(Received for publication, May 31, 1995)
From the
Mutations in the gene encoding p53 have been found to be the most common genetic alterations in human cancer. p53 is thought to exert its function of tumor suppression through inhibition of cell proliferation or induction of apoptosis in response to DNA damage. Although there have been no proteins homologous to p53 identified in lower eucaryotic organisms, it is known that overexpression of wild-type human p53 can inhibit cell growth of Schizosaccharomyces pombe and Saccharomyces cerevisiae (Bischoff et al., 1992; Nigro et al., 1992), suggesting that certain aspects of p53 function may manifest or exist in yeast. In an attempt to identify the p53-like proteins in the yeast S. cerevisiae, we isolated a mutant that requires wild-type p53 for its viability. The mutant, rft1-1, is defective in cell cycle progression and arrests before mitosis when p53 protein is depleted. Genetic and biochemical studies show that p53 suppresses the rft1-1 mutation by forming a protein-protein complex with the Rft1 protein.
The mammalian protein p53 has been in the spotlight of cancer
research in recent years because a deficiency in p53 function is linked
to many types of human cancer (Hollstein et al., 1991; Levine et al., 1991; Prives, 1993; Vogelstein and Kinzler, 1992). The
mechanism by which p53 suppresses tumorigenesis in normal animals is
now beginning to be understood. It has been suggested that p53 plays an
important role in mammalian cell cycle as a checkpoint, maintaining
genetic integrity in response to environmental cues (Hartwell, 1992;
Hartwell and Kastan, 1994; Lane, 1992). Treatment of wild-type cells
with DNA damaging agents such as ultraviolet (UV) and ionizing
radiation leads to elevation of p53 activity and cell cycle arrest in
G (Kastan et al., 1991; Kuerbitz et al.,
1992; Maltzman and Czyzyk, 1984), whereas cells defective in p53 are
unable to respond to DNA damaging agents in a similar manner (Kastan et al., 1991; Kuerbitz et al., 1992). High expression
of wild-type p53 in the absence of DNA damage also results in cell
cycle arrest in G
(Lin et al., 1992). DNA
damage-induced p53 acts as a transcriptional activator to enhance
expression of a group of genes, some of which are known to play
important roles in regulation of cell proliferation (Fornace et
al., 1989; Kastan et al., 1992). Of particular importance
among these is p21 (Cip1/Waf1/SDI1), an inhibitor of the
cyclin-dependent kinases that are required for cell cycle progression
in most if not all eucaryotic cells (Dulic et al., 1994;
El-Deiry et al., 1993; Harper et al., 1993; Xiong et al., 1993). p21 has also been found to bind to the
replication factor PCNA (for proliferating cell nuclear antigen)
directly to inhibit DNA synthesis (Flores-Rozas et al., 1994;
Waga et al., 1994).
In view of the conservation of cell
cycle machinery from human to yeast (Draetta, 1990), it is tempting to
search for p53-related proteins in lower eucaryotic organisms like
budding yeast where studies of cell cycle regulation have been well
established. Budding yeast responds to DNA damage with cell cycle
arrest in G, which is dependent on a number of checkpoint
genes such as RAD9, RAD17, RAD24, MEC1, etc. (Weinert et al., 1994). The RAD9 and RAD24 genes have also been shown to be responsible
for cell cycle delay in G
after DNA damage (Siede et
al., 1994). The yeast homolog of p53, assuming there is one, could
be expected to be a checkpoint gene as well, perhaps guarding the
G
/S boundary as in mammalian cells. A few laboratories have
reported that human p53 is able to inhibit the proliferation of Saccharomyces cerevisiae (Nigro et al., 1992) and Schizosaccharomyces pombe (Bischoff et al., 1992),
possibly in a manner similar to that in which it inhibits mammalian
cells. Whereas the growth inhibition of S. pombe by p53 might
not be restricted to certain specific cell cycle stages, the S.
cerevisiae cells co-expressing wild-type p53 and human CDC2, which
supposedly enhanced the activity of p53, were arrested in
G
, analogous to observations made with mammalian cells
(Nigro et al., 1992). Despite the above reports, however, the
attempt to search for a p53 homolog in yeast has so far proven futile.
