from 3.31 to 5.09 nmol PRPP min1
mg
1). The single deletant strains
prs1::loxP (YN96-66) and
prs3::loxP (YN96-67) produce 0.85 and 1.08 nmol
PRPP min
1 mg
1, respectively. The double and
triple deletants bearing disruptions in PRS1 or
PRS3 together or in combination with disruptions in the
other PRS genes possess an enzyme activity that varies
from 0.27 to 1.68 nmol PRPP min
1 mg
1.
Nucleotide Content--
PRPP is a precursor required for the
production of purine and pyrimidine nucleotides, and it is possible
that different combinations of disruptions in the PRS genes
could affect not only the amount of PRPP produced but also the way it
is channeled into the biosynthetic pathways. Strains containing
disruptions in PRS1 and PRS3 together or in
combination with disruptions in the other PRS genes are more
affected than the strains containing disruptions in PRS2, PRS4, or PRS5 (Table
IV). The nucleotide content of the
prs1
prs3
prs4 strain (YN97-170) was the most
drastically reduced in comparison with that of the wild type with
adenine ribonucleotides being 18% of the wild type levels, guanine
ribonucleotides 12% of the wild type levels, and uridine
ribonucleotides 9% of the wild type levels, respectively. The cytidine
nucleosides were below the level of detection. All the other strains
lacking Prs1p or Prs3p were significantly altered in their nucleotide
content with amounts of nucleotides varying from 19 to 39% of the wild type. Strains containing disruptions in PRS2,
PRS4, or PRS5 each had a nucleotide content
differing only slightly from the wild type (75-110% of the amount of
nucleotides detected in the wild type). It is unlikely that the
reduction in the nucleotide content observed for the strains containing
disruptions in PRS1 and PRS3 was caused by
degradation of nucleotides because known amounts of ATP and GTP added
to extracts of the wild type strain were not significantly degraded
(data not shown). These data are consistent with previous observations
on the nucleotide content of the single disruptants that indicated that
deletions of PRS1 or PRS3 significantly affect
the nucleotide content of the yeast cell, whereas the nucleotide content of strains deleted in PRS2, PRS4, or
PRS5 was slightly or not affected (30). Nucleotide pools
were essentially identical in cells grown in YEPD (data not shown).
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Transcription of PRS Genes in the Deletant Strains
Each PRS gene product shares between 40 and 90%
identity with the other members of the family, and this may imply
functional redundancy. One might expect the existence of a control
mechanism of gene expression such that the absence of certain
PRS mRNAs or their translation products may trigger an
alteration in the amount of transcript of one and/or another of the
remaining PRS genes. To test this possibility we performed
Northern blot analysis with the wild type strain and representative
disruptants. Northern blots were probed with 32P-labeled
DNA from the coding region of each gene. The filters were stripped and
reprobed with 32P-labeled actin-encoding DNA as the loading
control. The wild type strain contained transcripts of each
PRS gene (PRS1, 1.5 kilobases; PRS2,
PRS3, and PRS4, 1.3-1.2 kilobases;
PRS5, 1.6 kilobases). In the deletant strains we could
detect only the transcripts of the genes not disrupted therein. After
correcting for background hybridization the signal associated with each
PRS transcript was estimated by comparison of its signal
with the actin signal. There were no significant differences under the
experimental conditions used between mRNA levels from wild type and
prs strains (data not shown).
Analysis of Physical Interactions by the Yeast Two-hybrid System
We used the yeast two-hybrid system (48) to test for
protein-protein interaction between the PRS gene products.
HF7c transformants containing all possible combinations of
Gal4pDBD-Prs1-5p with Gal4pAD-Prs1-5p were obtained. The
-galactosidase activity associated with each pairwise combination is
shown in Fig. 1A.
Gal4pDBD-Prs1p shows interaction with Gal4pAD-Prs3p; this interaction
is also detectable, although less pronounced, in the opposite
combination. The interactions of Prs1p and Prs2p appear to be
independent of their combination with the Gal4p activation or
DNA-binding domain. Gal4pDBD-Prs5p showed interaction with
Gal4pAD-Prs2p and Gal4pAD-Prs4p; however, these interactions are not
detectable when Prs5p is fused to the Gal4pAD. Finally, Prs5p seems to
be the only PRS gene product that interacts with itself.
