* The Johns Hopkins University School of Medicine, Department of Molecular Biology and Genetics, Baltimore, Maryland
21205; Centre for Molecular Medicine and Therapeutics, University of British Columbia, Vancouver, British Columbia
V5Z 4H4, Canada; and § Carnegie Institute of Washington, Department of Embryology, Baltimore, Maryland 21210
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Abstract |
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A genetic synthetic dosage lethality (SDL) screen using CTF13 encoding a known kinetochore protein as the overexpressed reference gene identified two chromosome transmission fidelity (ctf) mutants, YCTF58 and YCTF26. These mutant strains carry independent alleles of a novel gene, which we have designated CTF19. In light of its potential role in kinetochore function, we have cloned and characterized the CTF19 gene in detail. CTF19 encodes a nonessential 369-amino acid protein. ctf19 mutant strains display a severe chromosome missegregation phenotype, are hypersensitive to benomyl, and accumulate at G2/M in cycling cells. CTF19 genetically interacts with kinetochore structural mutants and mitotic checkpoint mutants. In addition, ctf19 mutants show a defect in the ability of centromeres on minichromosomes to bind microtubules in an in vitro assay. In vivo cross-linking and chromatin immunoprecipitation demonstrates that Ctf19p specifically interacts with CEN DNA. Furthermore, Ctf19-HAp localizes to the nuclear face of the spindle pole body and genetically interacts with a spindle-associated protein. We propose that Ctf19p is part of a macromolecular kinetochore complex, which may func- tion as a link between the kinetochore and the mitotic spindle.
Key words: centromere; kinetochores; chromosome segregation; mitotic spindle apparatus; Saccharomyces cerevisiae ![]() |
Introduction |
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MITOTIC cell division is a process which ensures
that a cell's chromosomes are faithfully replicated and equally segregated to two daughter
cells. The kinetochore (centromere DNA and associated proteins) is essential to the high fidelity of chromosome
transmission. The kinetochore mediates attachment of the
chromosomes to the spindle microtubules (MTs)1 and directs chromosome movement during mitosis and meiosis
(reviewed in Bloom et al., 1989; Koshland, 1994
). The kinetochore-MT interaction is monitored by at least one
centromere-based mitotic checkpoint system which delays onset of anaphase until stable bipolar attachment is
achieved (Gorbsky, 1995
; Rudner and Murray, 1996
).
Thus, kinetochores play both important structural and regulatory roles in ensuring faithful chromosome segregation
during mitosis.
The kinetochores of Saccharomyces cerevisiae are relatively simple compared with the large trilaminar structures
seen in multicellular eukaryotes (Rieder, 1982; Pluta et al.,
1990
). Major advances in defining components necessary
for a functional kinetochore in yeast have implications for
understanding the corresponding elements in more complex eukaryotes. The minimal functional centromere of S.
cerevisiae (~125 bp) is comprised of three conserved centromere DNA elements (CDEs). The central CDEII element consists of 78-86 bp of AT-rich DNA, and is flanked
by two highly conserved palindromic motifs, CDEI (8 bp)
and CDEIII (25 bp). Of these, CDEIII is absolutely essential for centromere function. In vivo, a unique nuclease resistant chromatin structure encompassing 160-220 bp is associated with CEN DNA and presumably corresponds to
the yeast kinetochore (Bloom and Carbon, 1982
; Funk et al.,
1989
). Seven genes that encode centromere proteins
have been identified. Four of these, NDC10/CTF14/CBF2
(Goh and Kilmartin, 1993
; Jiang et al., 1993
), CEP3/CBF3
(Lechner, 1994
; Strunnikov et al., 1995
), CTF13 (Doheny et al., 1993
), and SKP1 (Connelly and Hieter, 1996
; Stemmann and Lechner, 1996
) are essential for viability and encode the components of a multisubunit complex, CBF3,
that binds to CDEIII DNA in vitro (Lechner and Carbon,
1991
). CBF1/CEP1/CPF1 encodes a nonessential basic helix-loop-helix protein which binds to CDEI (Baker and Masison, 1990
; Cai and Davis, 1990
; Mellor et al., 1990
). In
addition, MIF2, a homologue of mammalian CENP-C, has
been shown to interact with CEN DNA in a CDEIII- and
CDEII-dependent manner (Meluh and Koshland, 1995
,
1997
), and CSE4, a homologue of mammalian CENP-A, is
suggested to be involved in a specialized centromeric nucleosome (Meluh et al., 1998
). Further analysis of the architecture of the CBF3 complex using DNA protein cross-linking (Espelin et al., 1997
) suggests that three subunits of
CBF3, Ndc10p, Cep3p, and Ctf13p, are in direct contact
with CDEIII over a region spanning 80 bp of DNA.
The binding of the CBF3 complex to CDEIII is necessary, but not sufficient for attachment of chromosomes to
MTs. Several studies indicate the existence of other factors
which are required to link the CBF3-DNA complex to polymerized MTs (Kingsbury and Koshland, 1991; Sorger et al.,
1994
). The CBF3 subcomplex is thought to act as the nucleation site onto which these as yet unidentified components assemble and form active MT-binding complexes. A
rich resource for identification of these additional kinetochore components is the chromosome transmission fidelity
(ctf) mutant collection, which was isolated by the sole criterion of an increased rate of missegregation of a nonessential chromosome (Spencer et al., 1990
). Previously, two
secondary in vivo genetic screens, the centromere transcriptional readthrough assay and the dicentric chromosome stabilization assay, were used to detect potential kinetochore mutants from this collection (Doheny et al.,
1993
). A subset of 12 ctf mutants screened positive for the
transcriptional readthrough assay. Three of these also
tested positive for the dicentric stabilization assay. Two
mutants were characterized in further detail, ctf13-30 and
ctf14-42, and were shown to correspond to CTF13 and
CTF14 (NDC10/CBF2), genes which encode essential kinetochore proteins (Doheny et al., 1993
).
To identify other proteins involved in kinetochore structure or function, a third in vivo genetic assay was developed
as a tertiary screen. This novel screen, which we have called
synthetic dosage lethality (SDL; Kroll et al., 1996), involves
overexpressing a wild-type reference gene in a collection of
target mutants. CTF13, encoding a known kinetochore protein (Doheny et al., 1993
), was used as the inducibly overexpressed reference gene to screen for synthetic interactions
within a subset of 12 potential kinetochore ctf mutants.
Overexpression of CTF13 caused SDL in combination with
four ctf mutants; one corresponded to a known kinetochore component, (CTF14/NDC10), and two were independent
alleles of a previously uncharacterized gene, CTF19. We report here the cloning and detailed characterization of the
CTF19 gene. The genetic and biochemical results presented
here indicate that Ctf19p represents a new kinetochore protein which may play a role as a molecular link between kinetochores and the mitotic spindle.
