(Received for publication, July 21, 1995; and in revised form, December 20, 1995)
From the
We have isolated a conditional lethal mutant mts3 in
the fission yeast Schizosaccharomyces pombe which at the
permissive temperature is resistant to the mitotic poison MBC and at
the restrictive temperature is defective in metaphase to anaphase
transition. The predicted amino acid sequence of mts3 is 36% identical with the budding yeast
gene NIN1. NIN1 cloned into a fission yeast expression vector
can rescue both mts3 temperature-sensitive and null alleles
demonstrating that NIN1 is the budding yeast homologue of the
fission yeast mts3
gene. The phenotype of the mts3 null is identical with the mts3 ts mutant
demonstrating that the phenotype of the mts3 ts mutant is due
to loss of mts3
function. The deduced amino
acid sequences of both mts3
and NIN1 show homology to peptide sequences obtained from subunit 14 of the
26 S protease purified from bovine or human cells.
It is becoming increasingly recognized that the stability of proteins is an important factor in the regulation of gene expression. The main nonlysosomal intracellular proteolysis pathway in the cell is the ubiquitin pathway. The ubiquitin pathway has been implicated in the instability of a number of important regulatory proteins such as p53, c-Myc, c-Mos, and the mitotic cyclins. Ubiquitin is a 76-amino acid protein, that when covalently attached to a lysine residue of a protein, targets it for destruction (reviewed in (1, 2, 3) ).
Biochemical analysis has determined that a multiprotein complex called the 26 S protease degrades proteins that have been targeted for destruction by the ubiquitin pathway. The 26 S protease is made up of two multiprotein functional components, the catalytic 20 S proteasome and the regulatory complex. The 20 S proteasome is a cylindrical structure composed of 14 different protein subunits and contains all the proteolytic activities of the complex. In vitro analysis on purified 20 S complexes has shown that proteins are degraded in a completely unregulated manner. Regulated proteolysis requires the addition of another multiprotein complex composed of at least another additional 15 different subunits to each end of the 20 S proteasome cylinder to form the 26 S protease. The different subunits of this regulatory complex are named according to their size on an SDS-polyacrylamide gel electrophoresis gel, S1 being the largest and S15 the smallest. With the formation of the 26 S protease, degradation of proteins is now carried out in a highly regulated manner. Certain substrates are degraded only if they have been polyubiquitinated and proteolysis is now ATP-dependent(4, 5) .
Recently, we isolated a
conditional lethal mutant, mts2, in subunit 4 of the 26 S
protease regulatory complex in the fission yeast Schizosaccharomyces pombe. Characterization of the phenotype
of mts2 strain at the restrictive temperature demonstrated
that the cells arrested at a particular point in the cell cycle, the
metaphase stage of mitosis(6) . In this paper, we describe
another conditional mutant, mts3, which was isolated in the
same screen. We show that mts3-1 arrests with a phenotype
similar to the mts2 mutant and that the S. cerevisiae NIN1 gene is the budding yeast homologue of the mts3 gene and
that both the mts3 and NIN1 gene
products are similar to subunit 14 of the regulatory complex of the 26
S protease purified from mammalian cells.
Figure 5:
A,
the cDNA and putative amino acid sequence of mts3. The bold nucleotides represent
the oligonucleotides to the complementary strand that were used to
construct the mts3 disruption. The asterisk represents the stop codon. B, a comparison of the Mts3
protein and the budding yeast NIN1 amino acid sequences. The mts3
amino acid sequence is on the top
line, and the NIN1 sequence is on the bottom
line. A bold line indicates identity. The asterisk represents the stop codon.