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Endogenous levels of PRS polypeptides in HF7c could interact with the
Gal4p-Prsp fusion proteins and prevent them from entering the nucleus
and thereby reducing interactions detected in the two-hybrid system. To
investigate this possibility we constructed HF7c-derivative strains
containing disruptions in each PRS gene as described under
"Experimental Procedures" (Table I). Each HF7c prs
strain was transformed with the series of the PRS two-hybrid plasmids, and the
-galactosidase activity associated with each combination was assayed (Fig. 1, B-F). In general the
interactions detected in the wild type are reproduced in the deletant
strains, albeit with
-galactosidase activity at least 10-fold
higher. There are two main profiles: one associated with deletion of
PRS1 (YN96-25), PRS3 (YN96-56), or
PRS5 (YN96-22) and the other associated with deletion of
PRS2 (YN97-140) or PRS4 (YN96-79) (Fig. 1,
B-F). In the
prs1,
prs3, and
prs5 backgrounds there is strong interaction between
Prs1p and Prs3p. The interaction between Prs1p and Prs2p is still
detectable but has not increased with respect to the wild type, and
Prs5p interactions vary from strain to strain. In the
prs1 and
prs5 deletant backgrounds Prs5p
interacts with all others, particularly with Prs4p and Prs2p, and the
Prs5p interaction with itself observed above can no longer be measured.
In the
prs3 background Prs5p interacts clearly with Prs2p
and Prs4p. Protein-protein interactions in
prs2 and
prs4 backgrounds are very similar; again we can detect
strong interaction between Prs1p and Prs3p. In these backgrounds we can
detect clear interaction between Prs1p and Prs2p. We can also detect
interaction between Prs1p and Prs4p, and the strength of interaction of
Prs5p with Prs2p and Prs4p has also increased. This enhancement of PRS
polypeptide interactions in strains depleted for endogenous PRS
polypeptides suggests that in vivo the PRS gene
products interact to form multimeric complex(es).
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DISCUSSION |
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In S. cerevisiae we have identified five PRS genes that constitute the PRS gene family. The existence of paralogous genes has been explained in different terms for different gene families. In some cases it has been shown that the product of each member contributes to the total activity of the corresponding enzyme, as is the case for the three members of the ATP1 gene family (49). In other cases, the products of the various members of the gene family are distributed to different cell compartments, for example the HSP70 gene family (50). A third explanation for a gene family is that the gene products may have different substrate specificities, as is the case for the five genes encoding acyl-CoA synthetases (51, 52). To elucidate the nature of the functional roles of the members of the PRS gene family, we have created a collection of strains containing all the possible combinations of disruptions in the five PRS genes and have performed a systematic phenotypic study of them.
This analysis has revealed three phenotypes: (i) the least affected phenotype associated with strains bearing combinations of disruptions of PRS2, PRS4, and PRS5 that are capable of synthesizing PRPP at about 17% of the wild type (Table III) sufficient to support growth and nucleotide production as in the wild type; (ii) a severely affected phenotype that corresponds to strains containing disruptions in PRS1 and PRS3 together or in combination with any of the others. These strains produce between 1 and 6% of the wild type level of PRPP and are impaired in their growth and nucleotide production confirming previous results that PRS1 and PRS3 gene products apparently make a more important contribution to yeast metabolism than the other members of the family (30); and (iii) a synthetic lethal phenotype that corresponds to a double disruption in PRS2 and PRS4 in combination with a deletion of either PRS1 or PRS3. Furthermore, a PRS5 null mutant is also synthetically lethal in combination with a disruption in either PRS1 or PRS3 (Table V). These findings suggest that the PRS gene products may be organized into two functionally discrete entities, one comprising Prs1p and Prs3p and the other comprising Prs2p, Prs4p, and Prs5p. In the absence of one entity or when some of its components are missing, the yeast cell could survive on the amount of PRPP produced by the other entity. However, when both functional entities are damaged, it represents such a burden for the cell that it is no longer able to survive. In this situation Prs2p and Prs4p, due to their high degree of identity, (89.3%) may be able to compensate functionally for each other, and only if the two are simultaneously absent would the functional entity be severely damaged. However, this interchangeability of Prs2p and Prs4p would only take place when the other entity is compromised because when either PRS1 or PRS3 is present, single and double disruptants of PRS2 and PRS4 all have a similar level of PRPP. There are three viable triple deletant combinations, and each of these defines a minimal functional entity (unit) capable of sustaining the PRPP requirement of S. cerevisiae. These three minimal functional units are: (i) Prs1p and Prs3p and (ii) Prs5p in combination with either Prs2p or Prs4p (Table V).