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Materials and Methods |
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Yeast Strains and Media
Table I lists the genotypes of all yeast strains used in this study. Media for
yeast growth and sporulation were as described (Rose et al., 1990), except as
otherwise indicated. For experiments monitoring the loss of a nonessential
chromosome fragment, adenine was added to 6 µg/ml minimal media to enhance the development of the red pigment in ade2-101 strains. For galactose
inductions, strains were grown on solid medium containing 2% raffinose as the sole carbon source, and then transferred to solid medium containing 2%
galactose, either with or without additional 2% raffinose. For experiments
using liquid cultures, strains were grown in liquid medium containing 2%
raffinose overnight, and galactose was added to a final concentration of 2%
for the induction times indicated. To inhibit MT function in liquid cultures,
nocodazole (NZ; Sigma Chemical Co.) was added to 20 µg/ml and cultures
incubated at 25°C for 2 h, or to 15 µg/ml at 30°C for 90 min. To inhibit MT function on solid medium, benomyl (DuPont) was added at 5, 10, and 20 µg/
ml as indicated. DMSO alone was added to the media as a control. All yeast
transformations were done by the method of Ito et al. (1983)
.
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Genetic Analysis
Synthetic Lethality (SL) Studies.
Strains containing mutations in the gene
of interest were mated to a ctf19 null strain to create the heterozygous diploids. These strains were sporulated, dissected, and cultured at 25°C. At
least two independent diploid clones were analyzed for each mating. To
test for conditional interactions, all double mutant spores recovered at
25°C were assayed for growth at higher temperatures, initially at 30°C and
37°C. Other temperatures were tested if there was an indication of a conditional effect at a permissive temperature to determine the lowest temperature at which lethality was observed. All synthetic lethal interactions
indicated by the tetrad data were confirmed by plasmid shuffle. CBF3 mutant strains which demonstrated potential SL with a ctf19 null were mated
to a ctf191::TRP1 or ctf19
1::HIS3 strain containing a wild-type copy of
CTF19 on a URA3-CEN plasmid (pKH7), sporulated and dissected. The
bub (budding uninhibited by benzimidazole) and mad (mitotic arrest deficient) deletion strains were transformed with the appropriate wild-type
gene (BUB1, BUB2, BUB3, or MAD2) on a URA3-CEN plasmid, mated
back to the ctf19 null strain, sporulated, and dissected. Double mutant
spores recovered containing a URA3-marked plasmid were streaked on
plates containing 5-FOA, which does not allow growth of cells expressing
URA3 (Boeke et al., 1987
). Therefore, only viable double mutants can
grow on 5-FOA, and true synthetic lethals can not.
SDL Studies.
Methods are as described previously (Kroll et al., 1996).
In brief, mutant strains were transformed under noninducing conditions, with overexpressing constructs containing either CTF13 (pKF88) or
CTF19 (pKH21) reference genes under transcriptional control of a GAL1
promoter, and the corresponding vector, p415GEU2. Expression of the
reference gene was induced on solid medium containing 2% galactose.
Growth was compared directly for each mutant containing either an overexpression plasmid or the respective vector alone. To test for conditional
effects, transformants viable at 25°C were also assayed at 30°C and 37°C.
Molecular Cloning and Characterization of CTF19
The chromosome missegregation phenotype of ctf19-58 was confirmed to
be due to a single genetic mutation through genetic analysis after backcrossing this mutant strain to its wild-type parent (YPH277). The CTF19
gene was cloned by complementation of the ctf sectoring phenotype from
a library containing 10-12-kb fragments of yeast genomic DNA inserted
into a LEU2-CEN vector (Spencer, F., and P. Hieter, unpublished results). Appropriate restriction fragments were used for subcloning the genomic DNA into pRS based vectors (Sikorski and Hieter, 1989). A 2-kb
MluI-NsiI fragment rescued the sectoring phenotype and was shown to
contain the CTF19 gene by genetic linkage analysis. CTF19 was physically mapped to chromosome XVI by hybridization of a [32P] labeled
SalI-NsiI fragment to filters containing overlapping lambda and cosmid
clones of the S. cerevisiae genome (Link and Olson, 1991
). This placed
CTF19 on the right arm of chromosome XVI, 40-50 kb from CEN6, distal to RAD1.
A complete deletion of the CTF19 open reading frame (ORF) was
generated using PCR-mediated gene disruption (Lorenz et al., 1995). Oligonucleotides for PCR were synthesized as follows. OKH1 (5'-GTGTGATCTTGTT GATAC TAGGT CGCAAAGAACGCAAATAGATTG-
TACTGAGAGTGCAC-3') has a 40-bp homology to the sense strand
upstream of the CTF19 ATG, followed by a 20-bp sequence from the
plus strand of pRS vectors (Sikorski and Hieter, 1989
) adjacent to the vector
selectable marker. OKH2 (5'-GTTTAAGCAAGCCGTCCAGTTGGCAATGGCAAATGGAACACTGTGCGGTATTTCACACCG-3') has a
40-bp homology to the antisense strand downstream of the stop codon followed by a 20-bp sequence from the minus strand of pRS vectors adjacent
to the vector selectable marker. OKH1 and OKH2 can be used to incorporate any one of the markers from the pRS vectors (URA3, HIS3, LEU2,
or TRP1) by PCR. These oligonucleotides were used to amplify either HIS3
from pRS303 or TRP1 from pRS304. The HIS3 PCR product was transformed into the haploid strain YPH877 and the diploid strain YJP57. The
TRP1 PCR product was transformed in the haploid strain YPH1125 and
the diploid strain YPH982 (Table I). Gene replacement was confirmed in
each case by Southern blot analysis.
Plasmids and Epitope-tagged Constructs
Plasmids containing CTF19 fused to the GAL1 promoter, either with or
without an in frame E1 epitope tag, were constructed from the plasmid
p415GEU1 and p415GEU2, respectively (Connelly and Hieter, 1996;
Kroll et al., 1996
). A PCR strategy was used to place the second codon of
CTF19 in frame with the ATG of p415GEU1 and p415GEU2, using a 5'
engineered Xho1 cloning site and a downstream engineered HindIII site
(Fig. 1 b). To avoid possible PCR errors, a wild-type PstI (bp 566 in ORF)
HindIII (in multiple cloning site) genomic fragment was used to replace
the 3' end of the PCR-generated sequence in pKH20. When transformed
into a ctf19 deletion strain containing pKF88 (CTF13 overexpression
construct; Kroll et al., 1996
), both pKH20 and pKH21 were able to rescue
the SDL phenotype. pKH20 and pKH21 were also able to stabilize a chromosome fragment in the ctf19 deletion strain when compared with the
vector alone control on galactose containing media.
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To make an HA epitope-tagged construct, a PCR based site-directed
mutagenesis method which utilizes Pfu polymerase was used to engineer a
StuI site directly 3' to the ATG of CTF19 in pKH6 (protocol from QuickChangeTM site-directed mutagenesis kit; Stratagene), creating pKH27.
