(Received for publication, April 4, 1997, and in revised form, May 23, 1997)
From the Cell Cycle & Signal Research Unit, A novel gene, psp1+,
which functionally complements a temperature-sensitive mutant defective
in cell cycle progression both in G1/S and G2/M
has been isolated from the genomic and cDNA libraries of
Schizosaccharomyces pombe. Disruption of this gene is
lethal for cell growth at 30 °C indicating that it is an essential
gene for vegetative cell growth. Western analysis of the protein by polyclonal antibody made from glutathione
S-transferase-Psp1 fusion protein indicated that the Psp1
protein exists in two different molecular weight forms depending on the
growth state of the cell. In vitro experiments with a
phosphatase showed that this difference is due to phosphorylation. The
dephosphorylated form of the protein is dominant in actively growing
cells whereas the phosphorylated form becomes the major species when
cells enter the stationary phase. The Cdc2-Cdc13 complex is shown to
phosphorylate the GST-Psp1 fusion protein in vitro, and
site-directed mutagenesis and phosphoamino acid analysis indicated that
the serine residue at position 333 in the carboxyl-terminal region is
required for phosphorylation. In situ fluorescein
isothiocyanate-conjugated antibody staining showed that this protein
tends to be localized to both ends of the cell upon entry into the
stationary phase of cell growth. However, overexpression of the novel
protein Psp1 in actively growing cells inhibits cell growth causing
accumulation of DNA (4n or 8n). Thus we speculate that Psp1 can
function at both G1/S and G2/M phases
complementing the defect of the new mutant we have isolated. It is
likely that Psp1 is required both for proper DNA replication and for
the process of mitosis.
DNA replication (S) and mitosis (M) are the two major events in
the eukaryotic cell division cycle and are proceeded by the two gap
periods, G1 and G2, respectively. Cell cycle
transition from G1 to S phase (G1/S) and from
G2 to mitosis (G2/M) occurs in a strict
sequence. Blocking S phase prevents onset of the subsequent mitosis,
which would be lethal if chromosome replication had not been completed,
and blocking mitosis prevents initiation of the subsequent S phase
which would otherwise lead to increase in ploidy. Dependences of S
phase and mitosis are examples of checkpoints that ensure orderly
progression through the cell cycle. Many genes are thought to be
involved in this process, and tight regulation of function or
expression of the relevant genes is required for proper cell cycle
progression. The checkpoint control at the two transition points,
G1/S and G2/M, ensures either progression or blocking of cell cycle according to the state of the cell. Several regulators such as cyclin-dependent kinases
(CDKs)1 function at these points (1, 2).
CDK activity is subject to regulation by association with positive
regulatory subunits known as cyclins, negative regulators known as CDK
inhibitors (CKIs), and by phosphorylation (reviewed by Pines (3),
Morgan (4), Elledge and Harper (5), and Harper and Elledge (6)). The
balanced function of these factors controls CDK activity and serves to
integrate signals intended to coordinate cell cycle transitions. The
levels of these proteins are tightly regulated both transcriptionally
and post-translationally as the cell cycle progresses. In yeast a
single CDK, Cdc2 in fission yeast and Cdc28 in budding yeast, is
required at the first checkpoint G1/S (7-12), while in
multicellular eukaryotes, several different CDKs, including CDK2, CDK4,
and CDK6, are involved at different stages of G1 and S
phase (13-17). Different G1 cyclins such as CLN1, CLN2,
and CLN3 of budding yeast or cyclin D and cyclin E of mammalian cells
and S phase cyclins such as CLB5, CLB6, or cyclin E and cyclin A are associated with these CDKs to ensure kinase function of the CDKs (18-22) at G1 to S phase progression. The phosphorylation
state of the CDK itself and proper timing of destruction of the cyclins are important for regulation of CDK activities (4). Thus checkpoint control at G1/S by these multiple elements is important for
the cell to commence DNA replication, to initiate the cell cycle, and
to integrate the positive and negative signals of the cell cycle. At
the second checkpoint, G2/M, p34cdc2, the
prototypic member of cyclin-dependent kinase, is known to be the major regulator both in yeast and mammalian cells. Association with G2 cyclins such as cyclin B is required to function as
an active protein kinase and to induce active mitosis (23, 24). The
phosphorylation state of Cdc2 directly affects its kinase function and
cell cycle progression to mitosis (4, 25, 26). Cdc25 dephosphorylates
Cdc2 at serine/threonine residues, and this activates the kinase
function of the Cdc2-Cdc13 (cyclin B) complex (27-29). Meanwhile,
phosphorylation of Cdc2 by Wee1 kinase results in inactivation of Cdc2
kinase function at G2/M (30-33), and destruction of
G2 cyclin by the ubiquitin-associated cyclosome complex
signals the cell to exit from mitosis and to prepare for the next round
of G1 to S phase progression (34). The CKIs such as p21,
p27, or p15, which have negative function to inhibit CDK activity (5,
35), counteract the cyclins and are required to prevent cell cycle
progression when cells are damaged. Mutations in CKI genes cause
unregulated cell cycle progression, and this results in abnormal cell
growth. In particular, deregulation of START at G1/S may
allow cell growth and division to become insensitive to external cues
(36-40). This can be a consequence of either the aberrant expression
of positive regulators, such as the cyclins, or the loss of negative
regulators, such as the CKIs. Thus proper regulation of cyclin-CDK-CKI
complex formation is critical for normal cell cycle progression.
Even though the global cell cycle controls operating at the S (22,
41-45) and M phase transitions (46-48) have been extensively analyzed, unknown relevant factors still remain to be identified. Fission yeast Schizosaccharomyces pombe has been a useful
model organism to study cell cycle control in eukaryotic cells. The relative ease of genetic manipulation of yeast has allowed detailed analysis of gene function in vivo, which has produced a
paradigm for cell cycle control applicable to higher organisms. In an
attempt to isolate factors involved in control of cell cycle
progression, we searched for new cell cycle-related genes in S. pombe. In this paper we describe isolation of a novel gene,
psp1+ (a gene encoding
phosphoprotein at stationary phase of S. pombe cell), essential for progression of cell cycle.
We used a strategy to isolate a new conditional mutant defective in
cell cycle progression by chemical mutagenesis and to find a clone
functionally complementing this mutant phenotype. Genetic and
biochemical studies have permitted us to identify the function of the
newly isolated psp1+ in S. pombe
cells.