In order to gain more insights into the function of p53 in relation to cell cycle regulation, we set out to isolate the putative yeast homolog of human p53 by the approach of functional substitution. In this report, we describe the isolation and characterization of a yeast mutant that requires human p53 for its viability. The mutant, rft1-1, arrests before mitosis when p53, or the wild-type Rft1 protein, is not provided. We also show that the RFT1 gene product interacts physically with p53. The Rft1 protein may represent a novel p53 binding factor yet to be identified from mammalian cells.
Figure 1:
The plasmids pMC136 and pMC147
contained the same pGAL1-p53 cDNA construct as described under
``Materials and Methods.'' The transcriptional termination
signal (GAL7 ter) was derived from the GAL7 gene.
pMC149 (see Fig. 2), which is not illustrated here, was
identical to pMC147 except that it contained the cDNA of p53 with Arg
at position 175 changed to His
(p53).
Figure 2: The plasmid shuffling assay in the rft1-1 mutant. The originally isolated rft1-1 mutant (YMC285) kept alive by p53 on a URA3-containing plasmid (pMC136) was transformed with LEU2-containing plasmids that carried either wild-type or mutant p53 under the inducible promoter pGAL1 (pMC147 and pMC149). The transformants grown up on galactose/leucine dropout (C-Leu + Gal) plates were patched again on such plates and after 3 days of incubation replica-plated on galactose/5-FOA plates (5-FOA + Gal) as well as C-Leu + Gal plates. The photograph was taken after another 3 days of incubation.
The GAL-RFT1 construct, pMC195, was made by partial digestion of the 5` region of the RFT1 gene with exonuclease III and fusion with the GAL1 promoter. pMC195 was selected based on its ability to rescue the viability of rft1-1::pMC136 on 5-FOA plates in a galactose-specific manner.
c-Myc epitope-tagged RFT1 construct was generated by first partially digesting the RFT1 gene with BglII and then inserting into this
site the oligonucleotide sequence encoding c-Myc epitope
(EQKLISEEDLNG). The correct construct with the epitope sequence
inserted into the BglII site near the N terminus of Rft1 was
identified by DNA sequencing. The epitope-tagged Rft1 was able to
support the growth of the rft1 strain when expressed from
a multicopy plasmid.
Disruption of the RFT1 gene by one-step gene replacement (Rothstein, 1983) was achieved by first deleting the 1.4-kb SalI-HpaI fragment from the RFT1 coding region and inserting the LEU2 marker into this position. This construct was used to transform a diploid yeast strain (YNN413). The deletion/disruption allele of rft1 was confirmed by Southern analysis.
Proteins were separated by
SDS-polyacrylamide gel electrophoresis (PAGE) and electroblotted to an
Immobilon-P membrane using the Bio-Rad transfer system as recommended
by the manufacturer. Blots were blocked over night by incubation in
PBS-T (137 mM NaCl, 2.7 mM KCl, 8.1 mM
NaHPO
, 1.5 mM
KH
PO
, pH 7.2, 0.05% Tween) containing 8% of
fat-free milk powder. Secondary antibodies were peroxidase-coupled
conjugates of either goat anti-mouse or goat anti-rabbit immunoglobulin
G (Jackson Laboratories) diluted into PBS-T containing 2% milk. Blots
were developed using a chemiluminescence detection system (Amersham
Corp).
We wished to use the colony color sectoring assay to screen for yeast mutants defining p53-related factors. The wild-type human p53 cDNA was placed under the inducible yeast GAL1 promoter in an ADE3/URA3-containing plasmid (pMC136, Fig. 1) and transformed into an ade2 ade3 strain (YMW2, see Table 1). The expression of p53 from the plasmid pMC136 was found to be dependent on galactose medium both at the RNA and the protein level (data not shown but see Fig. 8). A strong p53-specific signal was observed using a commercially available anti-p53 antibody. Cells carrying pMC136 were mutagenized with ethylmethanesulfonate to 70% lethality. Among 100,000 colonies screened, we obtained 200 or so red colonies, which showed little or no sectoring on galactose-rich medium (YEPG plates). These colonies were further tested for growth on galactose plates containing 5-fluoroorotic acid (galactose/5-FOA), a drug that kills cells with a functional URA3 gene (Boeke et al., 1984). Fifteen isolates were unable to grow on galactose/5-FOA medium.
