Nucleoside-diphosphate kinase (NDK; EC 2.7.4.6) (
)is
a ubiquitous enzyme that catalyzes transfer of the terminal phosphate
group of ribo- and deoxyribonucleoside 5`-triphosphates ((d)NTPs) to
nucleoside 5`-diphosphates ((d)NDPs) by a ping-pong
mechanism(1) . This enzyme functions as a hexamer, and two
kinds of subunits have been identified in higher eukaryotes including
human(2) . NDK has been believed to be involved in the
production of all kinds of nucleoside triphosphates except ATP and in
the maintenance of their levels in the cell.
Molecular cloning of
cDNAs encoding NDK has been performed in various
species(3, 4, 5) . Interestingly, recent
studies suggest that NDK may have a function other than as a simple
enzyme. In Drosophila melanogaster, the abnormal wing
discs gene (awd) encodes a NDK homolog(6) . The
null mutation in awd causes abnormalities in the development
of larvae, which result in their death at the prepupal
stage(7, 8) . The nm23 gene encodes NDK in
mammals(9) . It was originally cloned as a gene that was
differentially expressed in two lines of murine K-1735 melanoma cells,
which were distinct in their metastatic activities, and it was
considered to be a candidate suppressor of tumor metastasis (10, 11, 12) . Furthermore, human PuF, a
factor required for the transcriptional activation of c-myc in
vitro, and mouse I-factor, which inhibits differentiation of
murine leukemia M1 cells in the monocyte/macrophage pathway, are highly
similar to the product of human nm23-H2(13, 14) . NDK has been shown to be
able to interact with a GTP-binding protein G
,
microtubules, and succinyl-CoA synthetase, and it has been postulated
that NDK may regulate them(15, 16, 17) . It
has been also shown that the cytoplasmic membrane of Dictyostelium possesses NDK activity that is stimulated by a cAMP receptor and
that this activity in turn contributes to the activation of G proteins,
although precise mechanisms for this regulation remain
unknown(18) .
The fission yeast Schizosaccharomyces
pombe provides a useful model system for the molecular genetic
study of intracellular signal transduction. S. pombe haploid
cells proliferate mitotically under rich nutritional conditions. They
undergo sexual development, namely mating, meiosis, and sporulation,
when starved for nitrogen(19, 20) . Many genes have
been shown to be involved in this sexual process. They include the
genes for two kinds of heterotrimeric G proteins. One of them mediates
the nutritional signal and regulates the cAMP cascade(21) . The
other, together with the single ras homolog in fission yeast,
controls mating pheromone
signaling(22, 23, 24) . In this report we
describe cloning of a fission yeast gene encoding NDK. Molecular
genetic analysis of its physiological function suggested that fission
yeast NDK may have a regulatory role in the mating pheromone signaling.
EXPERIMENTAL PROCEDURES
Yeast Strains, Media, and Genetic
Procedures
S. pombe strains used in this study are
listed in Table 1. General genetic procedures for S. pombe have been described(25) . Yeast complete medium YPD (26) and three kinds of minimal medium, namely SD(26) ,
SSA(27) , and PM(28, 29) , were used. PM-N is
a nitrogen-free derivative of PM. SD, PM, and PM-N used in this study
contained 1% glucose instead of 2% described in the original recipes. A
lithium method was used for transformation of S.
pombe(30) . A semiquantitative assay of the mating
efficiency was done by staining colonies with iodine vapor, which
stains spores dark brown(25) .