The newly isolated temperature-sensitive
(ts To isolate ts To measure DNA content in each
cell cycle point, the cells were prepared by a procedure adapted from
Lew et al. (53) and Costello et al. (54). The
mutant cells were first grown in YEPD liquid medium at 23 °C to
midlog phase (A580 = 0.5) and transferred either
directly to fresh YEPD medium or to nitrogen-deficient EMM (0.003%
NH4Cl) which leads to cell cycle arrest at G1
in S. pombe. The cells transferred to EMM were incubated at
23 °C for 24-36 h to induce cell cycle arrest at G1,
and were then collected, washed, and resuspended in YEPD medium. The
cells transferred to YEPD medium were incubated at 36 °C for 4-8 h
to induce mutant phenotype. Aliquots of these cells (1 × 107 cells) were collected, fixed with 70% ethanol for at
least 12 h at 4 °C, and treated with RNase A (Sigma, 0.1 mg/ml)
for 2 h at 37 °C. After staining the cells with propidium
iodide solution (0.005% propidium iodide, 0.1% sodium citrate, and
0.03% Triton X-100), the DNAs in the cells were analyzed with Becton
Dickinson FACScan System (FACScanTM). Analysis was based on
the accumulation of 8,000-10,000 cells. As a reference, flow
cytometric analysis of the mutant cdc2 To examine the expression pattern
of the psp1+ gene during cell cycle progression,
synchronized cell cultures were used. The cells arrested at S phase by
hydroxyurea or M phase by thiabendazole were used (55-57). ED665 cells
were first grown in EMM to a cell density of
A580 = 0.5 (1 × 107 cells/ml)
and then in the presence of 12 mM hydroxyurea or 100 µM thiabendazole at 30 °C for an additional 4-5 h to
arrest them at S or M phase, respectively. The cells were then
collected by filtration, washed, transferred to fresh EMM, and
incubated at 30 °C to release them from S or M phase arrest.
Aliquots of the cells were collected every 20 min thereafter and
counted. Total RNA was prepared from these cell samples.
To identify a gene responsible for blockage of cell cycle
progression when mutated as shown in the mutant cyj92, a functional complementation test of the mutant with a S. pombe genomic
library was carried out. The mutant cyj92, showing an elongated
morphology and defect in cell cycle progression at 36 °C, was
transformed with a S. pombe genomic library prepared by
ligating genomic DNA partially digested with Sau3AI into the
BclI site of pWH5 vector (58,
59).2 The transformants showing
Leu+ at 23 °C were first selected, replica-plated onto
the plates containing phloxin B, and incubated at 36 °C. The pink
colonies that grew up and showed wild type morphology at 36 °C were
selected. Individual plasmid isolated from these transformants was
retransformed to test its ability to suppress the elongated
ts
To determine the transcription
start site of psp1+, the primer extension method
was used (62). The antisense primer sequence that corresponds to the
sequence from +150 to +169 (5 To
determine the location of the Psp1 protein in S. pombe
cells, membrane and cytosolic fractions of the cells were prepared (63). Cells were broken by the glass bead method (64) in bead buffer
(20 mM Tris-HCl, pH 7.5, 10 mM EGTA, 2 mM EDTA, 0.25 M sucrose, 20 µg of
leupeptin/ml, and 10 µM phenylmethylsulfonyl fluoride)
and centrifuged at 100,000 × g for 1 h at
4 °C. The supernatant was used as a cytosolic fraction. The pellet
fraction was treated with 1% Triton X-100 for 30 min at 4 °C and
used as a membrane fraction.
To prepare the
protein product of the psp1 gene, a coding region DNA of
psp1+ was fused to the
isopropyl-1-thio-
To generate polyclonal antibody
against Psp1 protein, affinity-purified GST-Psp1(w) protein was treated
with factor Xa enzyme and separated on SDS-polyacrylamide gel (65). The
Psp1 protein band on the gel was eluted, mixed with Freund's adjuvant
solution, and used to inject a rabbit. After two more successive
injections at 2-week intervals, serum was collected and used as a
source of antibody for Western blot analysis and immunocytological
experiments (74). For determining cellular localization of Psp1
protein, FITC-conjugated anti-rabbit IgG (Sigma, Catalog No. F7512) was used. Cells grown at log or stationary phase were collected, washed, and fixed with methanol (56, 66, 67). Antiserum prepared against Psp1
was mixed first with the fixed cell suspension and then treated with
FITC-conjugated anti-rabbit IgG in a concentration of 1/50 dilution.
Nuclei of the cells were counterstained with 4 Transcriptional
expression of psp1+ during cell cycle
progression was examined by Northern analysis of total RNA from cells grown synchronously by the arrest-release method with hydroxyurea. Total RNA was prepared from the cells collected every 20 min by the
method of Carlson and Botstein (68) and analyzed by hybridizing them
with the 1.5-kb PvuII-EcoRV DNA fragment
radioactively labeled by the random priming method. The expression
level of Psp protein was also examined with the same synchronized cells
released from the hydroxyurea arrest. Cell extracts were prepared, and
the level of Psp1 protein in both soluble and particulate fractions was examined by Western analysis using the polyclonal antibody prepared from the GST-Psp1 fusion. The effect of overexpression of Psp1 protein
in S. pombe cells was examined using the
pnmt1-psp1 fusion construct. The coding region
DNA of psp1+ was amplified from S. pombe chromosomal DNA with the 5 To introduce
mutations substituting serine residues at the putative Cdc2 substrate
sites, 324LVQSPSC,
330CPPSPKN, and
338AHLSPGS to alanine, we used PCR
methods with mutated primer sequences (70). Change of the first T of
the triplet sequence encoding serine (TCA, TCC, TCT) to G allows
substitution of serine with alanine. Thus the following
oligonucleotides, corresponding to the amino acid sequences
LVQAPSC (m1), CPPAPKN (m2), and AHLAPGS (m3), were synthesized:
the sense strands of m1,
5 To examine the phosphorylation state of Psp1
protein at different stages of cell growth, cellular proteins were
treated in vitro with phosphatase and analyzed. Cells were
grown to log or stationary phase, collected, and broken in phosphatase
buffer (50 mM Tris-HCl, pH 7.8, 5 mM
dithiothreitol, 2 mM MnCl2, 100 µg/ml bovine
serum albumin) by the glass beads method (50). After removing the glass
beads by centrifugation at 500 × g, the whole cell
extract was centrifuged at 100,000 × g for 1 h.
The supernatant was separated, and the pellet fraction was solubilized in phosphatase buffer containing 1% Triton X-100. 5-10 µl of
supernatant, solubilized pellet fraction, or whole cell extract was
mixed with phosphatase buffer and incubated at 30 °C for 30 min in
the presence of 400 units of To determine whether the
phosphorylated amino acid is serine as expected for phosphorylation by
the Cdc2-Cdc13 complex, phosphoamino acid analysis of Psp1 was carried
out. The protein labeled in vivo by
ortho[32P]phosphate was immunoprecipitated with Psp1
antibody and separated by SDS-PAGE. At the same time, GST-Psp1 protein
phosphorylated in vitro by the Cdc2-Cdc13 complex with
[ Through EMS mutagenesis of
S. pombe cells, several new temperature-sensitive mutants
defective in cell cycle progression were obtained (52). Morphological
analysis of one of the mutants, cyj92, showed a normal cell shape at
23 °C but showed an elongated cellular morphology at 36 °C (Fig.
