INTRODUCTION

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The hydrolysis of phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P₂] by phospholipase C (PLC) is a key early event in the regulation of diverse cell functions by many extracellular molecules. The reaction yields two prominent eukaryotic second messengers: 1,2-diacylglycerol (DG) and inositol 1,4,5-triphosphate [Ins(1,4,5)P₃] (15, 41, 44). The hydrophilic Ins(1,4,5)P₃ triggers the release of calcium from internal stores and thus modulates Ca²⁺- and calmodulin-regulated pathways (3), while the hydrophobic DG activates the phospholipid- and Ca²⁺-dependent protein kinase C (50). As a result, cytoplasmic PLC plays vital roles in the signal transduction cascades which ultimately regulate nuclear events.

Three classes of mammalian phosphatidylinositol (PtdIns)-specific PLC isoenzymes, designated , , and , were identified via biochemical, molecular, and immunological approaches (41). The Saccharomyces cerevisiae gene coding for PtdIns-specific PLC (PLC1) has also been cloned, and the protein product (Plc1p) is most closely related to mammalian  isoforms, both in terms of sequence identity and in arrangements of conserved domains (23, 54, 68). All isoenzymes of the three main families of PLC, as well as the yeast Plc1p, contain a series of common modules which facilitate the enzymatic reaction. The modular structure contains an N-terminal pleckstrin homology (PH) domain, an EF-hand calcium binding domain, an active site containing two conserved regions termed X and Y boxes, and a C-terminal C2 lipid binding domain (32, 35) (schematic representation of the modular structure of Plc1p is shown in Fig. 4).

While PLC1 is not an essential gene, its deletion results in a number of phenotypes, including slow growth (progressively exacerbated at higher temperatures), osmosensitivity, defective utilization of carbon sources other than glucose, altered cell morphology, and inability to complete cytokinesis (23). Flick and Thorner (24) identified two genes which, when present in high copy number, suppress the temperature sensitivity of cells in which the PLC1 gene has been deleted (plc1). One of these genes, PHO81, codes for an inhibitor of a cyclin-dependent protein kinase comprised of PHO80 and PHO85 gene products. The second suppressor is a novel gene, SPL2. In addition, Plc1p was previously shown to interact with the PtdIns kinase homolog Tor2p (43). Recent data implicate Plc1p in the regulation of several processes. Plc1p is required for synthesis of inositol hexakisphosphate, which supports efficient export of mRNA from the nucleus (70), and for oxidative-stress-induced destruction of cyclin Ume3p (12). In addition, Plc1p appears to be involved in signal transduction pathways responsible for the sensing of nutrients. Plc1p is required for glucose-induced activation of plasma membrane H⁺-ATPase (9) and is also a component of a nitrogen signaling pathway controlling the developmental switch from yeast-like to pseudohyphal growth (1). However, Plc1p's relationship to defined signal transduction pathways as well as the subcellular distribution of Plc1p in yeast remains unknown.

PLC1 was also identified in a genetic screen for mutants with increased frequencies of chromosome missegregation (54). The plc1-1 mutant, a temperature-sensitive mutant isolated in this screen, displayed a 32-fold increase in chromosome missegregation events. However, since plc1-1 cells appeared to have normal nuclei and spindle morphologies and were not supersensitive to the microtubule (MT)-destabilizing drug benomyl, Payne and Fitzgerald-Hayes reasoned that it was unlikely that the chromosome missegregation phenotype results from a defect in the components of the mitotic segregation apparatus (54). Rather, they concluded that plc1-1 affects chromosome segregation indirectly, possibly by interfering with proper timing of the cell cycle or by altering intracellular concentrations of calcium.

The kinetochore is a specialized organelle that mediates chromosome attachment to spindle MTs. Kinetochores are composed of centromeric DNA and a set of associated proteins. Whereas in animal cells the centromeres span several megabases, have complex organizations, and bind several MTs, in S. cerevisiae the centromere (CEN) is 125 bp long and engages only a single MT (67). CEN of S. cerevisiae includes three conserved elements, termed CDEI, CDEII, and CDEIII (22, 30). CBF3, a four-protein complex that binds the CDEIII sequence, is absolutely essential for kinetochore assembly and function in vivo (28). The four protein subunits of CBF3 have molecular masses of 110, 64, 58, and 23 kDa (11, 16, 40, 60). The genes borne by p110 (NDC10, CBF2, and CTF14) (16, 26, 33), p64 (CEP3 and CBF3b) (39, 61), p58 (CTF13) (16), and p23 (SKP1) (11, 60) have been cloned and characterized.

Traditionally, the PtdIns cycle and PLC activity have been thought to be associated with the plasma membrane. However, several reports suggest that an independently regulated PtdIns cycle operates also within the nucleus (4, 10, 13, 14, 45, 55). The regulation and downstream effectors of this nuclear PtdIns cycle are unknown. In this paper, we demonstrate that in S. cerevisiae Plc1p associates with the kinetochore and appears to affect the binding interaction between MTs and the kinetochore, which is essential for proper chromosome segregation and cell cycle progression. These results indicate a specific nuclear function for PLC in eukaryotic cells.