Cloning of ndk1
Two sets of degenerated
oligonucleotides, namely 5`-GCNAT(T/C/A)AA(G/A)CCNGA(T/C)GGNGT-3` and
5`-CC(T/C)TCCCA(A/T)ACCAT(G/A/T)G(G/C)NACNACNGGNCC-3`, were prepared as
the primers for polymerase chain reaction (PCR). N in the primers
stands for a mixture of G/A/T/C. The former set corresponds to the
amino acid sequence AIKPDGV and the latter, GPVV(L/A/P)MVWEG
(antisense), both of which are highly conserved among NDKs of various
species (see Fig. 2). PCR was carried out under the following
conditions. A reaction mixture (30 µl) contained approximately 50
ng of S. pombe genomic DNA as a template, 100 pmol of each
primer, 50 mM KCl, 100 mM Tris-HCl (pH 8.3), 2
mM MgCl
, 0.2 mM each dNTP, 0.01% gelatin,
0.4% Triton X-100, and 1 unit of Taq polymerase (Perkin Elmer
or Promega). This mixture was subjected to 45 cycles of 93 °C 1
min, 45 °C 1 min, and 72 °C 1 min. After one round of
amplification, an aliquot of the mixture was transferred as the
template to a fresh reaction mixture devoid of S. pombe DNA,
and another round of amplification was performed. The mixture was then
electrophoresed in a 4% agarose gel (NuSieve) and was stained with
ethidium bromide. DNA fragments were recovered from a band
corresponding to the size of 0.2 kb, using Mermaid kit (BIO 101, Inc.).
The fragments were cloned into the EcoRV site of pBluescript
SK(+) vector (Stratagene). The resulting clones were screened
using a part of the human nm23-H1 gene, which we amplified
from a HeLa cell cDNA library by PCR (data not shown), as the
hybridization probe. We obtained altogether eight positive clones in
this screen and sequenced their inserts. Four of them turned out to be
identical except for the terminal regions, which appeared to reflect
heterogeneity of the original PCR primers. Using one of these inserts
as a probe, a 6.0-kb EcoRI fragment that covered the entire ndk1 gene was cloned from the S. pombe genome into
pUC119.
Figure 2:
Comparison of amino acid sequences between
Ndk1 and NDKs in other species. S.c., S. cerevisiae Ynk(52) ; Mus.A, mouse Nm23-M1(10) ; Hum.A, human Nm23-H1(11) ; Rat A, rat
NDK-
(54) ; Mus.B, mouse Nm23-M2(55) ; Rat B, rat NDK-
(3) ; Hum.B, human
Nm23-H2(56) ; D.m., D. melanogaster Awd(6) ; D.d., D. discoideum NDK(4) ; and Myx., M. xanthus NDK (5) . Identical amino acids are shown in white against
black. Numbering of the amino acid residues is given on the right-hand side of the panel. The percentage given at the end
of each amino acid sequence stands for the degree of identity with
Ndk1. The histidine residue at the putative active center is marked
with an asterisk.
Southern Hybridization
Essentially, a standard
protocol (31) was followed. Hybridization using a part of the nm23-H1 gene as a probe was done at 42 °C overnight in 5
SSC (0.75 M NaCl, 0.075 M sodium citrate)
containing 0.1% N-lauroylsarcosinate, 0.02% SDS, 5% blocking
reagent (Boehringer Mannheim), and 20% formamide. Washing was carried
out in 2
SSC containing 0.1% SDS at room temperature for 5 min
twice and then at 42 °C for 30 min twice. In all other cases, the
concentration of formamide in the hybridization solution was raised to
50%, and additional washing was carried out with 0.1
SSC
containing 0.1% SDS at 42 °C for 30 min after the same washing as
above was done.
DNA Sequence Determination
Nucleotide sequences
were determined by the dideoxy chain termination method(32) .
DNA fragments to be sequenced were cloned into either pUC119 or
pBluescript. Subclones for stepwise sequencing were generated by
progressive deletion with exonuclease III and S1 nuclease
(Takara-shuzo)(33) . Single-stranded DNA was prepared by using
M13KO7 as a helper bacteriophage. The sequence shown in Fig. 1B was determined in both directions at least
once. A primer 5`-ATTCATTCGTAAATCCAAGG-3`, which is complementary to
the carboxyl-terminal region of ndk1, was used to determine
the sequence displayed in Fig. 5B.
Figure 1:
A restriction map and
the nucleotide sequence of the ndk1 locus. A, a
restriction map of the ndk1 locus. The arrow indicates the position and orientation of the ndk1 ORF.
The construct used for disruption of the chromosomal ndk1 gene
is shown underneath. Restriction sites are abbreviated as follows: E, EcoRI; EV, EcoRV; Sp, SphI; Hc, HincII; S, SalI; Pv, PvuI; and C, ClaI. B,
the nucleotide and deduced amino acid sequences of ndk1. The
nucleotide sequence of a 1.2-kb SalI-HincII fragment
and its 5`-flanking region is shown. Numbering starts at the putative
initiation codon for both nucleotide and amino acid residues. Underlines indicate highly conserved regions according to
which primers for PCR were designed.