1a). DNA content analysis of this mutant cell
indicated a defect in cell cycle progression (Fig. 1b). When
cyj92 cells were grown in rich medium at 23 °C, most of the cells
were at G2 phase as was the case for wild type S. pombe cells (Fig. 1b, 4, panel
1). But when they were shifted to a restrictive temperature
36 °C, DNA content increased and in the majority of cells the DNA
content was 4n (Fig. 1b, 4, panel 2)
indicating blockage of cell cycle after G2 phase. When the mutant cells were grown in nitrogen-deficient medium at 23 °C instead, cell growth was arrested at G1 phase as shown in
wild type cells (Fig. 1b, 3, panel 2).
However, if these G1-arrested cyj92 cells were shifted to
YEPD medium and incubated at 36 °C, unlike wild type cells, they
could not progress completely to G2 phase, and cell cycle
progression stopped at S phase (Fig. 1b, 3,
panels 3 and 4). A known G1/S mutant,
cdc10
Functional complementation of this mutant with a S. pombe
genomic library identified the two overlapping clones of 8.5 kb and 6.9 kb (pYJ1 and pYJ2). Subcloning and retransformation data confirmed that
the 4.2-kb PvuII-Sau3A fragment (pYJ4) was
sufficient to suppress the ts Northern analysis of total RNA isolated
from the wild type cells grown in EMM showed a 1.3-kb transcript, and
its level did not fluctuate much throughout the cell cycle. The level
of psp1 transcript in the synchronized cells released from
the S phase arrest was almost same as that in the cells progressing
through G2 and M phase (data not shown). The level of Psp1
protein detected by Western analysis in the same synchronized cells was
also constant throughout the cell cycle (data not shown). Thus, in
actively growing cells the amount of Psp1 protein as well as its
transcript is likely to be constant throughout the cell cycle. When the
location of Psp1 protein in cells was examined by Western analysis
using fractionated cell extracts and Psp1 antibody, Psp1 protein was found to be in the pellet fraction rather than in the soluble fraction
(Fig. 3a). In addition, two different
molecular weight forms of Psp1 protein were detected, and the ratio of
these two forms differed depending on the phase of cell growth. In
actively growing log phase cells (Fig. 3a, M-1,
and Fig. 3b, 1-5), the lower molecular weight
protein band was the major form. However, as soon as cells enter
stationary phase, the higher molecular weight species became
predominant (Fig. 3a, M-2, and Fig.
3b, 6-8). This result suggests that Psp1 protein
is modified to a higher molecular weight form upon entry into
G0-like stationary phase.
Localization of this protein in situ by FITC-conjugated
anti-rabbit IgG and Psp1 antibody demonstrated that Psp1 protein is present through out the whole cell in the actively growing stage (Fig.
3c, 1 and 3), whereas it is localized
to each end of the cell at stationary phase (Fig. 3c,
6 and 8). We suggest that when cells are actively
growing, Psp1 protein function may be required, and it is located
throughout the whole cell body. However when growth of cells almost
ceases at stationary phase, this protein may function only in
specialized areas of the cellular compartment or as a complex.
Detection of the different molecular weight forms of the
Psp1 protein indicated that the two proteins are either produced from
two different size transcripts or modified after translation. Therefore
we examined the size of psp1 transcript both by Northern analysis and determining the start site of the transcript using the
primer extension method. Total RNA prepared from the cells grown in EMM
showed only one major hybridizing band. However, the primer extension
experiment indicated that there are at least two transcription start
sites and the length of the 5 Phosphorylation of GST-Psp1
protein by the Cdc2-Cdc13 complex in vitro indicates the
presence of a specific sequence(s) recognized by Cdc2 in Psp1. Thus the
potential Cdc2 substrate sequences, (T/S)PX(K/R), (9, 76)
were searched. Three such sequences were found in the carboxyl-terminal
region of Psp1 (327SPSC, 333SPKN, and
341SPGS) (Fig. 2b). When we used a GST-Psp1
fusion construct devoid of these sequences (Nw), no phosphorylation of
the truncated Psp1 was observed (Fig. 4c, A,
lane 2). This suggests that the carboxyl-terminal region of
Psp1 is important for phosphorylation by Cdc2 kinase. The GST-Psp1
containing only the carboxyl-terminal region sequence (Cw) including
those potential Cdc2 kinase substrate sites was enough to serve as the
substrate molecule phosphorylated by Cdc2 (Fig. 4c,
A, lane 3). When we substituted each serine
residue at 327, 333, and 341 in the carboxyl-terminal region with
alanine (Cm1, Cm2, and Cm3) and examined the phosphorylation state of the truncated Psp1 protein by Cdc2-Cdc13 in vitro, the
serine at position 333 was the only critical one for phosphorylation by
Cdc2 kinase (Fig. 4c, A-lanes 4-6). Experiments with
in vivo labeled protein also indicated that the serine
residue at 333 is required for phosphorylation (Fig. 4c,
B). PCR-mediated site-directed mutagenesis of
psp1 generated a base change at the sequences encoding serines 327, 333, or 341, respectively, and resulted in their being
changed to alanine. When this mutated full-length psp1 was introduced in wild type cells as pnmt1-psp1 fusions
(pnmt1-m1, pnmt1-m2,
pnmt1-m3) (Fig. 4c, B, m1,
m2, m3) and the phosphorylation state of the
mutated Psp1 by in vivo phosphate labeling at stationary phase was examined, serine 333 was shown to be essential for
phosphorylation in stationary phase. The cells containing the unmutated
pnmt1-psp1 (w) produced more phosphorylated Psp1
than cells containing only the chromosomal copy of psp1
(lanes 1 and 0). This indicated that the Psp1
produced from the overexpression plasmid
pnmt1-psp1 is phosphorylated like that in the
chromosome. However, when the cells containing pnmt1-m2
(mutation at serine 333) was used, in vivo phosphate
labeling of the Psp1 greatly decreased (lane 3). This
decrease was not observed when pnmt1-m1 and
pnmt1-m3 (containing mutations at 327 and 341, respectively)
were analyzed instead (lanes 2 and 4). This
confirms the importance of serine 333 for phophorylation in
vivo. Phosphoamino acid analysis of the Psp1 protein
phosphorylated both in vivo and in vitro
confirmed that phosphorylation actually occurred at a serine residue
(Fig. 4d).
To examine whether phosphorylation of Psp1 by Cdc2 occurred through
direct binding of Cdc2 to Psp1, a yeast two-hybrid system was employed.
When the plasmids containing Gal4 binding
domain-cdc2 and Gal4 activation
domain-psp1 were introduced into a S. cerevisiae strain SFY526 and Since a gene disruption study indicated
that psp1+ is essential for cell growth (Fig.
2c), we examined the multicopy gene dosage effect of this
essential protein on cell growth. This was done by introducing
multicopy plasmid pnmt1-psp1 into wild type cells and measuring their growth rate in the presence and absence of thiamine. A wild type culture grown to log phase in the presence of
thiamine, which represses the nmt1 promoter function, was
split in half. We continued incubating one half at 30 °C in the same medium as before. The other half was transferred to medium devoid of
thiamine which induces nmt1 promoter function. With the
onset of induction psp1 at 12 h after transfer to
thiamine-less medium, growth of the cells in thiamine-less medium
became slower than those in thiamine-containing medium (Fig.