	MATERIALS AND METHODS

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Strains and media. Yeast strains used in this study (listed in Table 1) were grown in rich medium (YPD; 1% yeast extract, 2% Bacto Peptone, 2% glucose) or under selection in synthetic minimal medium containing glucose (SD) and supplemented with appropriate nutrients. Yeasts were manipulated as described previously (8).

Plasmids. Plasmids pAS2-1-PLC1 and pYX232-PLC1-3HA have already been described (43). Plasmids pGBT9-PLC1 and pACT2-PLC1 were constructed by ligating a PLC1-containing BamHI fragment from pAS2-1-PLC1 into pGBT9 and pACT2, respectively. To construct plasmid pRS413-PLCP, which contains the promoter region of PLC1, a 1,100-bp fragment of the PLC1 promoter region was amplified by PCR using the oligonucleotides PLCP5 (5'-GCGCGAATTCGGGCCCACTTTTGGACGCTGGCGTCGC-3'), which hybridizes 1,107 bp upstream of the ATG start codon and introduces an ApaI site, and PLCP3 (5'-GCGCGAATTCACACTGCGTGAATGACCTTACCG-3'), which hybridizes 6 bp upstream of the ATG start codon and introduces an EcoRI site. The amplified 1.1-kb fragment was digested with EcoRI and ApaI and ligated into EcoRI- and ApaI-digested pRS413. Subsequently, an EcoRI-SacI fragment from pYX232-PLC1-3HA was ligated into EcoRI- and SacI-digested pRS413-PLCP to construct pRS413-PLC1.

One-hybrid assay. The integrating reporter plasmid pHISi-1-CEN was constructed by annealing two 5'-end-phosphorylated oligonucleotides, CEN-5 (5'- [P]AATTCGCGCAGCTGTATTAGTGTATTTGATTTCCGAAAGTTAAAAAAGAAATAGTAAGAAATATATATTTCCC-3') and CEN-3 (5'-[P]GGGAAATATATATTTCTTACTATTTCTTTTTTAACTTTCGGAAAT CAAATACACTAATACAGCTGCGCG-3'), and by subsequent cloning into EcoRI- and SmaI-digested pHISi-1 (Clontech, Palo Alto, Calif.). Another reporter plasmid, pHISi-1-CEN-M53, was constructed in the same way as described above except that the CDEIII sequence with single base substitutions (underlined; CA and GT) was used. Both plasmids were linearized with XhoI and separately transformed into the YM4271 strain to construct two reporter strains, YM4271[pHISi-1-CEN] and YM4271[pHISi-1-CEN-M53]. Both reporter strains were individually transformed with the pACT2-NDC10, pACT2-CEP3, pACT2-CTF13, pACT2-SKP1, and pACT2-PLC1 plasmids, selected on plates coated with SD-Leu-His, and subsequently streaked on plates containing SD-Leu-His plus 50 mM 3-amino-1,2,4-triazole (3-AT).

Chromatin immunoprecipitation. In vivo chromatin cross-linking and immunoprecipitation were performed essentially as described previously (5, 47) with several minor modifications. Briefly, yeast cells were grown in 50 ml of YPD to an A₆₀₀ of 1.2 to 1.5, at which point they were fixed for 2 h by the addition of formaldehyde to a concentration of 1%. Subsequently, the cells were converted to spheroplasts with Zymolyase (8). Spheroplasts were washed sequentially in 2.5 ml of ice-cold phosphate-buffered saline (10 mM NaH₂PO₄, 150 mM NaCl at pH 7.4), 2.5 ml of buffer I (0.25% Triton X-100, 10 mM EDTA, 0.5 mM EGTA, 10 mM HEPES at pH 6.5), and 2.5 ml of buffer II (200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM HEPES at pH 6.5). Finally, the spheroplasts were resuspended in 300 µl of lysis buffer (1% sodium dodecyl sulfate, 10 mM EDTA, 50 mM Tris-HCl at pH 8.0) supplemented with protease inhibitors at the following concentrations: 10 µg/ml each for leupeptin, aprotinin, pepstatin, and antipain; 1 mM for phenylmethylsulfonyl fluoride; 2 mM for benzamidine; 0.5 mM for TLCK (N-p-tosyl-L-lysine chloromethyl ketone); and 100 µg/ml for TPCK (N-tosyl-L-phenylalanine chloromethyl ketone). The suspension was then sonicated 10 times for 10 s each to fragment chromosomal DNAs to an average size of ~500 bp. The suspension was centrifuged for 10 min at 10,000 × g, and the supernatant was diluted with 2.7 ml of dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl at pH 8.0) supplemented with protease inhibitors at the above-named concentrations. The resultant solubilized chromatin solution was divided in three aliquots (1 ml each), and each aliquot was precleared by adding 50 µl of 50% protein A-Sepharose 4B slurry and incubating for 10 min at 4°C with gentle rocking. Beads were then harvested by centrifugation, and the supernatant was incubated with 4 µg of 12CA5 antibody overnight on ice. Sonicated phage  DNA (2 µg) and protein A-Sepharose 4B beads (50 µl of 50% slurry) were added, and the incubation continued for 1 h. Beads were then harvested and washed, and the DNA was released and extracted as described previously (5).