Figure 5:
Characterization of the dominant- negative
allele of ndk1. A, JY450 cells transformed with
either pREP1, pREP-ndk1, or pREP-ndk1-dn were streaked on SSA medium
and incubated for 4 days. Cells were then exposed to iodine vapor,
which stains sporulated cells dark brown. B, the ndk1-dn allele was cloned in pBluescript, and the nucleotide sequence was
determined. The left panel shows a sequence ladder for the
wild type ndk1 gene, and the right panel shows the
counterpart for the ndk1-dn allele. The nucleotide and deduced
amino acid sequences were shown at the side of each panel. The arrow indicates the position where a mutation was found. C, NDK activity of the ndk1-dn gene product. Crude
extracts were prepared from JX23 cells carrying either pREP-ndk1 (ndk1
) or pREP-ndk1-dn (ndk1-dn),
and NDK activity in each sample was
assayed.
Disruption of the ndk1 Gene
One-step gene
disruption of ndk1 was done essentially as
described(34, 35) . A pUC-based plasmid was
constructed by inserting the following three fragments into the
multicloning sites of the vector in this order: a 2.0-kb SphI-EcoRV fragment carrying the region 5` upstream
to the ndk1 ORF, a 1.8-kb S. pombe ura4
cassette(36) , and a 3.0-kb PvuI-EcoRI
fragment carrying the 3`-terminal region of ndk1 and its
downstream vicinity, of which the PvuI end was blunted. The
resultant plasmid had a disrupted ndk1 allele, which lacked a
0.6-kb EcoRV-PvuI fragment covering most of the ndk1 ORF and carried the ura4
cassette in place. An SphI fragment that encompassed this
disrupted allele (see Fig. 1A) was introduced into a
diploid strain constructed by crossing JY878 with JY879 (h
/h
ade6-M210/ade6-M216 leu1/leu1
ura4-D18/ura4-D18). Ura
transformants
were selected, and genomic DNA was prepared from each of them. DNA
preparations thus obtained were digested with EcoRV and
examined by Southern hybridization. Diploid strains in which one ndk1 allele was properly replaced were chosen and subjected to
tetrad analysis.
Preparation of S. pombe RNA and Northern Blot
Analysis
S. pombe cells collected at the cell density
of 4-6
10
/ml were broken by vortexing
vigorously with glass beads in a buffer (0.2 M Tris-HCl (pH
7.5), 0.5 M NaCl, 0.01 M EDTA, 1% SDS). After two
rounds of extraction with phenol:chloroform (1:1), RNA was recovered by
ethanol precipitation. Each RNA preparation (10 µg) was denatured
with formamide, separated by formaldehyde gel
electrophoresis(37) , and blotted to a piece of Hybond-N
membrane (Amersham). DNA probes to detect transcription of ndk1 (1.2-kb SalI-HincII fragment in Fig. 1A), ste11(38) , mei2(29) , mat1-P(39) , and sxa2(40) were prepared by labeling appropriate fragments
described in the original reports. Labeling was done with
[
-
P]dCTP using random primers.
ndk1 Plasmids
pREP-ndk1 was constructed by
ligating a 1.2-kb SalI-HincII fragment that carried
the ndk1 ORF to the vector pREP1(41) , which was
digested with SmaI and SalI and treated with calf
intestine alkaline phosphatase. The SalI site covered the
first through third amino acid residues of Ndk1 (see Fig. 1B), and the exact amino-terminal sequence of Ndk1
was regenerated in pREP-ndk1. pREP-ndk1-dn was constructed by ligating
a 0.7-kb SalI-ClaI fragment that carried the ndk1 variant allele to pREP1 by a similar procedure.