Figure 1: First cycle arrest of mts3-1 strain after shift from the permissive to the restrictive temperature. Two early log phase wild type and mts3-1 cultures were grown at the permissive temperature of 25 °C. At time 0 min, one culture was kept at 25 °C while the remaining culture was shifted to the restrictive temperature of 35°C. The cultures were sampled at the times shown, and the cell number was measured using a Coulter counter and plotted on a logarithmic scale.
mts3-1 cells were also fixed for cytological examination as detailed under ``Experimental Procedures.'' The cells were stained with the DNA-specific stain DAPI and with the anti-tubulin antibody TAT1 to investigate the state of the DNA and microtubules. The DNA and microtubule structures seen in wild type cells are shown in Fig. 2a. After shift to the restrictive temperature, an increasing proportion of the population displayed a characteristic mutant phenotype in that the DNA becomes highly condensed (Fig. 2b, B). Associated with this condensed DNA is a short mitotic spindle (Fig. 2b, A). The condensed DNA is found to be located in the middle of the short spindle (Fig. 2b, C) consistent with these cells being at the metaphase stage of mitosis. After 4 h at the restrictive temperature, up to 75% of cells in the population are displaying this phenotype (Fig. 2c). On cells sampled at later time points, the DNA was never found to separate nor the short spindle elongate to a length typical of anaphase in S. pombe (Fig. 2a, C). The mts3-1 mutant therefore appears to be defective in the metaphase to anaphase transition. This metaphase arrest phenotype was found to be transient in that after further incubation at the restrictive temperature the short metaphase spindle was found to disassemble and the DNA was found to decondense to reform a nucleus of normal appearance. In most cases, the single nucleus became displaced to one end of the cell and a septum formed to divide the cell into an anucleate and nucleate half (Fig. 2b, E). This phenotype is very similar to that shown by the S. pombe mutant mts2-1 isolated in the same screen(6) . The only difference between the phenotypes displayed at the restrictive temperature is that a much higher proportion (75%) of the mts3-1 strain showed a metaphase arrest compared to 25% of cells in the mts2-1 culture. The septa that formed appeared aberrant when stained with the septum specific stain calcofluor and did not appear to be functional as cell number was not observed to increase (Fig. 1). Cytoplasmic microtubules were found to reform after the metaphase spindle disassembled (Fig. 2b, D). These microtubules, however, stained far less efficiently with the anti-tubulin antibody than wild type cells, implying that they could be defective. Interestingly, after extended incubation at the restrictive temperature, the culture did not go through another round of chromosome condensation as has been reported for some of the S. pombe cut mitotic mutants(17) .
Figure 2: Phenotype of mts3-1 at the restrictive temperature. a, wild type cells stained with the anti-tubulin antibody TAT1 and DAPI. The two images were merged on the computer to give the image shown. The DAPI signal was changed on the computer from blue to red to increase the resolution of the desired structures. A, interphase cells; B, mitotic metaphase cell; C, late anaphase cell; D, postanaphase. b, mts3-1 cells stained with TAT1 and DAPI after shift to the restrictive temperature. A, B, and C, cells sampled 4 h after shift. A, TAT1 staining of microtubules. B, DAPI staining of the DNA. C, the A and B images were merged on the computer to give the image shown. D and E, cells sampled 7 h after shift. D, cells stained with TAT1 and DAPI. The two images were merged as before to give the image shown. E, cells stained with DAPI and the septum specific stain calcofluor. The septum stains as a band located in the middle of the cell while the DNA is found in the ovoid-shaped nucleus located toward the end of the cell at a location similar to that in D. All the cells in A-E are printed at the same magnification. c, percentage of mts3-1 cells after shift to 36 °C that show the condensed DNA phenotype (open squares), the short metaphase mitotic spindle phenotype (open diamond), or the septated phenotype (filled circles).
Histone H1 kinase assay on an mts3 asynchronous culture shifted from the permissive to the restrictive temperature was performed (Fig. 3A, a) and indicated that H1 kinase activity increases after shift to the restrictive temperature. Quantitation on the PhosphorImager shows that activity peaks after a 2-h incubation at the restrictive temperature displaying approximately 3-fold more activity than cells growing at the permissive temperature. The increase in chromosome condensation in samples taken from the same culture occurs later, at 4 h (Fig. 2c), than the peak in H1 kinase activity. After the peak in activity, the level of H1 kinase activity decreased to a level that was much lower than the original value before shift to the restrictive temperature.