Figure 8:
Immunological detection of the Rft1
protein. Protein extracts were prepared from the wild-type (YMW2,
labeled as WT), YMC333::pMC195 (the rft1 strain carrying
the RFT1 gene under the control of the GAL1 promoter, labeled
as GAL-RFT1), or YMC333 kept alive by the
c-myc-tagged RFT1 gene under its native promoter
(labeled as RFT1-cMYC). The YMC333::pMC195 cells were
grown in liquid YEPG medium and shifted to YEPD medium for 4 or 8 h as
indicated. The WT and RFT1-c-myc cells were grown in
YEPD medium. The extracts were separated by SDS-PAGE, blotted, and
probed with affinity-purified polyclonal anti-Rft1 antibody as
described in detail under ``Materials and Methods.'' The same
band in the lane of RFT1-c-myc extracts was also
detected by the monoclonal antibody against c-myc (Data not
shown).
A plasmid shuffling assay was performed to distinguish the mutants that specifically depend on p53 for viability (class I) from those that require the plasmid for reasons unrelated to p53 (class II). The 15 strains that failed to sector on rich medium and were unable to grow on galactose/5-FOA were transformed with a second p53 expression plasmid (pMC147, Fig. 1) that differs from pMC136 in that it contains the LEU2 marker instead of URA3, and it does not contain the ADE3 gene. After receiving pMC147, the class I mutants should be able to lose pMC136 and hence grow on galactose/5-FOA and sector, whereas the class II mutants will remain non-sectoring on rich medium and nonviable on galactose/5-FOA. Among the 15 candidates, only one was found to be capable of growing on galactose/5-FOA after pMC147 was introduced into the cell. This mutant, therefore, fulfilled the criteria of a p53-dependent mutant. The mutation was named rft1-1 for requiring fifty-three.
We
then tested to see if a mutant p53 found to be defective in mammalian
systems could complement the rft1-1 mutation, using one of the
most frequently occurring p53 mutations in human tumors (Arg-175
His) in the plasmid shuffling assay described above. The wild-type p53
cDNA in pMC147 is replaced by the cDNA of p53
to
generate the plasmid pMC149 (see legend of Fig. 1). As shown in Fig. 2, this mutant p53, unlike its wild-type counterpart, could
not rescue the rft1-1 mutant from death on the galactose/5-FOA
medium. This result indicates that the yeast mutant rft1-1 probably requires functional p53 for its viability.
Figure 3:
The sequence of the RFT1 gene. A, the restriction map of the 2.5-kb XhoI/EcoRI fragment containing the RFT1 gene. The direction of transcription is indicated by the arrow. The acidic domain is shown as the dottedbox and the basic domain as the stripedbox. The N-terminal BglII site was used to
insert the c-Myc epitope tag (see text). B, the complete
sequence of the 2,550 base pair XhoI/EcoRI fragment.
This fragment was sufficient to complement both the rft1-1 point mutant and the rft1 deletion
mutant.
Figure 4: Sequence alignment between Rft1 and the large T antigens of SV40 and polyoma virus. The amino acid sequences in single-letter code are from 372 to 401 (Rft1), 364 to 393 (SV40 T) (Fanning and Knippers, 1992), and 365 to 394 (POV T) (Frisque et al., 1984). Identical residues are boxed.
The DNA sequence comprising the RFT1 locus has been published by Hahn et al.(1988), who studied the HAP3 gene, which is located 5` adjacent to the RFT1 coding sequence in the yeast genome. This allows mapping of the RFT1 locus to chromosome II. A 3-kb transcript from this region, synthesized constitutively in glucose and lactate media and independently of the hap3 mutation, had already been noticed (Hahn et al., 1988) and was confirmed in our Northern blot experiment to be the transcript of the RFT1 gene (data not shown). However, due to some sequencing errors in the RFT1 locus reported by these authors, the complete and correct open reading frame of RFT1 was not identified in their publication.