Mutagenesis of PLC1 and gap repair. We employed the PCR procedure for localized mutagenesis and gap repair as described previously (49). PLC1 was first amplified by PCR with a slightly error-prone Taq polymerase, using the primers PLC-M5 (5'-CCTTGACATGATTTTGAAAATGG-3') and PLC-M3 (5'-TAGAGGTGTGGTCAATAAGAGCG-3') and with pGBT9-PLC1 as a template. The PCR product (100 ng) was cotransformed with 100 ng of EcoRI- and BamHI-digested pGBT9 into the CG1945 strain harboring the pACT2-NDC10 plasmid. About 10⁵ transformants were selected on SD-Leu-Trp plates and subsequently replica plated onto plates containing SD-Leu-Trp-His plus 2.5 mM 3-AT. The transformants that were unable to grow on these plates were identified, and pGBT9-PLC1 mutant plasmids were isolated and transformed back into the CG1945 strain containing pACT2-NDC10 to verify the inability of the transformants to grow on plates containing SD-Leu-Trp-His plus 2.5 mM 3-AT. Twenty pGBT9-PLC1 plasmids were isolated in this way and transformed in plc1 cells. We took advantage of the fact that the pGBT9-PLC1 plasmid can suppress the temperature sensitivity, osmosensitivity, and nocodazole sensitivity of plc1 cells. We eliminated those pGBT9-PLC1 mutant plasmids, which suppressed none of these phenotypes and were not phenotypically distinguishable from plc1 cells. These plasmids probably encode mutated Plc1p with grossly reduced ability to function in vivo. All other transformants (plc1 cells harboring the mutated pGBT9-PLC1 plasmid) displayed temperature sensitivity and nocodazole sensitivity but osmoresistance (ability to grow in YPD medium containing 0.7 M NaCl). We selected three mutant plasmids with the tightest phenotypes and subcloned the coding regions of PLC1 as EcoRI-SalI fragments in the pRS413-PLCP plasmid (see above) to yield pRS413-PLC1-8, pRS413-PLC1-29, and pRS413-PLC1-39 (these plasmids express PLC1 from its natural promoter). The resultant plasmids were transformed in plc1 cells, and the temperature sensitivity, nocodazole sensitivity, and osmoresistance of the transformants were confirmed. Hereafter, these mutants are referred to as the plc1-8, plc1-29, and plc1-39 mutants.

Minichromosome stability assay. Mitotic minichromosome stability was measured as a fraction of cells that retained the plasmid after growth in nonselective medium (38, 46). Briefly, wild-type or plc1 cells containing the empty pRS413 plasmid, or plc1 cells containing PLC1, plc1-8, plc1-29, or plc1-39 in pRS413 (CEN HIS3) were transformed with the pRS414 plasmid (CEN TRP1). For each transformant, five single colonies were inoculated separately into medium nonselective for the pRS414 plasmid and grown for about 24 h at 28°C. At the end of the growth, the cultures were still in the exponential phase, as was determined by counting the cells with a hemacytometer and by measuring A₆₀₀. For each culture, the frequency of Trp⁺ cells was determined at the time of inoculation (F₀) and at the end of nonselective growth (F_end) by plating appropriately diluted cultures on YPD plates and subsequently replica plating them onto SC lacking tryptophan. The rate of plasmid loss per generation was determined as described previously (38, 46), according to the equation F_end = F₀(F_D)^G, where F_D is the fraction of cells in each generation that retain the minichromosome and G is the number of generations. The fraction of cells that lose the minichromosome per generation is (1  F_D). The number of cell doublings was calculated by counting the total cell numbers at the beginning and at the end of nonselective growth.

Minichromosome-MT binding assay. Preparation of yeast lysates containing minichromosomes and the minichromosome-MT binding assay were done as described previously (36, 37). Briefly, yeast cultures were grown to an A₆₀₀ of ~0.6 after the cells were maintained in exponential growth for several generations. In experiments with nocodazole-arrested cells, nocodazole was added to the cultures to 15 µg/ml for 6 h and nocodazole was also present during the preparation of spheroplasts (36). Cells were spheroplasted by glusulase and osmotically lysed in EBB buffer (10 mM Tris-Cl [pH 7.4], 10 mM MgCl₂, 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.1 mM dithiothreitol). Minichromosomes were eluted from nuclei by adding 0.3 M NaCl. After 5 min of incubation, the extracts were diluted threefold by adding EBB buffer and subjected to two subsequent centrifugations, each at 15,000 × g for 20 min. The clear supernatant was removed, supplemented with 10 µM taxol, and used for the MT binding assay. Purified tubulin was polymerized in vitro into MTs with an average length of 2 µm as directed by the manufacturer (ICN Biochemicals, Inc.) and stabilized by addition of the MT-stabilizing drug taxol (10 µM final concentration). Different amounts of stabilized MTs were added to 500-µl aliquots of the cleared extracts to initiate the MT binding assay. After 15 min of incubation at room temperature, the reaction mixtures were centrifuged at 15,000 × g for 8 min. The supernatant and pellet fractions were separated, and the amount of minichromosomes in each fraction was determined by Southern blot using a TRP1 probe. The calibration of the band intensities was performed by loading different amounts of the TRP1 DNA fragment on the gels, and the band intensities of the scanned images were quantified using UN-SCAN-IT software (Silk Scientific).