Figure 3: Histone kinase activity in the mts3-1 mutant on shift to the restrictive temperature. A, a, at time 0 h, an early logarithmic growing culture of mts3-1 cells was shifted from 25 °C to 36 °C sampled at the times shown and assayed for H1 kinase activity. Lane c shows the H1 kinase activity obtained from an exponentially growing haploid wild type culture. b, mixing experiment to assay for possible inhibitor. To 50 µg of extract from t = 0 h, increasing amounts of extract from t = 8 h were added (0, 50, 100, and 150 µg) and then assayed for H1 kinase activity as before. B, steady state levels of Cdc13 protein in mts3-1 after shift to the restrictive temperature. The same extracts assayed in A, panel a, were probed by Western analysis for Cdc13 protein. 50 µg of each extract was separated on an 10% SDS-polyacrylamide gel electrophoresis gel. The samples were transferred to nitrocellulose and probed with an anti-Cdc13 antibody. The antibody was detected with a rabbit monoclonal antibody conjugated to alkaline phosphatase (Promega). The alkaline phosphatase activity was detected according to the manufacturer's instructions.
Recently, Schwob et al.(18) showed that the budding yeast SIC1 gene
encodes a specific inhibitor of the CLB5/CDC28 kinase and that
degradation of SIC1 is required to release the CLB5/CDC28 activity essential for G/S phase
transition(18) . In addition, the degradation of SIC1 protein was shown to be by the ubiquitin pathway. To investigate
if a similar inhibition event was occurring in the mts3 extracts at the restrictive temperature, mixing experiments were
carried out. Increasing amounts of crude extracts prepared from cells
sampled 8 h (t = 8 h) after shift to the restrictive
temperature were mixed with 50 µg of protein extract prepared from
cells before they were shifted (t = 0 h). No evidence
of any inhibiting activity could be found to be present in sample t = 8 h (Fig. 3A, b).
H1 kinase activity measures the amount of activity of the Cdc2 kinase when it is complexed to the S. pombe cyclin B homologue Cdc13. To investigate if the extracts were losing activity due to a decrease in the levels of Cdc13 protein, the extracts assayed in Fig. 3A, a, were subjected to Western blot analysis with an antibody against the S. pombe Cdc13 protein. As shown in Fig. 3B, the level of Cdc13 protein remained essentially constant throughout the course of the experiment. The differences in H1 kinase activity that were observed could be due to differences in the level of the Cdc2 protein or alternatively to differences in post-translational modification of either the Cdc2 or Cdc13 protein.
Figure 4: A, FACS analysis of mts3-1 strain shifted to the restrictive temperature. The top three panels show nitrogen (n) starved wild type haploids which have predominantly cells with a 1n DNA content (a(i)), exponentially growing wild type haploid which have a 2n DNA content (a(ii)), and N starved wild type diploid which shows a 2n and 4n peak of DNA (a(iii)). Panels b(i), (b)(ii), and (b)(iii) shows mts2 cells, panels c(i), (c)(ii), and (c)(iii) show mts3 cells, and panels d(i), (d)(ii), and (d)(iii) show wild type cells shifted from 25 °C to 36 °C and sampled at 0 h (i), 4 h (ii), and 8 h (iii) after the shift. For each sample, the 1n, 2n or 4n peaks obtained are indicated. In b, c, and d, the mts2, mts3, and wild type strains were all haploid strains growing exponentially prior to the shift. B, FACS analysis of cdc25mts3 strain shifted to the restrictive temperature. A cdc25mts3 double mutant strain was shifted to 36 °C, sampled at the times shown, and subjected to FACS analysis. The single 2n peak of DNA observed is marked.
At 25 °C, the cdc25mts3 double mutant grew very slowly compared to the cdc25 or mts3 single mutants and appeared elongated, being 2 to 3 times longer at division. At 20 °C, however, the double mutant was observed to grow normally. Consistent with this finding was the observation that cdc25mts3 cells growing at this temperature divided at the roughly same size as wild type cells. Therefore, for the cdc25mts3 double mutant, 20 °C instead of 25 °C was used as the permissive temperature.
An asynchronous
culture of cells was shifted from the permissive to the restrictive
temperature and sampled at the times shown (Fig. 4B).