One copy of the RFT1 gene in a diploid strain (YNN413, Table 1) was disrupted by one-step gene replacement (Rothstein, 1983) using the LEU2 marker. The diploid strain thus generated, YMC349, confirmed by Southern blotting to be heterozygous for the rft1 deletion/disruption, was subjected to sporulation. Tetrad dissection revealed 2:2 segregation for viability and the LEU2 marker co-segregated with the lethal spores (data not shown, but see Fig. 5). This demonstrated that the RFT1 gene is essential. Transformation of the YMC349 strain prior to sporulation with a plasmid containing the galactose-inducible RFT1 construct (pMC195) gave rise to two viable spores per tetrad after dissection on glucose plates (YEPD), but more than two viable spores on galactose plates (YEPG) (Fig. 5).
Figure 5: The RFT1 gene is essential for cell viability. The diploid strain YMC349 heterozygous for rft1 deletion mutation was transformed with pMC195, a plasmid carrying galactose-inducible RFT1 construct. After sporulation, tetrad dissection was performed on either glucose/rich plate (YEPD) or galactose/rich plate (YEPG). The photograph was taken after 4 days of incubation.
Figure 6:
The G arrest phenotype of the rft1 mutant. A, accumulation of large-budded cells
after shift from galactose to glucose medium. The YMC333 (rft1
::pMC195) cells grown in galactose-containing medium
were washed and diluted into glucose medium at OD
= 0.1. After incubation at 30 °C for 3 h, the culture
was sonicated and photographed under a microscope. B, cell
cycle analysis of the rft1
mutant (YMC333) by flow
cytometry. The cell culture incubated in glucose medium as in Fig. 5A was sampled at the time points
indicated to be processed for FACScan analysis (see ``Materials
and Methods''). Galactose medium was used as a control. The same
culture was also analyzed for cell morphology under a microscope as
shown in Table 2.
Figure 7:
The G arrest phenotype of the rft1
mutant as examined by fluorescent staining. YMC333
cell culture was shifted from galactose to glucose medium as indicated
in Fig. 5. Samples were taken at 0 h (A and B)
as well as 4 h (C and D) after the medium
shift and stained for DNA with DAPI (A and C), and
spindles with anti-tubulin antibody (B and D).
Prolonged incubation of rft1 mutants in glucose (9 h or longer) generated a population of cells with multiple buds which could reach to 35% of the total by 12 h (Table 2). These multi-budded cells proved to be dead cells because they could not divide again after being transferred to galactose medium by micromanipulation, whereas the single-budded cells revived well on galactose medium (data not shown).
As the size of the protein (55 kDa)
detected by the antibody raised against the C terminus of Rft1 is
considerably smaller than the calculated molecular mass of 66.2 kDa,
the possibility existed that the N terminus of the Rft1 protein is
processed in vivo. To examine this possibility, synthetic
oligonucleotides encoding an antigenic determinant from the c-myc proto-oncogene were inserted into the BglII site of the RFT1 gene at the N terminus. This epitope-tagged version of RFT1 was functional in complementing the rft1 mutation when expressed from a 2-µm plasmid (data not shown).
Using a commercially available antibody against the c-Myc epitope, we
could again detect a 55-kDa protein on Western blot (data not shown),
which was absent in a control strain containing no c-myc tag.
We therefore concluded that the 55-kDa species of Rft1 detected by the
antibody contained the entire predicted sequence.
Figure 9: Rft1 forms complex with human p53 in vivo. Lysates were made from wild-type cells (WT, lanes 2, 6, and 7) carrying the GAL-p53 construct on a multicopy plasmid or from the rft1-1 mutant (YMC298, lanes1, 3, 4, and 5) in the presence of 1% Triton. All cultures were grown on galactose/Ura dropout medium except the wild-type strain in lane7, which was grown in glucose/Ura dropout medium to turn off the p53 expression. The sample in lane3 was immunoprecipitated with preimmune serum (PRE). Samples in lanes 4-7 were immunoprecipitated with affinity-purified polyclonal anti-Rft1 antibody (K2). Lane5 is the immunoprecipitation carried out in the presence of competing Rft1 peptide (K2 + peptide). The immunoblot was probed with the monoclonal p53 antibody Ab1. The lower band in lanes 3-7 is due to cross-reaction of rabbit IgG heavy and light chains with anti-mouse secondary antibody used in the immunoblotting.