Enzymatic Assay of NDK
S. pombe cells
(1-3
10
) were collected by centrifugation and
disrupted in a lysis buffer (20 mM Tris-HCl (pH 7.4), 10
mM MgCl
, 1 mM EDTA, 5% glycerol, 1
mM dithiothreitol, 0.3 M
(NH
)
SO
) containing 1 mM phenylmethanesulfonyl fluoride. Cellular debris was removed by
centrifugation in a microcentrifuge. The protein concentration of each
preparation was determined by the BCA Protein Assay System (Pierce).
Measurements of NDK activity were carried out according to a previous
report (42) with a slight modification, as described below. 10
µl of each cell lysate diluted appropriately with TEA buffer (100
mM triethanolamine HCl (pH 8.0), 10 mM
MgCl
, 2 mM
-mercaptoethanol) was added to 20
µl of TEA buffer containing 12.5 mM [
-
P]ATP (2 mCi/mmol) and 10 mM dCDP. The mixture was incubated at 37 °C for 15 min and then
boiled for 5 min to inactivate the enzymatic activity. 3 µl of each
sample was spotted onto a polyethyleneimine TLC plate (Macherey-Nagel)
and developed in 0.85 M KH
PO
(pH 3.4).
Radioactivity in the resultant dCTP spots was either detected by
exposing an x-ray film to them or quantitated by using a Bioimage
Analyzer (BAS 2000, FUJIX).
Western Blot Analysis
Crude extracts were prepared
from S. pombe wild type cells, ndk1
cells, and
cells overexpressing ndk1, and their protein concentration was
determined as above. Each sample containing 50 µg of protein was
separated by SDS-polyacrylamide (14%) gel electrophoresis (43) and then electrotransferred onto a piece of Immobilon-P
membrane (Millipore) at 8 V/cm in a transfer buffer (24 mM
Tris, 192 mM glycine (pH 8.3), 20% methanol, 0.1% SDS) at 0
°C for at least 3 h. The membrane was incubated in TBST buffer (20
mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% Tween 20)
containing 3% skim milk for 1 h at room temperature and washed a few
times with TBST buffer. The membrane was then incubated with anti-rat
NDK polyclonal antibodies (44) in TBST buffer containing 3%
skim milk for 1 h at room temperature. After washing, alkaline
phosphatase-conjugated goat anti-rabbit IgG (Sigma), diluted with TBST
buffer containing 3% skim milk, was applied as the secondary
antibodies. The color reaction was performed in AP buffer (100 mM Tris-HCl (pH 9.5), 100 mM NaCl, 5 mM
MgCl
) using 5-bromo-4-chloro-3-indolyl phosphate and nitro
blue tetrazolium (Sigma), essentially as described(45) .
Hydroxylamine Mutagenesis
Hydroxylamine is a
mutagen known to introduce point mutations. We essentially followed a
standard protocol(46) . 350 mg of hydroxylamine was resolved in
0.56 ml of a freshly prepared 4 N NaOH, which was then diluted
with 5 ml of sterile water (Solution A). 0.5 ml of Solution A was mixed
with 5 µl of 0.2 M EDTA and 5 µg of pREP-ndk1. The
mixture was incubated at 37 °C for 20 h. 10 µl of 5 M NaCl, 50 µl of 0.1% bovine serum albumin, and 1 ml of cold
ethanol were then added, and DNA was recovered by centrifugation.
RESULTS
Cloning of the ndk1 Gene Encoding a Homolog of
NDK
To clone a gene encoding a NDK homolog in fission yeast, we
carried out the polymerase chain reaction using a pair of primers
corresponding to highly conserved amino acid sequences in NDK, as
detailed under ``Experimental Procedures.'' We eventually
obtained a 0.2-kb DNA fragment that could hybridize with a piece of
human nm23-H1 cDNA. Sequence analysis suggested that this
fragment carried part of the gene encoding a NDK homolog, which we call ndk1 hereafter. We then cloned a 6.0-kb EcoRI
fragment that appeared to contain the entire ndk1 gene, using
the 0.2-kb fragment as a probe. Fig. 1A shows a restriction
map of the ndk1 locus. Further Southern blot analysis suggested
that the ndk1 ORF is located on a 1.2-kb SalI-HincII fragment. We determined the nucleotide
sequence of this fragment and its vicinity (Fig. 1B).