Three tandem copies of the 9-amino acid HA epitope tag (Field et al.,
1988) were amplified by PCR from the vector pSM492 (a gift from Susan
Michaelis) with StuI sites engineered on the 5' and 3' ends. The triple HA
tag was ligated into the StuI site engineered at the NH2 terminus of
CTF19, and this epitope-tagged fusion construct was subcloned into
pRS313 and pRS315, generating pKH31 and pKH32, respectively (Fig. 1 c).
These constructs were transformed into ctf19
1::TRP1 (YPH1319) and
shown to rescue the chromosome missegregation phenotype. The CTF19-3HA fusion was integrated into the genome by gamma integration (Sikorski and Hieter, 1989
). A 1.4-kb XhoI-SmaI fragment containing CTF19-3HA from pKH32 was subcloned into a HIS3 marked gamma integration vector, p679 (Doheny et al., 1993
), which is designed to direct an integration event at the leu2
1 locus on chromosome III. The resulting integrating construct, pKH35, was linearized at the NotI site, between the targeting sequences to the left (5') and right (3') of the leu2
1 locus, and
transformed into a ctf19
1::TRP1 deletion strain (YPH1317), creating
leu2
1::CTF19-HA3 (YPH1327). Integration at the leu2
1 locus was confirmed by colony PCR analysis, using primers OMB-leu2
(5'-GTGTAGAATTGCAGATTCCC-3', provided by M. Basrai) and T7. An integration event targeted correctly to the genomic leu2
1 locus results in a 550-bp product. Both the plasmid based and genome integrated versions of
the CTF19-3HA epitope-tagged fusion produce the same 48-kD band by
Western blotting and probing with anti-HA antibody, which is not seen in
control strains containing a wild-type untagged copy of CTF19.
Quantitation of Chromosome Missegregation
Colony color half sector analysis was performed as previously described
(Koshland and Hieter, 1987; Gerring et al., 1990
). In brief, homozygous
diploid strains (YPH982, CTF19/CTF19; YPH1320, ctf19
::TRP1/ctf19
::
TRP1; YPH1321, ctf19-58/ctf19-58; and YPH1322, ctf19-26/ctf19-26) containing a single SUP11-marked chromosome fragment were plated to single colonies on solid media containing a limiting amount of adenine (6 µg/ml), and grown at 30°C. The red pigment was allowed to develop at
4°C before scoring the sectoring phenotypes. Colonies scored as half-sectored were
50% red. A 1:0 missegregation event (chromosome loss) in
the first division results in pink/red half-sectored colonies, whereas a 2:0
missegregation event (nondisjunction; chromosome gain) results in white/
red half-sectored colonies.
Flow Cytometry and Cytological Analysis
Cells were processed for flow cytometry as previously described (Gerring et
al., 1990), with a few modifications. In brief, logarithmically growing cells
were pelleted, resuspended in 0.2 M Tris, pH 7.5, containing 70% ethanol,
and fixed overnight at 4°C. Cells were treated with 1 mg/ml RNase A at 37°C
for 1 h. Proteinase K was added, and the cell suspension incubated at 55°C for 1 h. Cells were stained overnight at 4°C in 0.2 M Tris containing 3 µg/ml
propidium iodide (Sigma Chemical Co.). Before being subjected to flow cytometry, cells were sonicated, using a Branson sonifier 450 on a setting of
two for 2-5 s. Aliquots of cells fixed with 3.7% formaldehyde, as described for
immunofluorescence, were stained with 300 ng/ml 4',6-diamidino-2-phenylindole (DAPI) to analyze nuclear morphology, and anti-
-tubulin decorated
with fluorescein isocyanate, to analyze spindle morphology.
For experiments comparing ctf13-30 and ctf191 single mutants to
ctf13-30 ctf19
1 double mutants, cultures of wild-type (YPH501), ctf13-30/ctf13-30 (YPH1329), ctf19
1::HIS3/ctf19
1::HIS3 (YPH1330), and
ctf19
1::HIS3/ctf19
1::HIS3 ctf13-30/ctf13-30 (YPH1331, YPH1332) were
grown at 25°C to early log stage (~106 cells/ml), then split at time = 0, keeping half of the culture at 25°C and shifting the other half to 37°C.
Samples were taken at the start of the experiment (time = 0), and at 3 and
5 h after the temperature shift. For each time point, samples were assayed for viability, nuclear and spindle morphology, and cell DNA content by
flow cytometry.
Isolation of Minichromosomes and MT-Binding Assay for Yeast Centromeres
Isolation of minichromosomes from yeast cells and assaying their ability
to bind to MTs were performed as previously described (Kingsbury and
Koshland, 1991).
In Vivo Cross-Linking and Chromatin Immunoprecipitation
The methods used for chromatin immunoprecipitation and the PCR primers used to amplify both centromeric and noncentromeric test loci were as
described in Meluh and Koshland (1997). Purified HA mAb 12CA5 (Berkeley Antibody Co.) was added at either a 1:250 dilution (resulting in a
4 µg/ml final concentration), or 50 µl of HA antibody pre-cross-linked to
CNBr-Sepharose beads was added to the chromatin solution. Crude anti-Mif2p rabbit antisera, provided by Pam Meluh (Carnegie Institute of Washington, Baltimore, MD), was used at a 1:250 dilution and served as a positive control for this assay. For NZ treatment experiments, NZ was added to logarithmically growing cells at 15 or 20 µg/ml final concentration, and incubated for 90 min at 30°C to completely depolymerize MTs before fixation.
Immunofluorescence
Ctf19p was localized by indirect immunofluorescence microscopy as described by Pringle et al. (1989), with a few modifications. To visualize
MTs, cells were fixed with 3.7% formaldehyde for 90 min at 30°C. To visualize Ctf19-HAp or Tub4p, cells were fixed for 60 min at 25°C. Primary
antibodies were diluted in block solution (4% milk, 2% BSA, and 0.1%
Tween in 1× PBS) as follows: 1:50 for anti-
-tubulin (YOL1/34, Serra
Lab), 1:5,000 for anti-HA antibody (12CA5, Boehringer Mannheim
Corp.), and 1:500 for anti-Tub4p (provided by L. Marschall; see Marschall
et al., 1996
). Affinity-purified secondary antibodies (Cappel Research
Products) were used at 1:1,000 dilution in block solution. YOL1/34 was
detected with rhodamine conjugated goat anti-rat antibodies, anti-HA antibody was detected with fluorescein conjugated goat anti-mouse antibodies, and anti-Tub4p was detected with CY3 conjugated goat anti-rabbit
antibodies (Jackson ImmunoResearch Laboratories, Inc.). When costaining for Ctf19-HAp and either
-tubulin or Tub4p, primary and secondary
antibodies were applied in rounds, with anti-HA and goat anti-mouse applied first, to avoid background from secondary antibody cross-reactivity. Cells were examined using fluorescence microscopy with a Zeiss Axioskop fitted with UV, fluorescein isothiocyanate, and rhodamine/CY3 optics and a 100× objective. Digital images were captured using a cooled
charged-coupled device (CCD) camera (Photometrix) and IPLabSpectrum software (Signal Analytics).