5a). When cells grown for 12 h in the
above conditions were passaged into fresh medium at a lower cell
density, the growth difference between the two cultures was greater in
early log phase than that in late log phase (Fig. 5b). Cells
overexpressing Psp1 protein in early log phase, as evidenced by Western
analysis of the total protein with Psp1 antibody (Fig. 5b,
inset, A and a), showed half the
growth rate of the uninduced cells (Fig. 5b, A
and a). However differences in growth rate, caused by
psp1 overexpression, decreased as cells entered stationary
phase (Fig. 5b, B and b). The protein
produced in the cells from pnmt1-psp1 in log phase was
mainly the low molecular weight form whereas that in stationary phase
was the phosphorylated high molecular weight form (Fig. 5b,
inset, a and b) as for that produced
from the chromosomal copy of psp1+ (Fig.
5b, inset, A and B). The
cells with retarded growth following overexpression of Psp1 also
exhibited an altered cellular morphology (Fig. 5c). These
cells rounded up and DAPI staining of nuclear DNA revealed that the
area occupied by nuclear DNA enlarged, that is, the compactness of the
nuclear DNA was lost (Fig. 5c, 2, panels 3 and 4). Flow cytometric observation of DNA content in
the Psp1 overexpressed cells indicated doubled or tripled DNA contents (Fig. 5c, 3, panels 2-4). The changes
in DNA content and cellular morphology were maximum at 13.5 h
after transfer to fresh medium (4n or 8n, Fig. 5c,
3, panel 4). Growth rate at this hour was about
one-fourth of that in uninduced cells (Fig. 5b,
4). However, by 24 h after transfer to fresh media, the
growth difference became relatively small, and DNA content of the
induced cells tended to return to amounts found in uninduced cells (2n
or 4n). After 30 h, at which time cells entered the stationary
phase (Fig. 5b, 5), DNA content and morphology of
the cells overexpressing Psp1 were similar to those of the cells
containing only one copy of the psp1 gene (Fig.
5c, 3, panel 5). Thus maximal growth
inhibition and changes in DNA content on overproducing Psp1 was
observed in the actively growing cells rather than in the slow growing cells. However, the doubling time of the cells in which Psp1 was overexpressed was prolonged.
Temperature-sensitive mutants of S. pombe defective in
normal cell cycle progression provide a convenient system to identify the elements involved in cell cycle regulation. Random mutagenesis of
S. pombe cells enabled one to isolate such a mutant. Using the same mutagenic approach as Nurse et al. (51), we
isolated one mutant, cyj92, which possesses an elongated cellular
morphology similar to G1/S mutants of Nurse et
al. (51) at 36 °C. Comparative DNA content analysis of the
mutant in rich and nitrogen-starved media indicated that the mutation
in cyj92 hindered progression of S. pombe cell from
G1 to S phase and perhaps also from G2 to M
phase. The terminal phenotype of the mutation is accumulation of
abnormal DNA which is either incapable of completing DNA replication (Fig. 1b, 3) or overreplicated in the absence of
separation into daughter cells (Fig. 1b, 4). It
is likely that the mutation causes both blocking of S phase which leads
to the prevention of onset of the subsequent mitosis and a defect in
mitosis which prevents reinitiation of the subsequent S phase and leads
to an increase in ploidy.
The characteristics of the mutant phenotype enabled us to isolate a
novel gene, psp1+, which suppresses the
elongated mutant phenotype of cyj92 at 36 °C and made cell cycle
progression normal. The finding that psp1 gene function is
required for normal cell growth at 30 °C suggests that this gene is
essential for vegetative growth. However, the fact that its
overexpression in the actively growing cells (log phase cells) is
detrimental to cell growth indicates that excessive functioning of this
gene also causes a defect in normal cell growth. From the flow
cytometric DNA analysis data we speculate that this is due either to
accumulation of abnormal DNA or to inhibition of production of the
elements necessary for normal cell division. Since we were not able to
observe the accumulation of undivided cells, that is the cells attached
together without septum, we consider that overproduction of Psp1
protein does not affect the cytokinesis process itself but rather
affects the normal process of DNA replication. The accumulation of the
2n or 4n DNA content in the actively growing cells by overproduction of
Psp1 protein suggests that the signal for completion of DNA replication may not be transferred correctly to the elements working in
G2 or M. Alternatively, it may be possible that
overexpression of psp1 may lead to bypass or delay of the
mitosis process and thereby allow continued replication of DNA. The
fact that Psp1 protein is required for normal DNA replication but
excessive function of this protein causes delay of the cell cycle
suggests that this Psp1 may act at two different points in the cell
cycle. Identification of target molecules with which Psp1 protein
interacts will be necessary to elucidate the detailed function of this
protein. The finding that the inhibitory effect on cell growth becomes less severe upon entry into the stationary phase indicated that this
protein is in its active form in actively growing cells but is
inactivated when cell growth slows down. However, we do not know
whether inactivation of this protein is a prerequisite for entry into
the stationary phase or conversely entry into stationary phase leads to
inactivation of this protein.
The results that Psp1 is a phosphoprotein and that its dephosphorylated
form is the major one in actively growing cells indicated that the
function of this protein depends on phosphorylation. If this protein
functions as a positive element, required in the cell cycle
progression, the dephosphorylated form is active in actively growing
cells and the phosphorylated Psp1, which is the major form in
stationary phase cells, is inactive. However, if Psp1 functions as a
negative element, the dephosphorylated form is inactive in actively
growing cells, and the phosphorylated form is its active form in the
stationary phase. Although we don't know where in the cell cycle Psp1
acts, the result that this protein is phosphorylated by the Cdc2-Cdc13
complex in vitro suggests that this protein could be one of
the substrates for the Cdc2-Cdc13 complex and hence Cdc2 kinase may be
associated with its function.
The carboxyl-terminal domain of this novel protein is phosphorylated at
serine 333 by Cdc2 kinase, and alteration of this serine to alanine
abolishes phosphorylation by Cdc2 kinase in vitro. However,
in vivo we were not able to detect a defect in phosphorylation in the cdc2 mutant at the restrictive
temperature. Because it showed the mutant phenotype within 4-5 h after
being shifted to restrictive temperature, this is not long enough for cdc2
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) L36906. We thank Dr. Kyu-chung Hur at Ewha Woman's
University for his kind help in phosphoamino acid analysis and Dr.
Chun-Jeih Ryu at KRIBB for his help in preparing polyclonal antibody
for Psp1. We appreciate Dr. Hyang-Bae Kim at Korea University for
providing us with the known cdc mutants we used in this study. We also
thank Dr. Terrance Cooper at the University of Tennessee, Memphis, for his critical reading of this manuscript.
While we were preparing this manuscript, Ishii
et al. (77) reported the sds23+ gene
which has the same DNA sequence as the psp1+
gene. The sds23+ gene is a multicopy suppressor
for mutation in pp1 and 20 S cyclosome/anaphase promoting complex.