The ndk1 gene appeared to have a coding capacity of 151 amino
acids with no intron. The deduced Ndk1 protein was more than 50%
identical with most of NDKs from other species. The highest identity
(62.3%) was observed with S. cerevisiae Ynk and the lowest
(41.7%), with one from Myxococcus xanthus (Fig. 2).
Disruption of ndk1 Results in No Obvious
Phenotype
S. pombe diploid strains in which one ndk1 allele was disrupted as schematically shown in Fig. 1A were constructed (see ``Experimental
Procedures''). Sporulation was induced in these strains, and
resultant asci were subjected to tetrad analysis. Four viable spores
were recovered in most asci, and Mendelian segregation of the disrupted
and normal ndk1 alleles among progeny spores was confirmed by
Southern blot analysis (data not shown). Haploid cells carrying the
disrupted ndk1 allele appeared to be normal in vegetative cell
growth and sexual development.
NDK Activity of the ndk1 Gene Product
A plasmid
named pREP-ndk1, which overexpresses ndk1 from the nmt1 promoter(41) , was constructed as described under
``Experimental Procedures.'' Crude extracts were prepared
from S. pombe strains JY450 (ndk1
),
JX23 (ndk1
), and JX23 transformed with pREP-ndk1. The NDK
activity in each extract was assayed, and the results are summarized in Fig. 3A. The activity in JX23 was only 20% of that in
JY450, whereas the activity in JX23 transformed with pREP-ndk1 was more
than 10 times higher than that in JY450. This strongly suggests that
the product of ndk1 is responsible for the NDK activity.
Furthermore, Western blot analysis of crude extracts prepared from
JY450, JX23, and JY450 transformed with pREP-ndk1 demonstrated that the ndk1 gene product cross-reacts with anti-rat NDK antibodies
previously described (44) (Fig. 3B). We thus
conclude that the ndk1 gene encodes NDK in fission yeast.
Figure 3:
Biochemical analysis of the ndk1 gene product. A,
-phosphate transfer activity of the ndk1 gene product. Crude extracts were prepared from JY450
(denoted wild type, closed bar), JX23 (disruptant, open bar), and JX23 carrying pREP-ndk1 (disruptant + pREP-ndk1, hatched bar). Given
amounts of each sample were assayed for NDK activity. B,
Western blot analysis of cell extracts with anti-rat NDK antibodies.
Crude extracts were prepared from JY450 (wild type), JX23 (disruptant), and JY450 carrying pREP-ndk1 (wild type
+ pREP-ndk1). 50 µg of protein of each sample was
separated in SDS-polyacrylamide gel electrophoresis, transferred to a
membrane, and analyzed using anti-rat NDK antibodies. The arrow indicates the ndk1 gene product cross-reacting with the
antibodies. The numbers on the left-hand side represent molecular mass markers
(kDa).
Expression of the ndk1 Gene
Expression of ndk1 was examined in S. pombe heterothallic haploid (h
and h
),
homothallic haploid (h
), and diploid (h
/h
) cells by
Northern blot analysis. The ndk1 gene was transcribed into a
0.7-kb-long transcript irrespective of the mating type of the cell (Fig. 4). The level of this transcript was increased upon
nitrogen starvation, suggesting that ndk1 may have a function
required under the starved conditions.
Figure 4:
Expression of the ndk1 gene.
Cells of JY334 (h
) (lanes 1 and 2), JY333 (h
) (lanes 3 and 4), JY450 (h
) (lanes 5 and 6), and JY362 (h
/h
) (lanes 7 and 8) were cultured in PM medium. Half of each culture
was harvested at midlog phase (4-6
10
cells/ml) (lanes 1, 3, 5, and 7). The remainder was transferred to PM-N medium, incubated
for 4 h, and collected (lanes 2, 4, 6, and 8). Total RNA was prepared from these samples. 10 µg of
each RNA preparation was separated in formaldehyde-agarose gel
electrophoresis, blotted onto a membrane, and analyzed with a
hybridization probe carrying the ndk1 ORF. rRNAs stained with
ethidium bromide are shown in the bottom panel to confirm that
nearly the same amount of RNA was loaded in each
lane.