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Results |
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Isolation and Characterization of the CTF19 Gene
An SDL screen (Kroll et al., 1996) was performed in which
CTF13 was inducibly overexpressed in a subset of 12 potential kinetochore ctf mutants, which tested positive for
the centromere transcription readthrough assay (Doheny
et al., 1993
). Overexpression of CTF13 caused SDL in
combination with four mutants within this set: ndc10-42, ctf17-61, ctf19-26 (YCTF26), and ctf19-58 (YCTF58). The
chromosome missegregation ctf phenotype (visualized by
the formation of red sectors in a white colony) of ctf19-58
was confirmed by genetic analysis to be due to a single nuclear mutation, and the corresponding gene was cloned by
complementation of this phenotype (see Materials and
Methods; Fig. 1 a). CTF19 was localized to the right arm of chromosome XVI using physical mapping methods. Nucleotide sequencing of the 2-kb CTF19 clone revealed a
previously unidentified 1.2-kb ORF that encodes a protein
of 369 amino acids with a predicted molecular mass of 40 kD. Database searching revealed no significant overall homology at the amino acid level. Protein motif searching (ProSite) revealed a putative leucine zipper beginning at
amino acid 131 (Fig. 1 d). The sequence and mapping data
of CTF19 (YPL018W) was corroborated upon release of
the complete sequence of the S. cerevisiae genome (Goffeau et al., 1996
).
ctf19-26 was confirmed to be an independent allele of CTF19 using four lines of evidence: first, the cloned CTF19 DNA complements the ctf19-26 sectoring phenotype; second, the ctf19-58/ctf19-26 diploid exhibits a chromosome missegregation phenotype; third, when this diploid is sporulated, all spores which bear a chromosome fragment display the sectoring phenotype (Basrai, M., personal communication); and fourth, when ctf19-26 is crossed to a wild-type strain with the LEU2-marked genomic CTF19 locus (YPH1313), the ctf phenotype always segregated away from the LEU2 marker.
A complete deletion of the CTF19 ORF was generated
using PCR-mediated gene disruption (Lorenz et al., 1995).
The CTF19 ORF was replaced with two different marker
genes, creating ctf19
1::HIS3 and ctf19
1::TRP1. The deletion strains are viable, display no temperature conditional phenotypes, and exhibit a chromosome missegregation phenotype similar to that seen in strains containing
either of the original mutant alleles, ctf19-58 or ctf19-26.
Upon overexpression of CTF13 under the control of a
GAL1 promoter, SDL was observed in the deletion strain,
comparable to that seen with ctf19-58 and ctf19-26, albeit
somewhat more pronounced.
ctf19 Mutants Display Phenotypes Consistent with a Role in Kinetochore Function
Strains carrying the ctf191, ctf19-58, or ctf19-26 mutations were tested for sensitivity to benzimidazole compounds. These agents cause depolymerization of MTs, and
it has been observed that kinetochore mutants, as well as
mitotic checkpoint mutants, are sensitive to compounds
such as benomyl (Spencer et al., 1990
; Hoyt et al., 1991
; Li
and Murray, 1991
). All three strains are highly sensitive to
10 µg/ml of benomyl at 25°C, and slightly sensitive at the
level of 5 µg/ml (Fig. 2 a). Isogenic wild-type strains grow
well with 10 µg/ml of benomyl at 25°C, and are sensitive to
20 µg/ml. As an interpretive note, the bub (Hoyt et al.,
1991
) and mad (Li and Murray, 1991
; Hardwick and Murray, 1995
) mutants are also sensitive to 10 µg/ml of
benomyl.
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Examination of possible cell cycle defects revealed that
a ctf191/ctf19
1 mutant shows a G2/M accumulation
with 2C DNA content in a logarithmically growing culture
(Fig. 2 b). Cytological analysis of these cells reveals an accumulation of large budded cells with the nucleus at or
near the neck (29% in ctf19
1 versus 6% in wild-type). A
similar G2 delay is seen in ctf13-30, ndc10-42 (Doheny et
al., 1993
), cep3-1/-2 (Strunnikov et al., 1995
), and skp1-4
(Connelly and Hieter, 1996
) kinetochore structural mutants at their nonpermissive temperatures. While there is
an accumulation of large budded uninucleate cells, the
number of telophase cells in the ctf19 null mutant remains
similar to that seen for wild-type, 17% versus 16%, respectively. In contrast, the percentage of telophase cells in a
ctf13-30 mutant at the nonpermissive temperature drops
to 3%. This is consistent with the notion that ctf13-30 cells
experience a strong arrest at G2/M at the nonpermissive
temperature, whereas ctf19 null cells experience a shorter
delay, most likely because the lesion is less severe.
Chromosome missegregation in the ctf19 mutants was
quantitated in homozygous diploid strains using colony
color half sector analysis (Koshland and Hieter, 1987).
Chromosome loss (1:0 segregation) results in a pink/red
half-sectored colony, whereas nondisjunction (2:0 segregation) results in a white/red half-sectored colony. The rate
of chromosome loss in a ctf19
1/ctf19
1 mutant is ~100 times greater than that of wild-type, and the rate of nondisjunction is 60-fold higher than wild-type (Table II). For
ctf19 mutant alleles, the rates of chromosome loss and
nondisjunction are 77- and 17-fold higher than wild-type
for ctf19-58/ctf19-58, and 37- and 13-fold higher for ctf19-26/ctf19-26, respectively. Thus, both chromosome loss and
gain are occurring in ctf19 mutant diploids, and the deletion strain is affected the most severely. Similar rates of
chromosome loss and nondisjunction have been reported for essential kinetochore mutants ctf13-30 (Doheny et al.,
1993
) and skp1-4 (Connelly and Hieter, 1996
).
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Ctf19p Interacts Genetically with Components of the Kinetochore and the Mitotic Spindle Checkpoint
To explore a potential role of Ctf19p in kinetochore function, genetic analysis was used to look for synthetic phenotypes between ctf19 mutants and known kinetochore mutants. As our first genetic test, we used the SDL screen
(Kroll et al., 1996) to assay the effect of overexpression of
CTF19 in a wild-type background, and in strains containing mutations in each of the four subunits of the CBF3
kinetochore complex. CTF19, the reference gene, was placed under a GAL1 promoter (pKH21, Fig. 1 b) and this
plasmid was transformed into a set of target mutants
(ndc10, cep3, ctf13, and skp1). Growth was assessed upon
galactose induction, as previously described (Kroll et al.,
1996
). Results of this dosage study are summarized in Table III. SDL was seen when CTF19 was overexpressed in
the background of two independent mutant alleles of
NDC10, ndc10-42, and ndc10-1, thus providing another genetic link between CTF19 and the kinetochore. Moreover,
overexpression of NDC10 results in lethality in ctf19-26
and ctf19-58 mutants. The plasmid expressing CTF13 under a GAL1 promoter (pKF88) was also transformed into these mutant strains and viability assessed. Overexpression of CTF13 results in SDL in ndc10-42 (as previously
described), and also in ndc10-1 at elevated temperature
(30°C), as well as cep3-1 and cep3-2 (Table III).