Department of Biological
Sciences,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
Addendum
REFERENCES
Strains and Media
) mutant was derived from a haploid strain, ED665
(h
, ade6-M210, ura4-D18,
leu1-32). For gene disruption analysis and back-crossing
test, a S. pombe diploid strain, SP286
(h+/h+,
ade6-M210/ade6-M216,
ura4-D18/ura4-D18,
leu1-32/leu1-32) and a haploid strain, ED668
(h+, ade6-M216, ura4-D18,
leu1-32) were used. For genetic complementation tests and
comparative flow cytometric analysis, the known cell cycle mutants,
cdc2
, cdc10
,
cdc20
, and cdc22
and
wee1
were used. YE-glucose (YEPD) agar medium
and Edinburgh minimal medium (EMM) were used as described by Gutz
et al. (49). When necessary, 75 µg/ml each of adenine and
uracil or 250 µg/ml leucine was added as a supplement. Phloxin B
(Sigma) was added to the YEPD and minimal agar medium at a
concentration of 20 mg/liter after autoclave. 50-500 µM
thiamine was added to EMM to repress the nmt1 promoter
function, and it was excluded from EMM when induction of the
nmt1 promoter function is necessary. For in vivo cell labeling with ortho [32P]phosphate,
phosphate-free minimal medium (MMP) in the presence of 1 mM
phosphate (added from a 0.5 M
NaH2PO4 stock solution) or 50 µM
phosphate was used (50).
mutants of
S. pombe, ED665 cells grown in YEPD medium were mutagenized
with ethylmethanesulfonate (EMS) to 40% survival as described
previously (51, 52). The mutagenized cells were spread on YEPD plates
and incubated at 23 °C for 4-6 days. The cells grown on the plates
were replica-plated onto YEPD plates containing phloxin B, and the
plates were incubated at 23, 36, and 39 °C for 3-4 days. The cells
that grew at 23 °C but did not grow at 36 or 39 °C were selected.
Out of 2.5 × 105 cells mutagenized, 180 candidate
colonies were first selected and examined for their morphological
changes at the restrictive temperature (36 °C) under a light
microscope. The 51 colonies that showed defects in cell division were
further characterized by back-crossing to the parent strain ED665 or
ED668 and by genetic complementation test with the known cell division
cycle mutants. Their DNA contents at 36 °C were determined by flow
cytometrical analysis as described in Jang et al. (52). One
mutant, cyj92, that showed an elongated morphology at 36 °C and a
defect in cell cycle progression both at G1/S and after
G2, was selected and used to clone a gene recovering the
mutated phenotype.
or
cdc10
was carried out simultaneously. To
measure DNA content in cells overexpressing
psp1+, the cells containing
pnmt1-psp1 plasmid were first grown in EMM
containing 100 µM thiamine and then transferred to EMM
devoid of thiamine and grown for 12 h. Aliquots of the cells were
reinoculated into fresh EMM lacking thiamine to a cell density
A580 = 0.1 and incubated at 30 °C. Cell
samples were collected thereafter every 3 h and subjected to flow
cytometric analysis.
phenotype of cyj92 (50). Two such plasmids, containing
8.5-kb and 6.9-kb insert DNA, were isolated and characterized by
restriction enzyme mapping and Southern hybridization. The 4.2-kb
Sau3AI-PvuII fragment present in both of these
plasmids and possessing the suppressive function was subcloned (Fig.
2a, pYJ4) and sequenced by the dideoxy method of Sanger
et al. (60) with USB Sequenase (U. S. Biochemical Corp.).
cDNA clones were also isolated by screening a S. pombe
cDNA library constructed in the
-ZAP vector (61) with the
labeled 1.5-kb PvuII-BamHI fragment encoding most
of the coding sequences. The DNA containing open reading frame
sequences was designated as psp1+
(phosphoprotein of stationary phase of S. pombe). The disruption construct of the
psp1 gene, with the ura4+ as the
selectable marker, was made as follows (Fig. 2a, pYJ5). The
PvuII-EcoRV fragment containing most of the Psp1
coding sequences, including 5
-nontranslating region DNA, was deleted
and replaced with the 1.8-kb ura4+ fragment. The
resulting 3.5-kb BamHI-ApaI fragment was used to transform a diploid strain SP286. Transplacement of the genomic psp1 gene with the ura4+ disrupted
copy was confirmed by Southern analysis. The disrupted diploid strain
was then sporulated according to the methods of Gutz et al.
(49), and viability of the resulting meiotic products was analyzed
(Fig. 2c).
Fig. 2.
Sequence of psp1+
gene complementing the mutant phenotype of a novel ts
mutant cyj92. a, map of genomic clones and their subclones complementing the mutant phenotype of cyj92 and a disruption clone. + indicates suppression of the mutant phenotype. b, nucleotide and deduced amino acid sequence of psp1+ gene.
Lowercase and uppercase letters indicate genomic
and cDNA sequences, respectively. The arrowheads
indicate the start sites and the end of two cDNAs. The short intron
sequence at the 5
-nontranslating region is absent in the cDNA
clone 1. The potential Cdc2 substrate sequences, 327SPSC,
333SPKN, and 341SPGS were
underlined. c, effect of disruption of
psp1 on cell viability. Diploid SP286 strain containing
psp1 gene disrupted with ura4+ as
evidenced by the Southern analysis of its chromosomal DNA (lane
2 in 1) produced two viable and two nonviable spores at 30 °C on tetrad analysis (2). These sequence data are
available from GenBankTM under accession number L36906 and
were deposited in August, 1994.
[View Larger Versions of these Images (53 + 99K GIF file)]
-GAGCAATTTCATTATCATG-3
) of the
amino-terminal coding region was synthesized and labeled with T4
polynucleotide kinase (New England Biolabs) and
[
-32P]ATP. The labeled primer was then hybridized with
5-10 µg of poly(A) RNAs isolated from the ED665 strain grown in EMM.
Moloney murine leukemia virus reverse transcriptase (New England
Biolabs) was added to the hybridization mixture and incubated at
37 °C for 30 min. The position of transcription start site was
determined by comparing the size of the extended fragment with the
sequenced DNA using the same primer.
-D-galactopyranoside-inducible glutathione S-transferase (GST) of fusion vector pGEX-3X.
The GST-psp1 fusion constructs that encode different
portions of Psp1 were prepared as follows (Fig. 4c,
A). The DNA fragment containing the whole coding region (w),
amino-terminal sequence 1-307 (Nw), or carboxyl-terminal sequence
323-408 (Cw) was cloned into the BamHI site of
plasmid pGEX-3X. The DNA fragments encoding carboxyl-terminal sequences
323-408 (Cm1), 330-408 (Cm2), and 338-408 (Cm3), bearing substitutions of serine residues at 327, 333, and 341 with alanine, respectively, were fused into the same vector. Escherichia
coli strain DH5
transformed with these fusion plasmids was
grown in LB medium containing ampicillin, and production of GST-Psp1
fusion proteins was induced by growing cells in the presence of 0.1 mM isopropyl-1-thio-
-D-galactopyranoside at
30 °C. Each GST-Psp1 fusion protein was purified on
glutathione-agarose beads as described previously from E. coli crude cell extracts (65). For in vitro phosphorylation of Psp1 protein by Cdc2 kinase, GST and GST-Psp1 fusion
proteins purified on glutathione-agarose beads were eluted with 10 mM reduced glutathione in 50 mM Tris-HCl, pH
8.0.