The ndk1-dn Allele Interferes with Sexual Development in
the Fission Yeast
We postulated that the ndk1-disrupted
strains showed no obvious phenotype because the remaining NDK activity,
although very small, was sufficient for cell growth and sexual
development. Postulating further that NDK subunits other than Ndk1,
which can complex with Ndk1 to form a hexamer, are responsible for this
residual activity, we suspected that it might be possible to obtain a
dominant-negative (dn) allele of ndk1 that completely
diminishes cellular NDK activity. Alternatively, if Ndk1 has any
regulatory function in S. pombe cells, it may also be possible
to obtain a dn allele of ndk1 that blocks the
function of its target molecule. We thus set out to screen for such an ndk1-dn allele. pREP-ndk1, which carried the ndk1 gene, was mutagenized with hydroxylamine, and a homothallic
haploid strain JY450 was transformed with the mutagenized plasmid
library. As it was suggested that ndk1 may play a significant
role under the starved conditions (Fig. 4), we examined 5,000
transformants by iodine staining for their ability to mate and
sporulate. Three transformants were found to be poor in this ability.
We could recover a plasmid from only one of them. The ndk1 ORF
on this plasmid was excised and recloned into pREP1 that was not
treated with hydroxylamine. The resultant plasmid (pREP-ndk1-dn) could
inhibit mating and sporulation upon transformation (Fig. 5A), confirming that a mutation(s) in the ndk1 gene is responsible for the sterility of the
transformant. The mating efficiency of JY450 cells carrying
pREP-ndk1-dn was about 20%, while that of the cells carrying the vector
was about 70%. Similarly, the sporulation efficiency of JY362 cells
carrying pREP-ndk1-dn was about 15%, while that of the control was 45%.
Characterization of the ndk1-dn Mutation and the Mutant
Gene Product
We determined the nucleotide sequence of the ndk1-dn allele. One point mutation, which replaces the
cysteine residue at position 116 with tyrosine, was found (Fig. 5B). This mutation site was next to the putative
active center for the enzymatic activity (His
). To
examine NDK activity of the mutant gene product, the ndk1-dn allele was overexpressed from the nmt1 promoter in ndk1
cells. However, the overexpression caused no
increase in the NDK activity, clearly indicating that the ndk1-dn gene product does not have the enzymatic activity (Fig. 5C). We tried to determine the level of the
residual NDK activity in the ndk1-disrupted cells transformed
with pREP-ndk1-dn. Despite repeated trials, we were unable to reach any
firm conclusion concerning whether the cells transformed with ndk1-dn have a lower level of NDK activity than the ndk1
cells, because the level was very low in both types
of cells and the detected difference was within the range of
experimental fluctuation.
The ndk1-dn Allele Decreases the Level of Transcripts
Induced by Mating Pheromone Signaling
Effects of ndk1-dn on the expression of genes involved in the regulation of sexual
development in fission yeast were examined. Characteristics of the
genes we examined are summarized briefly below. The ste11 gene
encodes a transcription factor that induces a number of genes required
for sexual development, which include mei2, mat1-P, mat1-M, and ste6(38) . Ste11 carries an high
mobility group motif and binds to the TR box (TTCTTTGTTY) commonly
found in the 5`-upstream of these genes. The mei2 gene encodes
an RNA-binding protein, which is essential for the initiation of both
premeiotic DNA synthesis and meiosis I, and forms a complex with meiRNA
to promote meiosis I(47) . The mat1-P gene has two
transcription units, termed mat1-Pc and mat1-Pi (m), the former of which is required for mating whereas
the latter is required for meiosis(39) . The sxa2 gene
encodes a protease that is supposed to degrade mating pheromone
P-factor(40) . As shown in Fig. 6, transcripts of mat1-Pi and sxa2 were less abundant in cells
transformed with pREP-ndk1-dn than in cells transformed with the
vector, while transcripts of ste11 and mei2 were
equally abundant. The expression of mat1-Pi and sxa2 calls for both mating pheromone signaling and nutritional
signaling(23, 40, 48) , while that of ste11 and mei2 depends only on nutritional
signaling(29, 38) . Thus, our results suggest the
possibility that the ndk1-dn allele interferes with the mating
pheromone signaling pathway.