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To look for additional genetic interactions between
CTF19 and CBF3 kinetochore mutations, double mutants
were made between a ctf19 null mutant and the same
CBF3 subunit mutations used in the dosage studies above.
The heterozygous diploids were sporulated at 25°C and
tetrads analyzed (Table IV). To detect any conditional interactions, all double mutants recovered at 25°C were assayed for growth at successively higher semipermissive
temperatures. The ctf191 ctf13-30 double mutant showed
conditional SL at 30°C, which is lower than the nonpermissive temperature of ctf13-30 alone (35.5°C; Doheny et al.,
1993
). ctf19
1 ndc10-42 double mutants could not be recovered at 25°C, although ctf19
1 ndc10-1 double mutants
were viable at all permissive temperatures tested, demonstrating allele specific SL. ctf19
1 skp1-4 double mutants were conditionally synthetic lethal at 28°C, whereas
ctf19
1 skp1-3 double mutants were viable at all permissive temperatures. This allele specificity is significant since
the skp1-4 allele arrests in G2, and the skp1-3 allele arrests
in G1 at the respective nonpermissive temperatures (Connelly and Hieter, 1996
), suggesting that the interaction detected between CTF19 and SKP1 is within the G2/M
phase of the cell cycle. Similarly, a conditional synthetic lethal interaction was detected between ctf19
1 and sgt1-3,
but not with sgt1-5 (SGT1 is a suppressor of skp1-4; Kitagawa, K., personal communication). sgt1-3 arrests at G2/M,
whereas sgt1-5 arrests at G1/S. Therefore, this allele specificity is analogous to the ctf19
1-skp1-4 interaction. Finally, both mutant alleles of CEP3 tested, cep3-1 and cep3-2,
were synthetically lethal with ctf19
1 at 25°C. Thus,
CTF19 genetically interacts with all four components of
the CBF3 CDEIII-binding complex.
|
To address the specificity of these genetic interactions, a
ctf191 strain was crossed to strains containing cbf1::
TRP1, cse4-1, or mif2-3 mutations, representing other proteins which function at the kinetochore, as well as tub1-1
or tub2 tubulin mutations. No synthetic lethal interactions
were detected in any of the corresponding double mutants,
except for ctf19
1 mif2-3, which is inviable (Table IV).
Interestingly, Meluh and Koshland (1997)
have shown
that Mif2p interacts with CEN DNA primarily through
CDEIII. Thus, the genetic interactions identified between CTF19 and the kinetochore appear specific for CBF3 and
other CDEIII-associated components.
The ctf191 ctf13-30 double mutant was a valuable reagent, because double mutants could be recovered at
25°C, but were conditionally synthetic lethal at 30°C. It has
been shown that ctf13-30 alone causes initiation of the mitotic checkpoint at the nonpermissive temperature, resulting in delay at the G2/M point of the cell cycle (Doheny et
al., 1993
). When ctf13-30 is combined with mad2 or bub1
mitotic checkpoint mutants, the cells do not arrest upon shift to the nonpermissive temperature, and rapid death
ensues (Wang and Burke, 1995
; Pangilinan and Spencer,
1996
). In contrast, when ctf13-30 is combined with a
ctf19
1 mutant, although a rapid loss in viability is observed upon shift to the nonpermissive temperature, the
checkpoint appears to be intact judging by a G2/M delay
visible in flow cytometry profiles at 25°C and 37°C (data
not shown). This G2/M delay in the double mutant is accompanied by an accumulation of large budded uninucleate
cells with short spindles. The inability of the ctf19
1 ctf13-30 double mutant to recover after exposure to the nonpermissive temperature suggests that there is a more severe
structural aberration than in the ctf13-30 mutant alone.
In light of the observations that the mitotic spindle
checkpoint responds to impaired kinetochore function
(Wang and Burke, 1995; Pangilinan and Spencer, 1996
;
Wells and Murray, 1996
), we asked directly whether
CTF19 genetically interacts with BUB1, BUB2, BUB3, or
MAD2 mitotic checkpoint genes. To do this, we again used the SDL assay. The CTF19 overexpression plasmid
(pKH21) was transformed into mitotic checkpoint target
mutants, bub1
, bub2
, bub3
, and mad2
. Upon induction of CTF19 on galactose-containing medium, growth
was assessed as previously described. SDL was observed in
the bub1
and bub3
strains, but no defect of growth was
seen with mad2
or bub2
. To interpret these results, we
also determined the effect of overexpressing CTF13 in the
same target mutants, especially since the ctf13-30 mutation
triggers the mitotic checkpoint resulting in a G2/M delay,
as described above. When CTF13 was overexpressed in
combination with bub2
, bub3
, and mad2
, SDL was
observed with bub3
, but not with mad2
or bub2
.
To test for synthetic lethal interactions, crosses were
made between strains carrying a ctf19 null mutation and
strains carrying either bub1, bub2
, bub3
, or mad2
mutations (Table V). A rad9
mutation, previously characterized as defective in monitoring incomplete replication and DNA damage (Weinert and Hartwell, 1988
; Lydall and Weinert, 1995
), was also tested. SL was seen when
ctf19
1 was combined with bub1
, bub3
, or mad2
, but
not with bub2
or rad9
(Table V). Bub1p, Bub3p, and
Mad2p are all necessary for the initiation of the mitotic
checkpoint pathway and are sufficient for shorter delays,
as seen with low levels of NZ, whereas Bub2p is required
for maintaining longer delays, as seen with high levels of
NZ (Wang and Burke, 1995
; Pangilinan and Spencer,
1996
). These synthetic dosage lethal and synthetic lethal
genetic interaction results are consistent with the notion
that ctf19 mutations invoke a mild premitotic delay, which
does not require Bub2p. Taken together, these genetic
studies strongly suggest that Ctf19p plays a structural role
at the kinetochore.
|
Ctf19p Interacts with Centromere DNA In Vivo
Sucrose gradient sedimentation analysis revealed that
Ctf19-HAp sediments with an S value of 20 (data not
shown). This suggests that Ctf19p may be part of a large
protein complex in the cell. There is no evidence that
Ctf19p is part of the CBF3 CDEIII-binding complex, as
Ctf19p is not required for the normal CEN DNA bandshift in vitro, and antibodies to an epitope-tagged Ctf19p
do not result in a supershift of the CBF3-CEN DNA complex (data not shown; Stemmann et al., 1999). In addition,
coimmunoprecipitation experiments using crude yeast cell
extracts were unable to detect a direct interaction between
Ctf19p and CBF3 proteins.