It has recently become clear that the role of p53 in tumor suppression or prevention depends, at least in part, on its function in cell cycle regulation in response to DNA damage. Although significant progress has been made in the field of p53 research, there are still important questions that remain unanswered. For example, what is the mechanism that controls the level of p53 in response to DNA damage? Why do p53-deficient cells survive better than wild-type cells after DNA damage (Lee and Bernstein, 1993)? How is the signal for DNA damage transmitted to, and detected by, p53? Does p53 have any functions in regulation of cell cycle progression in the absence of DNA damage when its activity is not elevated? Identification and characterization of p53-related protein(s) in classical genetic systems like yeast will no doubt help elucidate these mechanisms.
As the previous attempts to probe for the yeast p53 homolog by immunological and recombinant DNA technologies failed (Nigro et al., 1992), we tried to identify p53-related proteins in yeast by using the colony color sectoring assay. This approach is ideal for identifying the yeast homolog of a protein from an evolutionarily distant organism (Kranz and Holm, 1990). It does not rely on the presence of extensive homology between the two proteins because it works on the basis of functional substitution. Therefore, functional homologs with low levels of sequence homology unsuitable for detection by the conventional techniques of DNA hybridization and cross-species antibody reaction may be obtained with this approach. This approach, however, is limited to essential genes. It is known that p53 is not essential for viability in mammals (Donehower et al., 1992). Nevertheless, it is still possible that the yeast p53 homolog, or a protein whose function can be replaced by human p53, may be encoded by an essential gene in yeast which therefore we could isolate using this assay. Additionally, we may also use this approach to isolate essential genes in yeast whose protein products are recognized by p53 such that the mutants are dependent on p53 for viability. In this case, one would expect to screen a large number of colonies considering the possibility of allele specificity. Indeed, the rft1-1 mutant we isolated this way, as we have described, is lethal in the absence of p53 because p53 complexes with the Rft1 protein in vivo. This single allele was isolated from over 100,000 mutagenized colonies, a frequency much lower than that of simple loss of function mutations (Kranz and Holm, 1990).
Nigro et al. described the growth-inhibitory effects of p53 on yeast cells, which were dependent on the strain being protease-deficient (Nigro et al., 1992). With a non-protease-deficient strain, these authors could not detect the expression of p53 by Western blot assays and observed little or no differences in growth between cells containing wild-type p53 and no p53 (Nigro et al., 1992). All strains in our experiments were non-protease-deficient. Like these authors, we observed no growth retardation upon p53 induction by galactose. We even tried using a multicopy plasmid (2 µm) to carry the GAL-p53 construct and obtained the same results (data not shown). However, the p53 protein was readily detectable in our strains by Western blotting. Although we did not try co-expression of p53 with CDC2 like these authors did, it is certain that the overexpression of p53 alone is not sufficient to cause significant growth-inhibitory effects in our strains.
We have demonstrated that the rft1-1 mutant depends on p53 for viability through the plasmid shuffling
experiment. The finding that a mutant p53, p53, cannot
support the growth of the rft1-1 mutant suggests that rft1-1 cells require active p53 protein. The p53
mutation is one of the most frequently observed p53 alterations
in human cancers (Hollstein et al., 1991; Levine et
al., 1991). The mutant protein is found to be associated with the
heat shock protein hsc70 (Hinds et al., 1987), recognized by
the antibody that is specific for denatured p53 (Gannon et
al., 1990), and unable to transactivate p53-dependent promoters
(Ory et al., 1994). It has a much longer half-life than that
of the wild-type protein (Levine et al., 1991). The
p53
mutant is dominant to the wild-type protein since
it can efficiently transform primary rat cells in culture in
co-operation with the oncogene ras (Levine et al.,
1991). The p53
cDNA used in this study has been
previously shown to be capable of overriding the growth-inhibitory
effects imposed by the overexpressed wild-type p53 in the yeast S.
pombe (Bischoff et al., 1992). Although we did not test
to see if the p53-supported growth of the rft1-1 mutant can be
abrogated by expression of the p53
cDNA, it is clear
that the p53
cDNA is unable to suppress the rft1-1 mutation. In this regard, the rft1-1 mutant may be
valuable for identifying molecules that can convert the mutant p53 to
wild-type, by screening for chemicals that are able to support the
growth of the rft1-1 mutant containing p53
.