Figure 6:
Expression of genes required for sexual
development in ndk1-dn-transformed cells. JY450 cells
transformed with either pREP1 or pREP-ndk1-dn were grown to midlog
phase in PM medium. A portion of each culture was harvested (0 h), and
the remainder was transferred to PM-N medium. Sampling was done at 4,
6, and 8 h after the transfer. Total RNA was extracted from each
sample, electrophoresed in formaldehyde-agarose gel, blotted onto a
membrane, and analyzed with appropriate hybridization probes for ste11, mei2, matP, and sxa2. rRNAs
stained with ethidium bromide are shown in the bottom panel to
confirm that nearly the same amount of RNA was loaded in each
lane.
Other Point Mutations in ndk1
NDK mutations that
affect biological processes have been identified in higher organisms.
The Killer of prune mutant of D. melanogaster has a
point mutation in the awd gene(49) . In human nm23-H1, a deletion and a point mutation were discovered (50, 51) . We carried out site-directed mutagenesis of ndk1 to obtain analogous mutations. The proline residue at
position 95 was substituted with serine, mimicking the mutation in awd
, and the serine residue at position 119 was
substituted with glycine, mimicking the mutation in nm23-H1 in
neuroblastoma. However, overexpression of these ndk1 derivatives did not cause any specific phenotype (data not shown).
Unlike the dominant-negative Ndk1, the mutant products of the fly and
human genes have been reported to retain the NDK activity (49, 51) , which may suggest that the ndk1-dn mutation is either qualitatively different from or quantitatively
more effective than these mutations.
DISCUSSION
In this study we identified the ndk1 gene encoding a
NDK homolog in S. pombe. The ndk1 gene product
cross-reacted with anti-rat NDK antibodies and apparently possessed the
enzymatic activity as NDK. The deduced amino acid sequence of Ndk1 was
highly similar to those of NDKs from other species (Fig. 2).
Ndk1 showed the highest similarity to S. cerevisiae Ynk, and
it was also similar to mammalian NDK nearly as well. The histidine
residue known to be phosphorylated when the enzyme assumes a high
energy intermediate form was conserved in Ndk1 as His
.
The residual NDK activity in ndk1-disrupted cells was less
than 20% of the wild type level. However, cells defective in ndk1 showed no obvious phenotype. This indicates that S. pombe cells naturally have NDK activity in large excess, most of which
is dispensable for the usual life cycle. The same situation was
observed when S. cerevisiae YNK was disrupted (52) .
What is responsible for the remaining NDK activity, then? It is
suggested that (d)NTPs can be produced by the function of pyruvate
kinase(53) . However, it may be more likely that S. pombe has another gene encoding a NDK homolog, as is the case in
mammals, although this has not been proven yet.
As described in the
introduction to the text, cases are accumulating in which NDK appears
to function not as a simple enzyme. This study presents evidence that
NDK may play a role in promotion of sexual development in S.
pombe. Introduction of a dominant-negative allele of ndk1, termed ndk1-dn, into wild type cells blocked
their sexual development. The induction of mat1-Pi and sxa2 expression, but not that of ste11 and mei2 expression, was inhibited in these cells. This suggests that ndk1-dn may interfere with the mating pheromone pathway,
because the former two genes are induced in response to mating
pheromone
signaling(23, 39, 40, 48) , as
discussed in more detail under ``Results.''
Although ndk1
strains showed no obvious phenotype, the ndk1-dn allele could inhibit sexual development. One obvious explanation
will be that the dominant-negative Ndk1 depletes the residual NDK
activity provided by the NDK subunits other than Ndk1, by binding to
them. However, we had difficulties in proving this experimentally, and
there remains an alternative possibility that Ndk1-dn may sequester its
target(s). This target will be essential for sexual development and
will have a direct physical contact with the enzyme. In particular, an
interesting possibility will be that S. pombe NDK regulates
the activity of either Ras1 or Gpa1, both of which are GTP-binding
proteins and are essential for the mating pheromone signaling in S.
pombe(22, 23, 35) . Further analyses of
Ndk1 and related proteins are required to clarify how NDK controls
mating pheromone signal transduction in S. pombe.