Knowing that Ctf19p is not a part of the CDEIII-binding complex detected by CEN DNA bandshift in vitro, we
investigated other ways in which it could be involved in kinetochore function. One possibility is that Ctf19p plays a
role in binding of MTs to kinetochores. To test this hypothesis, an in vitro assay was used which measures the
ability of yeast centromeres to bind to MTs (Kingsbury
and Koshland, 1991). In this assay, minichromosomes
containing a centromere are introduced into wild-type or
mutant strains, and purified chromatin is prepared. Taxol-stabilized bovine MTs are added to the lysate and sedimented. The supernatant and pellet are separated, DNA
from each fraction is extracted, and the relative amount of the minichromosome in each fraction is determined. In
wild-type cultures with wild-type centromeres on the minichromosomes, the minichromosomes sediment with the
MTs. CEN DNA mutations in CDEII and CDEIII abolish
the centromere's ability to bind MTs (Kingsbury and Koshland, 1991
). Similarly, trans-acting mutations in CEN-binding proteins also inhibit the ability of the minichromosome
to bind MTs, exemplified by the cep3-1 mutant (Strunnikov et al., 1995
). Two independent alleles of CTF19 were
tested in this assay, ctf19-58 and ctf19-26, and both were
shown to exhibit a dramatic defect in centromere-dependent binding of minichromosomes to MTs (Fig. 3). Therefore, knowing that Ctf19p interacts genetically with the
CBF3 complex, is important for binding MTs to centromeres of minichromosomes, and sediments with an S
value consistent with being part of a protein complex, we
next investigated whether Ctf19p is involved in a higher
order centromere complex.
|
To examine whether Ctf19p physically interacts with a
kinetochore macromolecular complex, we used an in vivo
cross-linking and immunoprecipitation strategy (Meluh
and Koshland, 1997). Formaldehyde cross-linked chromatin (2 h fixation) prepared from an epitope-tagged CTF19-3HA strain and a wild-type untagged control strain were sonicated to shear the DNA and immunoprecipitated with
anti-HA antibody. After reversing the cross-links, the
presence of specific DNA sequences in the immunoprecipitates was assessed by PCR analysis. Mif2p, which was previously shown to associate with CEN DNA in vivo (Meluh
and Koshland, 1997
), served as a positive control for this
assay. Two CEN DNA sequences were tested, CEN3 and
CEN16, and both were found to be present in the Ctf19-HAp immunoprecipitate, but not in the untagged control
strain or in the mock-treated control (Fig. 4 a). The anti-Mif2p immunoprecipitate also contained these CEN DNA
sequences. To test if the interaction detected is specific for
CEN DNA, two noncentromeric loci, PGK1 and HMRa, which are AT-rich intergenic regions located on yeast
chromosome III, were analyzed. Both of these sequences
were not found in the Ctf19-HAp immunoprecipitate, as
was the case with the Mif2p positive control (Fig. 4 a).
Thus, as predicted by the numerous genetic interactions
with kinetochore protein components, Ctf19p specifically
associates with the centromere in chromatin preparations.
|
It is plausible that the association of Ctf19p with the centromere is transient and contact may be made with kinetochore proteins upon MT attachment. To test whether MTs are necessary for Ctf19p to associate with the centromere, the chromatin immunoprecipitation assay was performed with strains grown in the presence of NZ, a MT depolymerizing agent. Results from this experiment show that, as with Mif2p and Ndc10p (not shown), Ctf19p specifically immunoprecipitates CEN DNA, and not noncentromeric loci, even in the presence of NZ (Fig. 4 b). These data further support the conclusion that Ctf19p is in fact part of a centromere-protein complex.
Ctf19p Localizes Near the Spindle Pole Body (SPB)
The localization of Ctf19p in yeast cells was determined by
indirect immunofluorescence using an HA epitope fusion
construct (pKH32). To avoid potential copy number effects,
the CTF19-3HA fusion was integrated into the genome at
the leu21 locus (leu2
1::CTF19-3HA, YPH1327). For immunofluorescence studies, an asynchronous population of
cells from strains containing CTF19-3HA (either plasmid-based or integrated, YPH1324 or YPH1327) or an untagged
control (YPH1323 or YPH500) were formaldehyde-fixed,
and stained with anti-HA antibody and anti-
-tubulin,
which stains spindle MTs. The HA antibody detected a
bright dot at the vertex of the MTs in interphase cells, and
at the ends of the spindle in mitotic cells (Fig. 5). This staining pattern, reminiscent of SPB staining, is similar
to that seen with other centromere proteins, including
Ndc10p (Goh and Kilmartin, 1993
), Mif2p (Meluh and
Koshland, 1997
), and Cse4p (Meluh et al., 1998
), and is
consistent with the notion that centromeres display a nonrandom localization throughout the cell cycle, clustering near the SPB during interphase (G1) and late mitosis
(anaphase and telophase; Guacci et al., 1997
; Straight et
al., 1997
). A key difference between the staining pattern
observed for Ctf19-HAp and those reported for other centromere proteins occurs during early mitosis. Both Ndc10p
and Cse4p are reported to stain the spindle MTs, evident
as a short bar, in cells with short spindles. Ctf19-HAp
staining, however, resolves into two distinct foci even in
cells with short spindles.
|
To examine the specificity of this localization, we obtained antibodies against Tub4p (provided by L. Marschall, Stanford, CA), which served as an SPB marker (Sobel and Snyder, 1995; Marschall et al., 1996
; Spang et al.,
1996
). Tub4p, the
-tubulin homologue in S. cerevisiae, is
part of a complex that localizes to the inner and outer
plaques of the SPB (Geissler et al., 1996
; Knop et al., 1997
), where nuclear and cytoplasmic MTs are organized,
respectively. Costaining experiments revealed that Ctf19-HAp localizes to the same region as Tub4p (Fig. 6, a and
b). Further analysis of cells stained for both Ctf19-HAp
and Tub4p revealed that the Ctf19-HAp signal appears to
be just interior to (on the nucleoplasmic side of) the
Tub4p signal in mid-mitotic cells with short spindles (Fig. 6
d). This observation was examined in more detail by measuring the distance between the Ctf19-HAp signals and
Tub4p signals in individual cells. The average distance between Ctf19-HAp signals for 50 mitotic cells was 1.5 µm ± 1.1, whereas the comparable distance between Tub4p signals was 1.8 µm ± 1.1. These measurements correspond to
an average distance of 0.2 µm between Ctf19p and Tub4p
in mitotic cells. To interpret the significance of this difference, it should be considered that Tub4p resides on both nuclear and cytoplasmic surfaces of the SPB and that the
fluorescent signal observed for Tub4p is an average of
both signals. Therefore, the distance from the Ctf19-HAp
signal to the nuclear side of the SPB may be less than indicated by these measurements. These data suggest that
Ctf19p localizes near the SPB, but, as expected, is not
likely to be an integral component of the SPB. Consistent with this hypothesis, we were unable to demonstrate a direct interaction between Ctf19-HAp and Tub4p by coimmunoprecipitation (data not shown). It is possible that
Ctf19p is part of a complex which resides at the nucleoplasmic surface of the SPB and transiently interacts with
kinetochores. However, it is more likely that the staining
pattern observed for Ctf19-HAp corresponds to a centromere localization, as noted above for other kinetochore
proteins, and is thus compatible with data implicating a kinetochore function for Ctf19p.