Fig. 4.
Phosphorylation state of Psp1. a,
phosphatase treatment of Psp1 protein in vitro. Proteins in
the membrane (M) and total fraction (T) of the
cells from log and stationary phase (sta) were treated
in vitro with -phosphatase (PPase), separated on SDS-PAGE gels, and analyzed by Western blotting with Psp1 antibody.
and + indicate the untreated and phosphatase-treated proteins, respectively. b, in vitro phosphorylation of Psp1
with the Cdc2-Cdc13 kinase complex. The GST-Psp1 fusion protein
purified from E. coli was mixed with the mitotic Cdc2-Cdc13
complex in the presence of [
-32P]ATP and analyzed by
SDS-PAGE and autoradiography. The bands in lanes
1-3 showed the phosphorylated proteins, and those in lanes
4-6 are the same amount of the proteins as in lanes
1-3 stained with Coomassie Blue. c, identification of
the serine residue phosphorylated in vitro by Cdc2-Cdc13
(A) and in vivo (B). Three serine
residues (327, 333, and 341) at the potential Cdc2 substrate sites in
the carboxyl-terminal region of Psp1 protein were mutated to alanine
and tested for phosphorylation by Cdc2-Cdc13. Lanes 1-6 in
A showed the phosphorylated part of the GST-Psp1 fusion protein. The whole Psp1 protein (w), carboxyl-terminal
protein (Cw), and carboxyl-terminal protein mutated at
serine 327 (Cm1) showed phosphorylation by Cdc2, but the
carboxyl-terminal protein mutated at serine 333 (Cm2,
lane 5) did not show phosphorylation by Cdc2. Lanes
0-4 in B showed the phosphorylated Psp1 protein produced in vivo from the pnmt1-psp1 plasmid. The
entire psp1 gene mutated at the base encoding serine residue
was fused to pnmt1 vector and was expressed in wild type
ED665 cells. The cells grown to late log phase in the absence of
thiamine were labeled in vivo as described under
"Experimental Procedures." Cell extracts were prepared and
immunoprecipitated with Psp1 antibody and analyzed on SDS-PAGE gels.
The open arrowhead indicates the protein produced from the
chromosomal copy of the psp1+ gene, and the
filled arrowhead indicates that from pnmt1-psp1. The asterisk indicates the mutation point. d,
phosphoamino acid analysis of Psp1 protein labeled in vitro
by Cdc2 (left) and in vivo in the presence of
ortho[32P]phosphate (right). S
indicates phosphoserine detected by autoradiography; T and
Y correspond to the positions of phosphothreonine and phosphotyrosine, respectively, detected by ninhydrin staining as shown in 2.
e, direct interaction of Psp1 with Cdc2 kinase shown by the
yeast two-hybrid method. psp1+ fused to the Gal4
binding domain sequence (pGBT9) and cdc2+ fused
to the Gal4 activation domain sequence (pGAD) were introduced into
yeast S. cerevisiae strain SFY526, and expression of the lacZ gene in yeast was examined. 1 and
2, cells containing pGBT9-psp1 and
pGAD-cdc2 alone, respectively; 3, cells
containing both pGBT9-psp1 and pGAD-cdc2;
4, cells containing pCL1 of the whole Gal4
gene.
[View Larger Version of this Image (36K GIF file)]
,6
-diamidino-2-phenylindole (DAPI) (56).
- and 3
-end primer sequences and
ligated into the SalI site of plasmid vector pREP1
containing thiamine-regulated nmt1 promoter (69). The
resulting plasmid pnmt1-psp1 was transformed into
S. pombe cells, and expression of Psp1 protein was induced
by growing cells in the absence of thiamine for more than 16 h.
-CTTGTGCAGGCACCTTCTTGC-3
, m2,
5
-TGCCCTCCTGCCCCAAAAAAT-3
, and m3,
5
-GCACATCTTGCCCCTGGATCC-3
and antisense
strands of m1, 5
-GCAAGAAGGTGCCTGCACAAG-3
, m2,
5
-ATTTTTTGGGGCAGGAGGGCA-3
, and m3,
5
-GGATCCAGGGGCAAGATGTGC-3
. Also, the sense
strand of the amino terminus, 5
-ATGCCTTTGTCAACTCAATCG-3
, and the
antisense strand of the carboxyl terminus, 5
-TCACCGACGTGGTGTATCTAC-3
were synthesized. To introduce point mutation at the sequence
encoding serine 327 (Ser-327- Ala), the two primer pairs, sense
strand of amino terminus and antisense strand of m1 and sense strand of
m1 and antisense strand of carboxyl terminus were used for the first
PCR. Then the second PCR was carried out using the first PCR product as
a template and sense strand sequence of amino terminus and antisense
strand sequence of carboxyl terminus as the primers. Amplified DNA was
then cloned into vector plasmid pTZ18U. The change in the sequence at
residue 327 was confirmed by sequencing the amplified fragment (Fig.
4c, B). Mutations at Ser-333 and Ser-341 were
introduced by similar PCR methods. The primer pairs of the sense strand
of amino terminus and antisense strand of m2 or m3 were used for the
first PCR and followed by the second PCR with the amino- and
carboxyl-terminal sequence pairs. For overexpression of these mutated
Psp1 in ED665, amplified DNAs were cloned into plasmid vector pREP1
containing the thiamine-inducible nmt1 promoter
(pnmt1-m1, pnmt1-m2, and
pnmt1-m3).
protein phosphatase (
-PPase, New
England Biolabs) (71, 72). The reaction mixture was boiled in
SDS-polyacrylamide gel (SDS-PAGE) loading dye for 5 min. The proteins
were then separated on SDS-PAGE and electrotransferred to a
nitrocellulose paper for Western analysis with the polyclonal antibody
against Psp1. To determine whether Psp1 could be phosphorylated
in vitro, 1-2 µg of purified GST-Psp1 protein was mixed
with 10 units of mitotic Cdc2-Cdc13 complex (New England Biolabs) and
incubated at 30 °C for 30 min in the presence of 40 µCi of
[
-32P]ATP. The resulting protein was then resolved in
SDS-PAGE and analyzed by autoradiography. As a standard substrate of
Cdc2 kinase, 2 µg/ml histone H1 (Boehringer Mannheim) was used (73).
In vivo labeling of the protein was carried out with
ortho[32P]phosphate according to the procedure of Moreno
et al. (50). Cells were first grown to late log phase
(A580 = 2.5-3.0) in 5-10 ml of low phosphate
medium (MMP plus 1 mM phosphate) and then transferred to
the same volume of fresh MMP containing 1 mCi of ortho[32P]phosphate (Amersham, Catalog No. PBS13). After
incubation at 30 °C for 5-6 h, cell extracts were prepared from the
harvested cells as described above. For immunoprecipitation,
800-1,000-µl aliquots of the cell extracts were mixed with 20 µl
of antisera of Psp1 (final 1/40 dilute) and 50 µl of protein
A-agarose (Sigma). The immunoprecipitate was analyzed on SDS-PAGE and
by autoradiography (74).