Other methods. Two-hybrid screening, expression and purification of the glutathione S-transferase (GST)-Ndc10_(194-446) fusion protein, Western blotting, and coprecipitation assays were carried out as described previously (8, 43). The DNA contents of cells were determined by flow cytometry of propidium iodide-stained cells (64).

	RESULTS

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Plc1p interacts with two components of the kinetochore complex. Because the gene encoding PtdIns-specific PLC, PLC1, was isolated in a genetic screen of mutants that exhibit defects in chromosome segregation (54), it seemed to us that Plc1p might be involved in kinetochore function. To test this hypothesis, we used the two-hybrid assay (21) to determine whether Plc1p interacts with Ndc10p, a component of the CBF3 complex of the kinetochore. We found that Plc1p interacts with full-length Ndc10p (Fig. 1A). We also constructed several truncation alleles of Ndc10p and identified amino acids 194 to 446 as the essential domain required for interaction with Plc1p (data not shown). To confirm the interaction between Plc1p and Ndc10p by an independent biochemical approach, we expressed the domain of Ndc10p encompassing amino acids 194 to 446 in Escherichia coli as a fusion with GST, purified the fusion protein on glutathione (GSH)-Sepharose 4B, and tested its ability to interact with Plc1p. Because Plc1p is expressed at a very low level in S. cerevisiae (23), we overexpressed Plc1p from a high-copy-number vector with a strong, constitutive triose phosphate isomerase promoter as a C-terminal fusion with three copies of the hemagglutinin (HA) epitope (plasmid pYX232-PLC-3HA) in a protease-deficient strain, BJ5465 (43). Cell lysate prepared from this strain was incubated with GST-Ndc10p_(194-446) fusion protein or with GST protein as the control. The resulting protein complexes were isolated using GSH-Sepharose 4B beads. GST-Ndc10p_(194-446) fusion protein (but not GST protein) was able to precipitate Plc1p-3HA from the yeast cell lysate (Fig. 1B), confirming that Plc1p can interact with Ndc10p.

View larger version (106K): [in this window] [in a new window]   FIG. 1.   Plc1p associates with the kinetochore complex CBF3. (A) Two-hybrid assay of the Plc1p and Ndc10p interaction. Cells of strain CG1945 harboring the following plasmids were streaked on plates containing SD-Trp-Leu-His plus 2.5 mM 3-AT and incubated for 4 days at 30°C: pAS2-1-PLC and pACT2-NDC10 (sector 1), pAS2-1-PLC and pACT2 (sector 2), pAS2-1 and pACT2-NDC10 (sector 3), and pAS2-1 and pACT2 (sector 4). The above-named strains grew equally well on SC-Leu-Trp plates. (B) GST-Ndc10p specifically coprecipitates Plc1p. Plc1p was expressed in yeast as a C-terminal fusion with three copies of the HA epitope (Plc1p-3HA), and the total cell extract (lane 1) was incubated with GST-Ndc10p(194-446) fusion protein (lane 2) or with GST protein (lane 3). The protein complexes were isolated on GSH-Sepharose 4B, and the samples were analyzed by Western blotting with 12CA5 antibody recognizing the HA epitope. The amounts of samples loaded onto all three lanes correspond to the amount of the total cell extract (1%). Thus, the difference in the intensities of the Plc1p-3HA bands in lanes 1 and 2 reflects recovery of Plc1p-3HA. The amounts of Plc1p-3HA in the cell lysate and in the GST-Ndc10p(194-446)-precipitated sample were compared quantitatively by Western blot analysis using several different amounts of both samples (data not shown). From the band intensities of scanned images, we estimate that under the conditions of the experiment, GST-Ndc10p(194-446) precipitates approximately 10% of Plc1p-3HA in the cell lysate. (C) Two-hybrid assay of the Plc1p and Cep3p interaction. Cells of strain CG1945 harboring the following plasmids were streaked on plates containing SD-Trp-Leu-His plus 10 mM 3-AT and incubated for 4 days at 30°C: pAS2-1-PLC and pACT2-CEP3 (sector 1), pAS2-1-PLC and pACT2 (sector 2), pAS2-1 and pACT2-CEP3 (sector 3), and pAS2-1 and pACT2 (sector 4). The above-named strains grew equally well on SC-Leu-Trp plates. (D) Plc1p associates with the assembled CBF3 complex. Strain YM4271 was transformed with either pHISi-1-CEN or pHISi-1-CENM53 integrative plasmids to create two reporter strains, JC107 and JC108, respectively. The JC107 strain (sectors 1, 3, and 5) and the JC108 strain (sectors 2, 4, and 6) harboring the following plasmids were streaked on plates containing SD-Leu-His plus 50 mM 3-AT and incubated for 4 days at 30°C: pACT2-NDC10 (sectors 1 and 2), pACT2-PLC1 (sectors 3 and 4), and pACT2-CEP3 (sectors 5 and 6). The strains grew equally well on SC-Leu plates.