|
To test if we could differentiate between these two hypotheses, we asked whether Ctf19p requires MTs for its localization. Assuming that kinetochores are tethered to the
SPB in a MT-dependent manner (Guacci et al., 1997), cells
were treated with 15 µg/ml NZ (a concentration that depolymerizes all MTs) and examined by immunofluorescence. For proteins that are known to be integral components of the SPB, localization is unchanged by the
presence of NZ (Marschall et al., 1996
). Fluorescent in situ
hybridization analysis has revealed that centromere localization is, however, changed in the presence of NZ as they
are no longer clustered (Guacci et al., 1997
). It should be
noted that, although NZ and other benzimidazole drugs
depolymerize most of the MTs, it is possible that a small
amount of the polymer which is undetectable by immunofluorescence remains (Marschall et al., 1996
; Jacobs et al.,
1988
). Interestingly, Ctf19p localization in most cells remained near the SPB, visualized as one (~66% of cells) or
two (~27% of cells) brightly staining dots (Fig. 6 c). This
compares to 69% and 31%, respectively, in cells not
treated with NZ. However, ~7% of the cells (out of 500 cells analyzed) contained three or four discrete signals for
Ctf19-HAp. There were also cells observed in which two
signals were seen for Ctf19-HAp, but only one signal was
seen for Tub4p. These observations indicate that the majority of Ctf19p remains near the SPB in the absence of
MTs, however, a portion of the protein in some cells has
an altered localization in the presence of NZ. These results
may indicate that Ctf19p is present both at the centromere
and near the nucleoplasmic surface of the SPB. Whether
or not these localization data are suggestive of a function
for Ctf19p at the SPB, or merely a consequence of the limitations of the assay, has yet to be determined.
CTF19 Interacts Genetically with a Spindle- and Pole-associated Protein
As a preliminary investigation into a potential role for
Ctf19p at the SPB, we tested for genetic interactions between CTF19 and several genes encoding either integral
SPB proteins or proteins involved in SPB duplication. No
synthetic lethal interactions were detected in any double
mutants created between ctf191 and mps2-1, ndc1-1,
cdc31-1, spc42-10, spc110-1, tub4-32, or tub4-34 (data not
shown). In comparison to the unequivocal genetic interactions detected between CTF19 and the genes encoding the
CBF3 kinetochore components, we conclude that the
function of Ctf19p lies at the kinetochore. We also tested
for a genetic interaction between CTF19 and NDC80,
which encodes a spindle- and pole-associated protein. Interestingly, a conditional synthetic lethal effect was detected in ndc80-1 ctf19
1 double mutants at 28°C. This genetic interaction is allele specific, as no conditional SL was
detected in ndc80-2 ctf19
1 double mutants. NDC80 mutants display phenotypes very similar to a temperature-sensitive mutant of NDC10, which encodes a component
of the CBF3 kinetochore complex (Wigge et al., 1998
). Although the phenotypes and immunofluorescent staining pattern of Ndc80p are consistent with localization to the
kinetochore, no genetic interactions have been detected
between NDC80 and CBF3 kinetochore components
(Wigge et al., 1998
). These authors have concluded that an
indirect interaction may exist between Ndc80p and the kinetochore. It is quite possible that such an indirect interaction may occur through Ctf19p.
![]() |
Discussion |
---|
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---|
We have identified and characterized the CTF19 gene of
S. cerevisiae, and show that it encodes a novel protein
which is required for faithful chromosome transmission
and efficient binding of centromeres to MTs, associates
with a centromere DNA complex in vivo, and localizes to
the SPB region. Previously, two uncharacterized mutations, ctf19-58 and ctf19-26, were classified as putative kinetochore mutants by an in vivo centromere transcription
readthrough assay (Doheny et al., 1993). ctf19 mutants
were further implicated in kinetochore function by our
SDL screen (Kroll et al., 1996
), in which overexpression of
the CTF13 reference gene, encoding an essential CBF3 kinetochore protein, resulted in lethality in the context of
the ctf19-58 and ctf19-26 mutations. The phenotypes described here for ctf19 mutants, including chromosome missegregation, increased sensitivity to benomyl, and a G2/M
accumulation of logarithmically growing cells, are all consistent with a defect in kinetochore function.
Extensive genetic analysis revealed that CTF19 exhibits
strong genetic interactions with both kinetochore structural mutants and mitotic checkpoint mutants. Genetic interactions (SDL, SL, or both) were detected between the
ctf19 null mutation and mutant alleles of all four subunits
of the CBF3 kinetochore complex, as well as with Mif2p,
which has been shown to interact with CEN DNA in a
CDEIII-specific manner (Meluh and Koshland, 1997). We also demonstrate that cells defective in, or overexpressing
Ctf19p require a functional mitotic checkpoint pathway, as
do cells defective in, or overexpressing Ctf13p. The extent
and specificity of these interactions strongly implicates
Ctf19p as playing a role in kinetochore structure or function.
Ctf19p Functions as Part of a Macromolecular Kinetochore Complex
Consistent with the numerous genetic interactions linking
Ctf19p to the kinetochore, as well as the sedimentation
data, we demonstrate that Ctf19p associates specifically
with centromeric DNA in vivo through formaldehyde
cross-linking followed by chromatin immunoprecipitation.
Two known kinetochore proteins, Ndc10p and Cbf1p, as
well as two implicated kinetochore proteins, Mif2p and Cse4p, have been shown to cross-link wild-type CEN
DNA in vivo (Meluh and Koshland, 1997; Meluh et al.,
1998
). Ndc10p and Mif2p both demonstrate a specific dependence on the integrity of CDEIII (Meluh and Koshland, 1997
). A similar specificity of interaction between
Ctf19p and CDEIII is described by Lechner and colleagues, who have independently identified Ctf19p as part
of a complex of proteins that interacts with CBF3 (Stemmann et al., 1999
). These data are consistent with genetic
interactions reported here, which are also specific for
CBF3 components.