-32P]ATP was also separated by SDS-PAGE. After being
transferred to a polyvinylidene difluoride membrane (Millipore) and
localized by autoradiography, the phosphorylated Psp1 band was excised
and subjected to phosphoamino acid analysis (64, 75). The protein on
the membrane was eluted and hydrolyzed in 6 N HCl for
1 h at 110 °C. After being dried in a Speed Vac evaporator, the
sample was dissolved in 6-10 µl of water containing 100 µg/ml each
of phosphoserine, phosphothreonine, and phosphotyrosine. The sample was
subjected to two-dimensional thin layer electrophoresis (HTLE 7000 apparatus, CBS Scientific). The first dimension was electrophoresed for
20 min at 1.5 kV in pH 1.9 buffer (50 ml of 88% formic acid, 156 ml of
glacial acetic acid, and 1794 ml of water). The second dimension was
electrophoresed for 16 min at 1.3 kV in pH 3.5 buffer (100 ml of
glacial acetic acid, 50 ml of pyridine, and 1880 ml of water).
32P-Labeled individual phosphoamino acids were identified
by aligning the phosphoamino acid markers stained with ninhydrin to the
signals obtained by autoradiography.
Characteristics of a Novel Temperature-sensitive (ts)
Mutant Defective in Cell Cycle Progression and a Gene Complementing
This ts
Phenotype
, and G2/M mutant,
cdc2
, showed cell cycle arrest at
G1 after nitrogen starvation at permissive temperature
23 °C also (Fig. 1b, 1 and 2,
panels 2) when they were tested simultaneously with cyj92.
They showed their mutant phenotypes at the restrictive temperature
(36 °C); that is, blockage of cell cycle progression to
G1/S phase and after G2 phase, respectively, as
expected (Fig. 1b, 1 and 2,
panels 3 and 4). Back-cross of the mutant strain
cyj92 to wild type strain ED668 revealed 2+:2
segregation of the mutant phenotype at 36 °C which indicates that
the mutation shown in cyj92 is a single gene mutation (Fig. 1c). Genetic complementation tests of cyj92 with known cdc
mutants of G1 and S such as cdc10
,
cdc20
, or cdc22
and
G2/M phase mutants cdc2
and
wee1
showed that the mutation in strain cyj92
was not allelic with any of them. Thus we concluded that the mutant we
had isolated is a new ts
mutant possessing a defect in
cell cycle progression after the G2 phase at restrictive
conditions and also at G1/S phase when cells pass the
G2 phase normally. It is likely that once the mutant cells
progressed to G2 phase, the mutation in cyj92 can cause blockage of further progression to M or cytokinesis. If mutant cells
were in G1, this progression through the next S phase was also adversely affected. It is alternatively possible that the mutation
carried in strain cyj92 causes blockage of cell cycle progression both
at G1/S and G2/M.
Fig. 1.
Characteristics of the newly isolated
temperature-sensitive mutant cyj92 defective in cell cycle
progression. a, cellular morphology of the mutant cyj92:
wild type morphology at 23 °C and an elongated cell shape at the
restrictive temperature 36 °C. b, flow cytometric
analysis of cyj92 (3 and 4) together with
G1-specific mutant cdc10
(1) and G2/M-specific mutant
cdc2
(2). Panels 1 in
1-4, cells grown in YEPD medium at 23 °C, panels 2 in 1-3, cells transferred to nitrogen-deficient EMM
and incubated at 23 °C for 25 h; panel 2 in
4, cyj92 cells grown in YEPD at 36 °C for 8 h,
panels 3 and 4 in 1-3, cells arrested
at G1 phase (panels 2 in 1-3) were
transferred back to YEPD and incubated at 36 °C for 4-8 h; and
panels 3 and 4 in 4, cyj92 cells
containing the genomic clone pYJ1 which suppresses the elongated mutant
phenotype was grown in EMM at both 23 °C (panel 3) and
36 °C (panel 4). This showed that the mutant, cyj92, has
a defect both in completion of S phase (panel 3 in
3) and in proper mitosis (panel 2 in
4). The genomic clone pYJ1 suppresses this defect and leads
normal cell cycle progression (panel 4 in 4). The
abscissa indicates intensity of fluorescence per cell, and
the ordinate indicates cell numbers. The vertical
line at the right side corresponds to 2n
(G2), and the one at the left side corresponds
to n (G1). c, back-cross test of the mutant. The
mutant cyj92 was crossed to wild type ED668 cells, and the diploid was
sporulated. The resulting tetrads were incubated at 23 °C first and
replica-plated and incubated at 36 °C.
[View Larger Version of this Image (48K GIF file)]
phenotype (Fig.
2a). cyj92 mutant cells harboring plasmids
pYJ1 or pYJ4 showed normal cell morphology at 36 °C, and their DNA content analysis indicated normal cell cycle progression at the restrictive temperature, 36 °C. Increased ploidy seen in cyj92 mutant cells (Fig. 1b, 4, panel 2)
also disappeared in the transformed cells (Fig. 1b,
4, panel 4); transformants were mainly 2n like the wild type. Sequence analysis of this genomic DNA revealed an open
reading frame sequence of 408 amino acids (Fig. 2b).
Meanwhile, sequence analysis of several cDNA clones indicated the
presence of two different transcripts depending on whether or not a
short intron at the 5
-terminus was processed. As shown in Fig.
2b, one cDNA clone, which started at
90 (clone 1) did
not contain sequences between
57 and
2 (intron). However, the other
cDNA clone, which started at
88 (clone 2) contained these
sequences. Primer extension experiments using mRNA isolated from
cells grown in EMM also revealed two transcript start sites correlating
with the two cDNA clones that differed by the presence or absence
of the intron sequence (data not shown). Hydropathy plot analysis of
the deduced amino acid sequence of psp1+ showed
that it is very hydrophobic and contains a putative transmembrane domain at its carboxyl terminus (amino acids 353-369). Sequence homology comparison of this protein showed that no known protein sequences in the GenBankTM data base exhibited high
homology with it. Thus we concluded this gene to be a newly identified
gene and designated it psp1+
(phosphoprotein of stationary phase of S. pombe). Sporulation and tetrad analysis of a diploid
strain containing one copy of psp1 transplaced with
ura4+ (Fig. 2a, pYJ5) showed
2+:2
segregation for cell viability at
30 °C (Fig. 2c). All viable spores were Ura
indicating that psp1 is essential for vegetative cell growth at 30 °C.
Fig. 3.
Localization of Psp1 protein in S. pombe cells. a, location of Psp1 protein in cells. The
proteins in soluble (C) and pellet fraction (M)
of the cells grown to log (1) and stationary phase
(2) were loaded on SDS-PAGE gels and analyzed by Western blotting with Psp1 antibody. b, profile of Psp1 proteins
produced during cell growth. Proteins in the membrane fractions
prepared from the same number of cells at each time point during cell
growth were analyzed by Western blotting. c, in
situ localization of Psp1 protein. Wild type cells grown at
30 °C were fixed and stained with FITC-conjugated antibody. The
numbers in c indicate the same cells on the
growth curve in b. 1 and 3, log phase
cells; 6 and 8, stationary phase cells.