Plc1p associates with the assembled CBF3 complex of the kinetochore. The interaction of Plc1p with Ndc10p and Cep3p can be interpreted in two ways: (i) Plc1p interacts with Ndc10p and Cep3p prior to assembly into the kinetochore or (ii) Plc1p contacts the assembled kinetochore via Ndc10p and Cep3p. Additionally, it is also conceivable that the domains of Ndc10p and Cep3p responsible for the interaction with Plc1p are hidden within the CBF3 complex after assembly of the kinetochore and are not available for interaction with Plc1p. To resolve these issues, we determined whether the assembled CBF3 complex interacts with Plc1p. To this end, we used a modified one-hybrid assay. The one-hybrid system is an in vivo assay used to test binding of a protein to short target DNA sequences. When a DNA binding protein is fused to the Gal4 AD and expressed in yeast, it can bind to its specific recognition sequence inserted in the promoter region of a reporter gene (such as HIS3) and activate its transcription. We used a one-hybrid assay to test the binding of CBF3 proteins to the CDEIII sequence. The rationale was that reporter gene transcription would be activated only if the CBF3 complex was assembled on the CDEIII sequence. Conversely, cis-acting mutations in CDEIII would prevent proper assembly of the CBF3 complex (56), resulting in a failure to activate the transcription of the reporter gene. A similar one-hybrid assay was used previously by Ortiz et al. (51) in a screen which resulted in the identification of Ctf19p, Mcm21p, and Okp1p as components of the kinetochore.

Plc1p localizes to CEN DNA in vivo. To test whether the interaction between Plc1p and the CBF3 complex occurs in vivo at the CEN locus, we used in vivo cross-linking followed by chromatin immunoprecipitation. Cells expressing Plc1p-3HA were first treated with formaldehyde to cross-link the cellular structures and then lysed and sonicated to shear the chromatin. Subsequently, Plc1p-3HA was immunoprecipitated using anti-HA (12CA5) antibody and protein A-Sepharose. As a positive control, we also used a strain expressing an epitope-tagged allele of CSE4, CSE4-HA. Cse4p, a histone H3 variant, is a structural component of the yeast kinetochore (48). To exclude the possibility that CEN chromatin is preferentially precipitated with anti-HA antibody or protein A-Sepharose, we also performed the chromatin immunoprecipitation with extracts that contained no HA-tagged proteins. The immunoprecipitated DNA was analyzed by PCR for the presence of the CEN3 fragment and two noncentromeric control fragments. The CEN3 DNA fragment specifically coimmunoprecipitated with Plc1p-3HA and Cse4-HAp (Fig. 2). Immunoprecipitates from mock-treated extracts and from extracts of an untagged strain did not contain detectable amounts of the CEN3 fragment. In addition, noncentromeric loci were not also detectable in any immunoprecipitate. Thus, Plc1p localizes to the centromere in vivo, presumably by interactions with the CBF3 components Cep3p and Ndc10p.

View larger version (68K): [in this window] [in a new window]   FIG. 2.   Localization of Plc1p to the CEN3 DNA locus. Cross-linked chromatin was prepared from cells expressing Plc1p-3HA (HL1-1 strain harboring plasmid pRS413-PLC1) or Cse4-HAp (strain PM1201) and wild-type cells (WT) (W303-1a) expressing no tagged protein. Chromatin was immunoprecipitated with the anti-HA antibody (Ab) 12CA5 (lanes 3, 6, and 9) or mock treated (lanes 2, 5, and 8), and aliquots of total input material (lanes 1, 4, and 7; in an amount corresponding to 0.8 µl of the chromatin solution) and coimmunoprecipitated DNA (in an amount corresponding to 20 µl of the chromatin solution) were analyzed by PCR with primers specific for CEN3 (244 bp) and two noncentromeric loci on yeast chromosome III: non-CEN (L) (249 bp) and non-CEN (R) (278 bp).