These cells were then fixed, stained with propidium iodide, and
subjected to FACS analysis. During the course of the experiment, at the
restrictive temperature, the cells elongated to give the characteristic cdc phenotype typical of cdc25 cells. Consistent with
this observation, the DNA was not found to condense, implying that the
cells had all arrested at the cdc25 G block and no
4n peak was observed (Fig. 4B). In conclusion, the
observed re-replication in the mts3-1 strain appears to be due
to the indirect effect of the cells entering mitosis and not the result
of deregulation of the initiation of DNA replication.
The genomic clone was used to isolate a corresponding
cDNA clone carried in the S. pombe expression vector pREP1.
Overexpression of the mts3 cDNA in pREP1
complemented the ts mts3 mutation and resulted in no obvious
phenotype. The nucleotide sequence of the mts3
cDNA was determined and is shown in Fig. 5A.
Comparison of the mts3 coding sequence
with other DNA sequences in the EMBL data base revealed a substantial
homology with NIN1, a known S. cerevisiae gene. The
deduced amino acid sequence of mts3
is 36%
identical with the NIN1 amino acid sequence (Fig. 5B). The nin1 mutant is a conditional
lethal mutation in which cells at the restrictive temperature arrest in
mitosis with a single nucleus and a G
DNA
content(19) . The described phenotype is very similar to that
of the mts3-1 strain at the restrictive temperature. In
addition, the two proteins are very similar in size.
Figure 6:
The budding yeast NIN1 gene can
rescue the conditional lethality of the mts3 ts and null
alleles. A, the mts3leu1.32h and
the mts2leu1.32h
strains were transformed
with the S. pombe expression vector pSP1 or pSP1 carrying the mts2
(pmts2
), mts3
(pmts3
), or NIN1 (pNIN1) cDNAs. The top two plates show the mts3leu1.32h
strain carrying the plasmids
streaked out at the permissive temperature of 25 °C (left)
or the restrictive temperature of 36 °C (right). Only the mts3 cells carrying the plasmids containing the NIN1 or mts3
genes can grow at the
restrictive temperature. The bottom two plates show the mts2leu1.32h
strain transformed with the
same plasmids. In this case, only mts2 cells carrying the
plasmid containing the mts2
gene can grow at
the restrictive temperature. B, the mts3
/mts3
ura4
heterozygous diploid was transformed with pSP1,
pmts2
, pmts3
, and pNIN1. The diploid
was sporulated and by selecting simultaneously for the ura4
gene in the disrupted mts3 allele and the leucine marker carried on the S. pombe expression vector the ability of the different plasmids to rescue
the mts3 null allele could be investigated. Only the
pmts3
and pNIN1 plasmids could rescue the mts3 null allele.
Although the budding yeast NIN1 gene can rescue an mts3 ts mutant, a much more stringent test would be to ask if the NIN1 gene could rescue an mts3 deletion allele.
The cloned mts3 gene was used to make a
null allele of the mts3
gene as detailed
under ``Experimental Procedures.'' The mts3
gene was deleted by PCR and replaced
with the selectable ura4
marker. This
construct was used to transform an S. pombe diploid strain
selecting for uracil prototrophy. PCR analysis was used to confirm that
integration had occurred at the mts3 locus (data not shown).
The heterozygous diploid was sporulated and tetrad analysis carried
out on the resulting asci. Out of 20 tetrads, only two viable spores
were obtained in each ascus. In addition, these viable spores were all
uracil auxotrophs demonstrating that the mts3 gene is essential for growth.
To investigate if the NIN1 gene could also rescue a null allele, the mts3/mts3
ura4
leu1.32/leu1.32
ura4D18/ura4D18 heterozygous diploid was transformed with the
pNIN1 plasmid and sporulated. By selecting simultaneously for the ura4
gene in the disrupted mts3 allele and the leucine marker carried on the pNIN1 plasmid, the
ability of the NIN1 gene to rescue the mts3 null
could be investigated. The NIN1 gene could rescue the mts3 null allele as well as the S. pombe mts3
gene (Fig. 6B). As a control, both the mts2
cDNA and the vector itself could not
rescue the mts3 null allele. This functional complementation
of a mts3 null allele combined with the similarity between the
deduced amino acid sequences confirms that the NIN1 gene is
the budding yeast homologue of the fission yeast mts3
gene.