The fact that Ctf19p is able to specifically cross-link
CEN DNA does not differentiate between a direct and indirect association. Ctf19p is not a subunit of the CBF3
complex (Stemmann and Lechner, 1996), and is not necessary for the gel mobility shift seen with the assembled kinetochore complex on a CEN DNA fragment (data not
shown; Lechner, J., personal communication). Therefore
we propose that Ctf19p interacts indirectly with CEN
DNA, likely through interactions with the CBF3 complex,
or perhaps through a larger macromolecular complex
whose assembly is initiated by recruitment of CBF3 to
CDEIII (Sorger et al., 1994
; Meluh and Koshland, 1997
).
Given the ability of Ctf19p to cross-link to CEN DNA,
and the defect seen in the ability of ctf19 mutants to bind
centromeres of minichromosomes to MTs, Ctf19p would
be a good candidate for a factor which interacts with CBF3
and is necessary to form active spindle MT-binding complexes. The fact that Ctf19p is able to associate with centromeric DNA in the presence of NZ suggests that MTs
are not required for this association, and places Ctf19p at
the kinetochore instead of on the spindle. Similar results were observed for Mif2p and Ndc10p. We are currently
testing whether Ctf19p is required for attachment of reassembled kinetochore complexes in vitro to polymerized MTs.
Implications of a Kinetochore Protein with SPB Localization
The immunolocalization pattern of Ctf19p is consistent
with kinetochore proteins, which is strongly supported by
the genetic and biochemical data presented here. Other
proteins which have been biochemically placed at the centromere, including Ndc10p, Mif2p, and Cse4p, show immunofluorescent staining patterns similar to Ctf19p. The
SPB staining pattern observed in G1 cells and in late mitotic cells could be due to the effect of centromere clustering, a phenomenon seen in higher eukaryotes and demonstrated in yeast by fluorescent in situ hybridization
(Guacci et al., 1997). Studies on cell cycle dependent centromere positioning in S. cerevisiae reveal that in G1 cells,
centromeres are loosely clustered around the SPB, similar
to that reported for fission yeast and mammalian cells
(Ferguson and Ward, 1992
; Funabiki et al., 1993
; Vourc'h et al., 1993
). In mitosis before anaphase (mid M), centromeres are centrally localized within the nucleus, away
from the SPB, reminiscent of metaphase in larger eukaryotic cells. In anaphase and telophase cells, centromeres are
clustered tightly and proximal to the SPBs. One caveat to
interpreting the Ctf19p localization as consistent with it
being a CEN binding protein is that, unlike Ndc10p (Goh
and Kilmartin, 1993
) and Cse4p (Meluh et al., 1998
), Ctf19p does not display spindle staining, as may be expected for centromere associated proteins when kinetochores are forming bipolar attachments to the spindle
MTs. The absence of spindle staining for Ctf19p may reflect limitations of the immunofluorescence assay, as other
kinetochore proteins, including Ctf13p and Cep3p, have not been visualized in the cell by traditional immunofluorescent techniques, or it may reflect a unique role for
Ctf19p in mitosis, perhaps functioning to tether kinetochores to the SPB. Given the prominent role of anaphase
B in sister chromatid separation in budding yeast (Straight
et al., 1997
), Ctf19p may be important in stabilizing the
link between kinetochores and each SPB during spindle
pole separation.
The results of immunofluorescence with Ctf19-HAp in
the presence of NZ suggest that at least some amount of
Ctf19p localizes adjacent to the SPB, as opposed to Ctf19p
residing solely at the kinetochores, because the positioning
of kinetochores near the SPB is believed to be dependent
on MTs. In the presence of NZ, it has been observed that
centromeres are no longer clustered, but rather disperse
throughout the nucleus (Guacci et al., 1997), presumably
because MTs are no longer present for the kinetochores to
remain attached to the SPB. However, the procedures
used to fix and process nuclei for in situ hybridization in
yeast may be more disruptive than that used for immunofluorescence, and theoretically could contribute to an aberrant delocalization of centromeres after treatment with
NZ. Although Ctf19-HAp does maintain a discrete localization in the presence of NZ, we did observe a few examples (~7% of cells analyzed) in which more than two fluorescent signals were seen for Ctf19-HAp which did not
correlate with a Tub4p signal. We reason that, because a
small polymer of MTs may still be present at the face of
the SPB even after NZ treatment (Marschall et al., 1996
;
Jacobs et al., 1988
), Ctf19p may be part of a complex that
is peripheral to the SPB. The localization of this complex
may be less stable in the presence of MT depolymerizing agents, accounting for the small population of cells that exhibit more than two Ctf19-HAp signals. Wigge et al.
(1998)
have identified several new spindle pole and spindle-associated proteins through analysis of purified spindle preparations with MALDI mass spectrometry. Ndc80p
is of particular interest because it stains spindles as well as
poles, and ndc80-1 mutants display phenotypes similar to
yeast kinetochore mutants, including chromosome missegregation and anaphase defects. In addition, Ndc80p is a
potential homologue of human HEC protein, which localizes to the centromere. However, no genetic interactions
were detected between NDC80 and three of the CBF3
components tested. Ndc80p partially copurifies with the
factors which bind kinetochores to MTs (Sorger et al.,
1994
), but is absent from the final fraction (Wigge et al.,
1998
). Since Ctf19p interacts with the centromere, both
genetically and biochemically, and it genetically interacts
with Ndc80p, we propose that Ctf19p provides a link between the mitotic spindle and the kinetochore in budding yeast.
![]() |
Footnotes |
---|
Address correspondence to Philip Hieter, Centre for Molecular Medicine and Therapeutics, 980 West 28th Avenue, Vancouver, BC V5Z 4H4, Canada. Tel.: (604) 875-3826. Fax: (604) 875-3840. E-mail: hieter{at}cmmt.ubc.ca
Received for publication 24 November 1998 and in revised form 16 February 1999.
We thank O. Stemmann, J. Ortiz, S. Rank, and J. Lechner for communicating unpublished results; P. Meluh, J. Kingsbury, S. Strunnikov, D. Koshland, F. Pangilinan, F. Spencer, S. Michaelis, L. Marschall, M. Basrai, and J. Lechner for reagents and advice; M. Winey, J.V. Kilmartin, and T. Stearns for strains; and Hieter lab members for their helpful discussions and support.
This work was supported by a grant from the National Cancer Institute (CA16519) to P. Hieter.
![]() |
Abbreviations used in this paper: bub |
---|
, budding uninhibited by benzimidazole; CDEs, centromere DNA elements; ctf, chromosome transmission fidelity; 5-FOA, 5-fluoroorotic acid; mad, mitotic arrest deficient; MT, microtubule; NZ, nocodazole; ORF, open reading frame; SDL, synthetic dosage lethality; SL, synthetic lethality; SPB, spindle pole body.
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References |
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