plc1 cells are hypersensitive to nocodazole and display an increased rate of minichromosome loss. Nocodazole is an MT-depolymerizing drug that arrests cells at mitosis (52). Recent studies suggest that low concentrations of nocodazole alter MT dynamics and cause unstable attachment of the kinetochore to spindle MTs (63, 66). Since mutations in kinetochore-associated proteins can result in nocodazole hypersensitivity (25), we determined the resistance of plc1 cells to nocodazole. Disruption of PLC1 resulted in nocodazole hypersensitivity (Fig. 3A). When considered together with the previous report that mutation in PLC1 causes a chromosome segregation defect (54), our results suggest that Plc1p may affect the fidelity of chromosome segregation. Therefore, we tested the stability of minichromosomes in plc1 cells. The stability of the pRS413 minichromosome was measured as a fraction of cells that retained the minichromosome after growth in nonselective medium (38). The stability of the pUN20 minichromosome (17) was measured using the half-sectored colony color assay (29). The results of both methods were in good agreement and showed that the minichromosome loss rate in plc1 cells was about fivefold higher than in the wild-type cells (Table 2). Nocodazole (2 µg/ml) increased the minichromosome loss rate twofold in the wild-type strain but almost fivefold in plc1 cells (Table 2). This indicates that the deletion of PLC1 acts synergistically with nocodazole in destabilizing minichromosomes. It should be noted, however, that this high rate of loss (32%; Table 2) for plc1 cells in the presence of nocodazole is at the high limit of the plasmid loss rate measurable by this assay and corresponds to the loss rate of an acentric minichromosome or YRp plasmid (36, 38). For the same reason, we were unable to determine the plasmid loss rate of plc1 cells in the presence of nocodazole by the half-sectored colony assay. In this case, frequent sectoring made identification of half-sectored colonies impossible.

View larger version (58K): [in this window] [in a new window]   FIG. 3.   Nocodazole sensitivity of PLC1 mutants can be suppressed by NDC10 overexpression. (A) plc1 cells (HL1-1) are hypersensitive to nocodazole. Wild-type W303-1a (sectors 1 and 3) and plc1 (sectors 2 and 4) cells were streaked on a YPD plate containing 4 µg of nocodazole per ml (sectors 1 and 2; nocodazole was added to the medium from a stock solution of 3.3 mg/ml in dimethyl sulfoxide) or a YPD plate containing only a corresponding amount of the solvent (0.12% dimethyl sulfoxide; sectors 3 and 4). The plates were incubated for 3 days at 28°C. (B) Suppression of the nocodazole sensitivity of plc1-29 and plc1-39 mutants by NDC10 overexpression. plc1 cells (HL1-1) harboring the following plasmids were streaked on a YPD plate containing 4 µg of nocodazole per ml and incubated at 28°C for 3 days: pRS413-PLC1 and pYX242-HT-T7-NDC10 (sector 1), pRS413-PLC1 and pYX242-HT-T7 (sector 2), pRS413 and pYX242-HT-T7-NDC10 (sector 3), pRS413 and pYX242-HT-T7 (sector 4), pRS413-plc1-29 and pYX242-HT-T7-NDC10 (sector 5), pRS413-plc1-29 and pYX242-HT-T7 (sector 6), pRS413-plc1-39 and pYX242-HT-T7-NDC10 (sector 7), and pRS413-plc1-39 and pYX242-HT-T7 (sector 8).

Disruption of the interaction between Plc1p and Ndc10p results in temperature sensitivity, nocodazole sensitivity, and an increased rate of minichromosome loss. What is the biological significance of the interaction between Plc1p and the kinetochore? To answer this question, we generated three plc1 mutants (the plc1-8, plc1-29, and plc1-39 mutants) that do not interact with Ndc10p (see Materials and Methods). In addition, none of these mutants interacted in the two-hybrid assay with Cep3p or with the assembled kinetochore in the one-hybrid assay described above. All three plc1 mutants displayed slower growth at the permissive temperature (28°C), temperature sensitivity (inability to grow at temperatures higher than 36°C), nocodazole sensitivity (inability to grow in YPD medium containing more than 4 µg of nocodazole per ml), and a slightly higher rate of minichromosome loss (Table 2). All three mutants displayed the osmoresistant phenotype (ability to grow in YPD medium containing 0.7 M NaCl), indicating that a subset of Plc1p functions is not altered in these mutants. To determine if Ndc10p overexpression can suppress the temperature sensitivity and/or nocodazole sensitivity in these mutants, Ndc10p was overexpressed either as an N-terminal fusion with the His tag and T7 epitope (HT-T7) or as a C-terminal fusion with three HA tags. Overexpression of both fusion proteins suppressed the nocodazole sensitivity of the plc1-29 and plc1-39 mutants (Fig. 3B) and weakly suppressed the temperature sensitivity of the plc1-39 mutant (data not shown). These results provide genetic evidence for an interaction between Plc1p and Ndc10p.

View larger version (22K): [in this window] [in a new window]   FIG. 4.   Structural features of PLC1 and mutations in plc1 alleles. The positions of the amino acid residues of the PH domain, EF-hand calcium binding domain (EF), catalytic X and Y domains, PEST motif, and C2 domain in Plc1p are shown in parentheses. The positions of mutations in individual plc1 alleles are indicated (), along with corresponding amino acid and nucleotide changes.