Figure 7:
Phenotype of the mts3 null allele.
Disrupted spores were germinated selecting for the ura4 gene present in the disrupted gene as
detailed under ``Experimental Procedures.'' After shift to 32
°C, cells were sampled and stained with the TAT1 anti-tubulin
antibody and DAPI as before. a, cells sampled 4 h after shift
to 32 °C. b, percentage of mts3 null cells that
show the condensed DNA phenotype (open squares), short
metaphase mitotic spindles (open diamonds) and septa (filled circles). c, FACS analysis of germinated
spores sampled at the times (in hours) shown. The 1n, 2n, and 4n peaks
are marked.
Figure 8:
Homology of the fission yeast Mts3 protein
to subunit 14 of the 26 S protease. A, comparison of the mts3 amino acid sequence to the amino acid
sequence of peptide fragments made from purified subunit 14 purified
from human or bovine cells. Peptide sequence P31-4 and P31-6 are from (21) , while S13-3 is from (20) . B,
comparison of the budding yeast NIN1 amino acid sequence to
amino acid sequence of peptide fragments obtained from purified subunit
14. Peptide sequences P31-2, P31-4, and P31-5 are from (21) .
S13-1 is from (20) .
In this paper we describe the phenotype of the mts3 mutant of S. pombe. At the restrictive temperature, mts3 cells enter mitosis and arrest at metaphase. Spindle
elongation and DNA segregation characteristic of anaphase did not
occur, demonstrating that the mutant is defective in the metaphase to
anaphase transition. In the same original screen, we isolated another
conditional lethal mutant, mts2, which encodes subunit 4 of
the same regulatory complex(6) . The mts2 gene product belongs to a highly conserved family of
ATPases(22) , while the mts3
amino
acid sequence bears no homology to these ATPases nor to any other
protein domains found in the EMBL data base. Comparison of the Mts3
peptide sequence to the peptide sequence obtained from tryptic digests
of mammalian 26 S subunits indicated a high degree of homology to
peptide sequence from subunit 14. Consistent with this observation, we
have shown recently that the mts3
gene
product is present in the regulatory complex of the 26 S protease
purified from S.pombe.
The phenotype of mts3-1 at the restrictive temperature is very similar to that which we
have previously described for mts2-1. Therefore, the metaphase
arrest phenotype appears to be associated with lack of 26 S protease
function. In addition, the demonstration that an mts3 null
allele has the same metaphase arrest phenotype as the ts mutant at the
restrictive temperature shows that this phenotype is a result of lack
of mts3
(subunit 14) gene product function.
We have previously shown that overexpressing the mouse MSS1 gene, which encodes subunit 7 of the 26 S protease, could rescue
the mts2 ts mutation but not an mts2 null mutation (6) . This provided genetic evidence for a possible interaction
between subunits 4 and 7 in the 26 S complex itself. We could find no
such genetic evidence for an interaction between the mts2 (subunit 4) and mts3
(subunit 14) gene products either in the phenotype of the mts2mts3 double mutant strain (data not shown) or by rescue of
the temperature sensitivity by overexpression of mts2
in a mts3-1 strain or vice
versa.
When the mts3 peptide sequence was
compared to other sequences in the EMBL data base, it was found to have
36% identity to the S. cerevisiae gene NIN1. The
phenotype of the nin1 mutant at the restrictive temperature is
similar to that described for the S. cerevisiae ts mutants cim3(MSS1) and cim5(SUG1) which encode subunits 7 and
8 of the 26 S protease(23) . We show here that the S.
cerevisiae NIN1 gene can rescue both S.pombe mts3 ts and
null alleles demonstrating that NIN1 is the budding yeast
homologue of the mts3
gene.
Surprisingly,
the 36% identity found between the mts3 and NIN1 gene products is much lower than that found between the mts2
and corresponding human S4 homologue,
which show 73% identity with each other. Both subunits are present in
the same multiprotein complex so what model could account for the
difference in divergence between different homologues of one subunit
(subunit 4) compared to the other (subunit 14). Perhaps this could be a
consequence of where each protein is situated in the complex itself.