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The high degree of similarity of PRS polypeptides is suggestive of a certain degree of functional redundancy. However, our phenotypic analysis indicates that apart from the possibility of functional compensation between Prs2p and Prs4p, this is not the case. This interpretation is also supported by the apparent lack of a mechanism for the control of gene expression, at least at the mRNA level, which would compensate for the absence of a PRS gene transcript by increasing the amount of transcript from any of the remaining PRS genes. Nevertheless, we cannot exclude the possibility of post-transcriptional or post-translational modification that may be compensatory. In any case the compensation will never be to the full extent because we have shown that disruptions affecting the components of the Prs1p/Prs3p functional entity are associated with a more severe defect than when the deletions damage the other entity.
In rat four members of the PRS gene family have been
identified (17, 25, 26). PRSI and PRSII encode
the catalytic subunits (24), whereas PAP39 and
PAP41 gene products have been proposed to play a negative
regulatory role in PRS because their removal from the rat liver complex
causes an increase of PRS activity of the remaining subunits (25).
Interestingly, PAP39 and PAP41 polypeptides both contain a short NHR
(31 and 32 amino acids in length, respectively) in the same relative
positions as NHR1-1 and NHR5-1. This raises the possibility that as
Prs1p and Prs5p have NHR regions, they may play a regulatory role. If
this is the case it would be positive rather than negative as PRS
activity in prs1 and
prs5 strains is lower
than in the wild type. If Prs1p and Prs5p were to play a regulatory
role, it is possible that, in analogy to rat PRS, Prs3p, Prs2p, and
Prs4p could represent catalytic functions. In the absence of data on
the effect of nucleotides on PRS activity in either the wild type or
the mutant cell extracts, one cannot say whether the putative functions
of Prs1p and Prs5p extend to stabilization of the PRS enzyme
complex(es) as shown for PAP39 and PAP41 (53). Interesting in this
respect is that the three minimal functional units referred to above
would each consist of a putative catalytic and regulatory function.
If in the wild type PRS exists as two functionally different entities
it may be expected that they could be distributed to different
subcellular compartments as seems to be the case for the PRS family in
S. oleracea in which one PRS gene product has been shown to be imported into the chloroplast, whereas the deduced amino acid sequence of another indicates that it might be localized in
the mitochondria (16). However, we have searched for subcellular targeting signals in the amino acid sequences of the S. cerevisiae PRS polypeptides using the PSORT program package (54),
and this search has failed to identify any targeting signals. The data from the two-hybrid analyses suggest that PRS in S. cerevisiae could exist as two subcomplexes that may be
functionally different because we detected strong interactions between
the members of each entity that are clearly detectable in the wild type
and enhanced in the deletant strains (Fig. 1). However, these
subcomplexes would not be completely independent because there are also
interactions between members of the two putative functional entities,
viz. interactions of Prs1p with Prs2p are measurable in the
wild type background and are even more pronounced in the
prs2 and
prs4 backgrounds in which
interactions of Prs1p with Prs4p are also detectable (Fig. 1,
E and F). To find out whether the two putative PRS subcomplexes interact as a larger complex and to establish the
structure of a PRS complex, it will be necessary to purify the enzyme.
In other organisms PRS has been shown to be a multimeric complex of
varying composition. For example the rat liver enzyme exists in an
aggregate form composed of 34-kDa (PRSI and PRSII), 39-kDa (PAP39), and
41-kDa (PAP41) subunits in a ratio of 20:5:8:1 (55). In rubber tree
latex the enzyme seems to be a tetramer (56), although the crystal
structure of the Bacillus subtilis PRS has shown it to be
hexameric (57) and in Salmonella typhimurium the smallest
active form of PRS may be pentameric (58).
The work presented here represents a genetic dissection of the
PRS gene family in S. cerevisiae. The collection
of strains obtained in this study will be very useful in biochemical
and kinetic studies of PRS and will allow us to define precisely the structure, function, and regulation of the PRS enzyme.
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ACKNOWLEDGEMENTS |
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We thank B. M. Pearson for performing the tetrad dissection and S. S. Wolf for initial help with the two-hybrid analysis. We are grateful to J. H. Hegemann (University of Düsseldorf) for providing us with the plasmids for the Cre-loxP system.
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FOOTNOTES |
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* This work was supported by Biotechnology and Biological Sciences Research Council and a fellowship from the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (to Y. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 44-1603-255250; Fax: 44-1603-458414; E-mail: Michael.Schweizer{at}bbsrc.ac.uk.
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ABBREVIATIONS |
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The abbreviations used are:
PRS, 5-phosphoribosyl-1()-pyrophosphate synthetase;
PRPP, 5-phosphoribosyl-1(
)-pyrophosphate;
PAP, PRPP synthetase-associated
protein;
NHR, nonhomologous region;
5-FOA, 5-fluoro-orotic acid;
PCR, polymerase chain reaction;
bp, base pair(s).
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REFERENCES |
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