plc1 cells exhibit mitotic delay. In addition to the kinetochore-MT interactions, the fidelity of chromosome transmission is also controlled by the action of at least one of the kinetochore-based mitotic checkpoint control systems (27, 31, 42, 56). Mutations that weaken a single centromere or mutations in genes that encode centromere DNA binding proteins can delay the cell cycle in the G₂/M stage, suggesting a role for the kinetochore in checkpoint control systems in yeast cells (16, 53, 59, 64, 65). Despite the good potential for being the signal-transducing molecule of the kinetochore, Plc1p did not appear to be involved in mitotic checkpoint control. After treatment with nocodazole (15 µg/ml), the plc1 cells properly arrested as large-budded cells and maintained viability for at least 6 h (data not shown). However, if Plc1p is involved in attachment of an MT to the kinetochore, then plc1 cells would be expected to linger in the G₂/M stage of the cell cycle, as was observed for ctf13, cep3, and skp1 mutants (2, 11, 16, 61). We examined the cell and nuclear morphologies of wild-type and plc1 cells during exponential growth at 28°C (Table 3). The frequencies of large-budded cells (with the diameter of the bud being at least 75% of the diameter of the mother cell) with a single nucleus positioned within 50% of the mother cell proximal to the neck (53, 59) were 12% for the wild-type strain and 26% for the plc1 strain. Exponentially growing plc1 cells also displayed a larger amount of cells with a 2N DNA content than the wild-type strain (Fig. 5). Taken together, these results demonstrate that plc1 cells exhibit a relatively mild delay at the G₂/M stage of the cell cycle. About 8% of plc1 cells also displayed a two-budded cell phenotype (Table 3). Depending on the size of the second bud, it is either with or without a nucleus. The appearance of cells with two buds in the plc1 population suggests an additional partial defect or delay in cytokinesis. This observation is supported by the previous work of Flick and Thorner (23), who described the appearance of multibudded plc1 cells after 10 h of incubation at 37°C (restrictive temperature for the plc1 mutant). Perhaps in addition to its role in G₂/M transition, Plc1p has an additional role in cytokinesis, which is more pronounced during suboptimal growth conditions, such as high temperature.

View this table: [in this window] [in a new window]   TABLE 3.   plc1 cells display mitotic-delay morphology

View larger version (7K): [in this window] [in a new window]   FIG. 5.   Flow cytometry of wild-type W303-1a (WT) and plc1 (HL1-1) cells exponentially growing in YPD at 28°C. Cells were fixed with ethanol and stained with propidium iodide. The fractions of cells in G2/M are indicated in parentheses.

Minichromosomes in plc1 extract exhibit reduced MT binding. Previous work suggested that kinetochore function can be perturbed by a failure either to form the core CEN DNA-protein complex or to assemble components that interact with the MTs to ensure faithful chromosome segregation (36). We used an assay developed by Kingsbury and Koshland (36, 37) to measure the ability of minichromosomes formed in vivo to bind MTs in vitro. The wild-type and plc1 mutant strains were transformed with the centromeric plasmid (minichromosome) pRS414, which was then assayed in clarified extracts of these cultures for binding to taxol-stabilized MTs. In the control lysate without MTs, about 2 to 3% of the plasmid pelleted when it was subjected to centrifugation. However, in the presence of a saturating concentration of MTs, about 24% of the plasmid pelleted with MTs in the wild-type extract and about 13% pelleted in the plc1 extract (Table 4). When the same experiment was performed with the pRS424 plasmid, which does not contain the CEN sequence and which is segregated by a kinetochore-independent mechanism, the amounts of plasmid precipitated from the wild-type and plc1 lysates were nearly identical and increased from only 2% (control experiment without MTs) to about 4% (saturating concentration of MTs). To improve the sensitivity of the assay and to exclude the possibility that the observed difference in MT binding activities between the two strains was merely the result of different cell cycle profiles, the assay was also performed with lysates prepared from nocodazole-arrested cells (Table 4). The G₂/M transition is the phase when the kinetochore is required for progression of the cell cycle through mitosis. It is also when the kinetochore is in its most active state. Kinetochores of cells arrested with nocodazole at the G₂/M transition exhibit the highest MT binding activity (36). Our data for the wild-type strain correspond to these previous results. The saturating concentration of MTs pelleted about 48% of the plasmid in the wild-type strain but only 28% in the plc1 strain (Table 4). The amount of 2µm plasmid (pRS424) precipitated from nocodazole-arrested wild-type and plc1 cells did not significantly differ from the amounts precipitated from asynchronous cultures and ranged from 2% in experiments without MTs to about 4% in experiments with a saturating concentration of MTs (data not shown). Although the difference in MT binding between the wild-type and plc1 strains is relatively small, it is reproducible in both asynchronous and mitotic extracts and suggests that in plc1 cells the kinetochore function is indeed partially impaired.

View this table: [in this window] [in a new window]   TABLE 4.   plc1 extracts exhibit reduced MT binding to minichromosomes

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

It is generally accepted that PtdIns signaling pathways are present in nuclei (4, 14), and enzymes responsible for PtdIns turnover and signaling, such as PLC and protein kinase C, have been identified in the nuclear interior, associated with nonmembrane nuclear structures (10, 13, 45, 55). The nuclear PtdIns cycle is regulated independently of the PtdIns cycle at the plasma membrane (14). Nuclear PtdIns(4,5)P₂, for instance, decreases as cells progress through the S phase of the cell cycle (69), and nuclear PLC activity and DG levels increase at the G₂/M transition (62). However, the processes regulated by the nuclear PtdIns cycle have not been identified and the biological function of the nuclear PtdIns cycle remains unknown. Our data demonstrate that in S. cerevisiae Plc1p associates with kinetochores and appears to affect the binding of kinetochores to MTs.