Subunit 4, for example, could lie in the middle of the complex in
intimate association with a number of different proteins allowing
little chance of divergence while subunit 14 could be located at one
end of the complex interacting with fewer proteins. Whatever the
reason, when homologues of subunit 14 are isolated from other
organisms, this reduced degree of identity between homologues should
aid in the identification of functionally important domains critical
for subunit 14 activity.
The mts2 and mts3 mutants
were both isolated in a screen looking for mutants that were resistant
to the mitotic poison MBC. It is not apparent why such a screen should
seem to be specific for subunits of the regulatory complex of the 26 S
protease. One possible explanation is that the concentrations of MBC
used in the screen causes cells to make a slightly defective spindle at
the permissive temperature which although it is still able to function
relatively normally activates a checkpoint pathway to cause cell cycle
arrest. The mutants are defective in this putative checkpoint response
and are therefore able to grow at the permissive temperature in the
presence of the drug. Whether such a model accounts for the MBC found in the mutants and whether this is related to mitotic
arrest at the restrictive temperature will have to wait until further
experiments have been carried out.
The mts2 and mts3 mutants at the restrictive temperature both arrest at the metaphase stage of mitosis. In addition, both genes encode different subunits of the same regulatory complex of the 26 S protease. This implies that some substrate(s) has to be degraded by the ubiquitin pathway to proceed from metaphase to anaphase in mitosis. Recent experiments on frog extracts have essentially come to the same conclusion(24) . It was originally thought that cyclin B was one of these substrates and that it had to be degraded for cells to proceed from metaphase to anaphase in mitosis. More recent work has disproved this hypothesis and demonstrated that cyclin B destruction is in fact required for exit from mitosis(24, 25) . Recent work on the cyclin B destruction pathway has implied that some components involved in this pathway also seem to be required for the destruction of the substrate(s) to allow metaphase to anaphase transition. ts mutants in three genes were isolated in S. cerevisiae that were defective in the destruction of the budding yeast cyclin B homologue, CLB2. Surprisingly, at the restrictive temperature, all three mutants appear to be defective in metaphase to anaphase transition. When the wild type genes were cloned, all three mutations turned out to be in known genes, CDC16, CDC23, and CSE1. The CDC16 and CDC23 genes encode two members of the TPR family of proteins which are characterized by blocks of 34 amino acid tandem repeats repeats known as tetratricopeptide repeats(26, 27) . Antibodies that recognize these proteins and an additional TPR protein called CDC27 were used to show that they are present in a large multiprotein complex purified from Xenopus extracts. This complex was isolated by its ability to attach ubiquitin to cyclin B molecules in a cell cycle-dependent manner, an event which targets the cyclin B molecule for destruction(28, 29, 30, 31) . To explain the metaphase arrest phenotype in the cdc16 and cdc23 mutants at the restrictive temperature, it is postulated that, in addition to being required for the destruction of cyclin B, they are also required for the destruction of the putative substrate that has to be degraded for metaphase to anaphase transition to occur.
The S. pombe cut9 and nuc2 mutants are
temperature-sensitive mutants whose genes encode members of this TPR
family(17, 31) . nuc2 has
the highest degree of similarity to CDC27 while cut9
is most similar to CDC16. At
the restrictive temperature, like the mts2-1 and mts3-1 ts strains, both nuc2 and cut9 mutants are
defective in metaphase to anaphase transition. Genetic evidence
suggests that the nuc2
and cut9
proteins interact with each
other(17) . The mts2, mts3, nuc2,
and cut9 mutants could therefore all define genes required in
a pathway in S. pombe to degrade the putative substrate whose
destruction has been postulated to be required for sister chromatid
separation at the metaphase to anaphase transition in mitosis.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X92682[GenBank].
Addendum-While this manuscript was in the reviewing process, a study of the S. cerevisiae nin1 mutant was published(32) . One of the main conclusions was that the NIN1 gene product encoded a subunit of the 26 S regulatory complex consistent with the data published here.