[View Larger Version of this Image (42K GIF file)]
-nontranslated region is different
depending on the processing of a short sequence from
57 to
2 (Fig.
2b). Even though these two transcripts differ by a 56-base
pair sequence in the 5
-nontranslated region, they contained the same
ATG codon, and the proteins translated from these two transcripts seem
to be the same. Thus we examined the possibility of modification after
translation by phosphorylation. We tested whether the high molecular
weight Psp1 protein in stationary phase cells contained a phosphate
residue(s). Western analysis of proteins in the membrane fraction
treated with phosphatase in vitro showed a reduction in the
amount of the high molecular weight Psp1 protein (Fig.
4a, lane 4) and a concomitant
increase in the low molecular weight band instead. In contrast, the
lower molecular weight form of Psp1 present mainly in the log phase cells remained unchanged after phosphatase treatment (Fig.
4a, lane 2). This result suggests that Psp1
protein can be phosphorylated. To test this possibility, GST-Psp1
fusion protein was prepared and tested in vitro with the
mitotic Cdc2-Cdc13 complex or with mitogen-activated protein kinase.
When the Cdc2-Cdc13 complex was incubated with GST-Psp1 protein in the
presence of [
-32P]ATP in vitro, GST-Psp1
protein was phosphorylated as was histone H1 control which is a known
substrate of Cdc2-Cdc13 (Fig. 4b, lanes 2 and
3). Phosphorylation did not occur in the GST portion of the
fusion protein but only in Psp1. However, when mitogen-activated protein kinase was mixed with GST-Psp1 protein, phosphorylation did not
occur at all (data not shown). This suggests that phosphorylation of
the Psp1 protein by the Cdc2-Cdc13 complex was specific.
-galactosidase production was examined,
-galactosidase production was observed. This indicates interaction
between Psp1 and Cdc2 (Fig. 4e). The level of
-galactosidase produced was not as great as that from GAL4 itself
(lanes 3 and 4). However there is strong
indication that Cdc2 and Psp1 interact with each other.
Fig. 5.
Inhibitory effect of cell growth by
overexpression of psp1+. a and
b, ED665 cells containing pnmt-psp1
overexpression plasmid were first grown in EMM in the presence of thiamine, and Psp1
expression was induced by transferring the cells to EMM devoid of
thiamine. The closed circles indicate the growth curve of
the cells grown continuously in EMM containing thiamine (uninduced), and the closed squares indicate that of the cells grown in
thiamine-less EMM (induced). The arrow in a
indicates the point where the thiamine induction actually started,
approximately 12 h after transfer to the thiamine-less medium. The
growth curves in b were from the cells that were transferred
to fresh medium to a lower cell density at the point where thiamine
induction actually started in a. The bands in the
inset showed the amount of Psp1 proteins detected by Western
analysis of the proteins in the uninduced (A and
B) and induced cells (a and b).
c, DNA content and shape of Psp1 overexpressed cells. The
cells from each point in the growth curves in b were stained
with DAPI or analyzed flow cytometrically. 1 and
2, DAPI-stained cells from uninduced (1) and
Psp1-induced (2) cultures; 3, flow cytometrically
measured DNAs in uninduced (white) and induced
(black) cells. The numbers in the panels
correspond to the points in the growth curve in b: 0 (1), 7.5 (2), 10.5 (3), 13.5 (4), and 30 h (5) after transfer to the
fresh medium.
[View Larger Version of this Image (28K GIF file)]
cells to enter the stationary phase to
show its kinase function. It is only in the stationary phase of the
cell growth that Psp1 protein is phosphorylated and
cdc2
cells die after 4-5 h at the restrictive
temperature long before we were able to detect the defect in Cdc2
kinase function in the mutant. Thus it was not possible to determine
whether mutation in Cdc2 kinase affected the function of Psp1 in
actively growing cells. We consider that phosphorylation of Psp1
protein upon entering the stationary phase is one of the major events
for slowing cell growth, and Cdc2 kinase controls this process. The
yeast two-hybrid method confirmed that Cdc2 and Psp1 interact directly
in vivo. Thus we can suggest that when cell cycle progresses
actively in log phase, Psp1 protein is in its dephosphorylated state,
and Cdc2 function is not required. However, when cell cycle progression slows at the stationary phase, Cdc2 kinase phosphorylates Psp1 protein,
and the phosphorylated form of Psp1 facilitates entry into the
G0 phase of the cell cycle or inactivates elements required for active cell growth. Cdc2 protein function is required for modification of Psp1 protein at this stage and there must be
phosphatase(s), which removes the phosphate from Psp1 protein when
cells begins active growth. We did not find yet what gene product is
involved in dephosphorylation of Psp1 upon entry into active cell
growth cycle. It will be interesting to find a specific phosphatase for Psp1, and function of this phosphatase may be a key regulatory element
for activation of Psp1 protein function. Localization of this Psp1
toward each tip of the cell upon entry into the stationary phase may
also be related to phosphorylation of this protein. It could be
suggested that the phosphorylated Psp1 may have the property to form
protein complexes in the cells leading to translocation of the protein
in a certain compartment of cells and the dephosphorylated Psp1 may
exist in monomeric form and is localized throughout cells as an active
monomeric form. The following model for Psp1 protein function can be
drawn (Fig. 6). Psp1 protein is activated by
dephosphorylation with a phosphatase when the cells enter
G1 to S phase of the cell cycle and is inactivated by
phosphorylation with Cdc2 kinase upon entry into the stationary or
G0 phase. It is an essential protein for cell growth, and
regulation of this protein function by phosphorylation could be the key
feature in cell cycle progression from G1 to S. In addition
to Cdc2 protein, the other element(s) which interact directly with this
Psp1 protein should exist, and finding this element(s) will be
essential for further characterization of Psp1 protein function.
Fig. 6.
Working model for Psp1 function in actively
growing and stationary cells.
[View Larger Version of this Image (15K GIF file)]
*
This work was supported by Grants N80880, N81310, HS030M,
and HS1030 from the Ministry of Science and Technology of Korea. Part
of the grants was supported by Dae-oung Pharmaceutical Co.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.: 82-42-860-4170;
Fax: 82-42-860-4597; E-mail: yoohyang{at}kribb4860.kribb.re.kr.
1
The abbreviations used are: CDK,
cyclin-dependent kinase; CKI, CDK inhibitor; EMM, Edinburgh
minimal medium; MMP, phosphate-free minimal medium; EMS, ethyl
methanesulfonate; kb, kilobase(s); FITC, fluorescein isothiocyanate;
DAPI, 4,6-diamidino-2-phenylindole; PCR, polymerase chain reaction;
PAGE, polyacrylamide gel electrophoresis.
2
Jang, Y. J., Chung, K. S., Park, C. K., and Yoo,
H. S. (1997) Biochim. Biophys. Acta, in press.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.