Plc1p exhibits all three characteristics expected of a kinetochore protein (61). First, Plc1p associates with centromeric DNA through interaction with the CBF3 complex. Second, plc1 cells delay in the G₂/M stage of the cell cycle, are hypersensitive to nocodazole, and display a greater instability of minichromosomes. In addition, minichromosomes isolated from plc1 cells have a reduced ability to bind MTs. Third, in comparison with the wild-type cells, plc1-1 mutant cells display a 32-fold-higher frequency of missegregation of a chromosome fragment (cen3X69) carrying a destabilizing mutation in the CDEIII region of the centromere (54).

The CBF3 complex is essential for binding of kinetochores to MTs in vitro. However, CBF3 does not mediate efficient MT attachment on its own (57). Attachment requires the presence of additional cellular factors. It was proposed that CBF3 forms a DNA-binding scaffold onto which additional kinetochore components assemble, including proteins that directly bind to MTs (57). However, the identities of these proteins remain unknown. Our data suggest that Plc1p may be one of these accessory proteins of the kinetochore involved in binding MTs.

Since Plc1p is not essential for cell viability and does not contain a recognizable MT binding motif, it is more likely that Plc1p has a regulatory rather than a structural role in the MT-kinetochore interaction. An alternative explanation may be that CBF3 subunits bind MTs directly but that Plc1p is required to achieve an active MT binding conformation of the CBF3 subunits. Using in vivo cross-linking and immunoprecipitation, Meluh and Koshland (47) demonstrated the existence of centromeric subcomplexes which probably correspond to kinetochore assembly intermediates. In their model, kinetochore assembly initiates with recruitment of the CBF3 complex, and possibly Mif2p, to CDEIII. The resultant subcomplex then serves as the nucleation site around which a fully functional kinetochore is assembled if CDEI and CDEII are adjacent. Conceivably, Plc1p may function in this folding process, which may affect the ability of kinetochores to bind MTs.

The MT binding activity of kinetochore complexes is high in mitotic extracts and low in interphase extracts. However, the DNA binding activity of CBF3 is high in both stages (57). These results suggest that CBF3 is assembled on the centromeric DNA throughout the cell cycle but that the MT attachment is controlled by a cell cycle-dependent interaction of additional factors with a constitutively bound CBF3 scaffold. At present we do not know whether Plc1p remains stably localized at the CEN locus throughout the cell cycle or whether it localizes to the CEN locus only during a particular stage(s) of the cell cycle.

Currently, it is difficult to determine whether only the ability of Plc1p to bind kinetochores or this binding ability and Plc1p's enzymatic activity are required for the apparent mitotic functions of Plc1p. The sequencing of the plc1-29, plc1-39, plc1-8, and plc1-113 alleles revealed that they all acquired mutations which likely affect the enzymatic activity. Since all of these mutants failed to interact with Ndc10p and Cep3p in a two-hybrid assay and with the CBF3 complex in a one-hybrid assay, we believe that they do not interact with kinetochores. Therefore, the corresponding phenotypes can be attributed to either a loss of binding to kinetochores, a decrease (or loss) of enzymatic activity, or both. Since plc1 cells display only a mild mitotic delay and only a sixfold-increased rate of minichromosome loss, it seems likely that Plc1p is not a direct structural component of the kinetochore but rather a regulatory factor affecting some aspects of the kinetochore activity.

Since kinetochores are unlikely to contain any PtdIns(4,5)P₂ component which could be used as a substrate for Plc1p, it seems unlikely that Plc1p would hydrolyze PtdIns(4,5)P₂ at the kinetochore. It is possible that the interaction of Plc1p with the kinetochore is spatially and perhaps also temporally separated from its enzymatic activity. It remains to be determined which of these two events [binding to kinetochore and hydrolysis of PtdIns(4,5)P₂] is relevant for the observed mitotic functions of Plc1p. However, since nuclei of mammalian cells contain a pool of PtdIns(4,5)P₂ which is not associated with nuclear envelope (4, 6, 71), the possibility that the kinetochore-bound Plc1p hydrolyzes PtdIns(4,5)P₂ cannot be completely discarded. It will be important to determine whether Plc1p remains localized to the CEN locus throughout the cell cycle or whether it localizes to the CEN locus only during a particular stage(s) of the cycle and whether this localization and interaction with the CBF3 complex regulates the Plc1p-dependent hydrolysis of PtdIns(4,5)P₂. The possibility that PLC is regulated in a cell cycle-specific manner is supported by the observation that in mammalian cells nuclear PLC activity and DG levels are increased at the G₂/M transition (62). In this context, it will be important to explore further the role of PLC during mitosis in both yeast and mammalian cells.