Such molecules may lead to useful pharmaceuticals for cancer treatment.
The genetic and biochemical data obtained in this study indicate that the Rft1 protein forms a specific complex with p53 in yeast. The Rft1 protein contains a sequence that shares a significant homology with a motif in SV40 T and human polyomavirus T antigens (Fig. 4), which lies within the region of SV40 T known to be responsible for p53 binding (Fanning and Knippers, 1992). The monoclonal antibodies that recognize this region in SV40 T block the association of T antigen with p53 (Lane and Gannon, 1986). Point mutations at a residue immediately adjacent to this motif in SV40 T (402) abolish the p53-binding of T antigen completely (Lin and Simmons, 1991). Therefore, it is reasonable to assume that this motif in Rft1 may also participate in p53 binding, although no attempt has been made to verify this assumption.
Given the fact that Rft1 contains a putative p53-binding motif, it may be argued that the suppression of the yeast mutation rft1-1 by human p53 may simply be fortuitous. It is reasonable, for example, to suggest that the mutant protein (Rft1-1) is unstable, or improperly folded in the absence of p53. In the presence of p53, the Rft1-1 protein can be stabilized or correctly folded by the complex formation with p53 thanks to its T antigen-like motif that p53 recognizes. This interpretation, however, is not favored by the following observations. First, since the mutant protein binds to p53 equally well as, if not better than, the wild-type protein (Fig. 9), it is unlikely that the Rft1-1 protein is significantly different from the wild-type protein in conformation relevant to p53 binding. Second, the inviability of the rft1-1 mutant in glucose-containing media does not seem to be due to the instability of the mutant Rft1 protein, since in the absence of p53 (in glucose) the mutant Rft1 protein was as stable as the wild-type protein for at least 4 h (data not shown). Based on these observations, it is sensible to suggest that, in addition to its binding to Rft1, some aspects of the p53's function may be responsible for the suppression of rft1-1 mutation.
The
function of Rft1 in yeast cell cycle is still unclear at present. Two
lines of evidence invite the speculation that Rft1 may be involved in
the process of DNA replication. First, under nonpermissive conditions
(glucose medium), rft1 mutants stop or delay their cell cycle
at G, reminiscent of some cdc mutants defective in
DNA replication or initiation of mitosis (Pringle and Hartwell, 1981).
Second, the G
arrest of rft1 mutants in glucose
media is not dependent on the checkpoint gene rad9, which does
not monitor the process of DNA replication (Weinert et al.,
1994). The rft1-1 mutant, however, may form synthetic
lethality with the DNA replication checkpoint mutant mec1 (Weinert et al., 1994) since the double mutant could only
form very small spore colonies on galactose-containing tetrad
dissection plates and failed to grow upon inoculation into YEPG (data
not shown). Another DNA replication checkpoint mutation mec2,
on the other hand, has little effect on the rft1-1 mutant
(data not shown). The Rft1 protein is highly hydrophobic. It is,
however, a soluble protein owing to the presence of one acidic and one
basic regions. Since p53 has been shown capable of activating
transcription in yeast (Fields and Jang, 1990), it is plausible that
Rft1 may play a role in transcription, although the sequence of Rft1
shows no obvious similarity to classical transcriptional factors.
While there has been no concrete evidence to support the presence of a p53 homolog in yeast, the yeast p53-related protein may still exist in a structurally poorly conserved form and is likely to be encoded by a non-essential gene. The rft1-1 mutant may be useful for identifying it. It is possible, for example, that the yeast p53 homolog could be isolated as a multicopy suppressor of the rft1-1 mutant. Alternatively, purification of Rft1-binding factors from yeast may also lead to a p53-related protein. In view of the specific interactions between p53 and Rft1, the Rft1 protein may represent a novel p53 binding protein in mammalian cells. The identification and isolation of the putative human homolog of Rft1 may widen our knowledge on p53 and help better understand the function of this important protein.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U15087[GenBank].