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Address correspondence to Mark Winey, MCD Biology, UCB 347, University of Colorado, Boulder, CO 80309-0347. Tel.: (303) 492-3409. Fax: (303) 492-7744. E-mail: mark.winey{at}colorado.edu
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Key Words: budding yeast; spindle pole body; MPS3; CDC31; centrin
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The SPB is a multilayered cylindrical structure that is embedded in the nuclear envelope throughout cell division (Byers and Goetsch, 1975). Thin section EM revealed that SPBs consist of three distinct layers: a central plaque within the plane of the nuclear membrane; an outer plaque facing the cytoplasm; and an inner plaque facing the nucleoplasm. In addition, one side of the SPB is associated with an electron-dense region of the nuclear envelope called the half-bridge (Byers and Goetsch, 1974). More recent analysis of SPB structure by electron tomography and cryo-EM have confirmed the presence of these three SPB substructures and have suggested that two intermediate layers connect the central plaque to the outer plaque (Bullitt et al., 1997; O'Toole et al., 1999).
Structural components of the SPB have been identified both genetically and biochemically, and the arrangement of proteins within the organelle has been determined by a combination of two-hybrid and immuno-EM analysis (Wigge et al., 1998; Adams and Kilmartin, 2000; Schramm et al., 2001). Important to this study is the central plaque component Spc42p and one of its binding partners, Spc29p, as well as the yeast centrin homologue Cdc31p, which binds a membrane protein of the half-bridge, Kar1p (Baum et al., 1986; Spang et al., 1993; Biggins and Rose, 1994; Vallen et al., 1994; Spang et al., 1995; Donaldson and Kilmartin, 1996; Bullitt et al., 1997; Adams and Kilmartin, 1999; Elliott et al., 1999).
SPB duplication proceeds via a discrete series of intermediates that have been characterized by EM analysis (for review see Adams and Kilmartin, 2000). During the G1 phase of the cell cycle, a small satellite structure containing Spc42p, Spc29p, Nud1p, and Cmn67p assembles at the distal end of the half-bridge on its cytoplasmic face. Next, the satellite enlarges, presumably due to templated assembly of Spc42p, to form the duplication plaque. Beneath the duplication plaque, the half-bridge elongates and fuses via an unknown mechanism. Assembly of the SPB is completed in late G1 when the half-bridge retracts, allowing the duplication plaque to be inserted into the nuclear envelope where it can associate with additional SPB components that make up the inner plaque.
Analysis of SPB structure in SPB duplication mutants has defined three distinct steps in the pathway (see Fig. 8 A). Mutations in CDC31 and KAR1, which encode components of the half-bridge, cause a block early in SPB duplication before satellite deposition, resulting in cells with a single enlarged SPB lacking an associated half-bridge (Byers, 1981; Rose and Fink, 1987). The next step in SPB duplication is expansion of the satellite into the duplication plaque and is defined by mutants in SPC42, SPC29, and MPS1, an essential protein kinase (Winey et al., 1991; Donaldson and Kilmartin, 1996; Elliott et al., 1999). Cells with mutations in these genes also arrest with a single SPB. However, their function in SPB duplication can be separated from that of Cdc31p and Kar1p; they are required for SPB duplication after the mating pheromone arrest point in G1. Insertion of the duplication plaque into the nuclear envelope is the last genetically defined step of SPB duplication, and mutants in this step arrest in mitosis with duplicated SPBs, but the nascent SPB is not inserted into the nuclear envelope (Winey et al., 1991, 1993; Schutz and Winey, 1998).
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30% of cells, no Spc42p–GFP signal was detected (unpublished data). Using this assay, we found that assembly of Spc42p–GFP into the SPB also depends on the function of another protein required for SPB duplication, Spc29p (Fig. 1 A) (Adams and Kilmartin, 1999; Elliott et al., 1999). The requirement for Mps1p and Spc29p in our assay suggests that assembly of Spc42p–GFP into the SPB after its overexpression occurs through a similar pathway used for SPB duplication in G1 and is a method to analyze the genetic requirements for SPB duplication.
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or YJL019W were still ts for growth, suggesting that one or both of these genes is mutated in mps3-1. Neither ORF could rescue mps3-1, but a plasmid containing both ORFs was able to complement the mps3-1 growth defect (Fig. 2 A). We sequenced the region of overlap between YJL018W and YJL019W from three different laboratory yeast strains, W303, S288c, and SK-1, and found an extra G/C base pair at position 1852 in YJL019W in all three strains (Fig. 2 B). This extra base pair results in a frame shift that joins YJL018W and YJL019W into a single, contiguous 2,049–base pair ORF. We sequenced the entire YJL019W/YJL018W gene from mps3-1 and found a single G to A transition at nucleotide 1415, resulting in a change of serine 472 to an asparagine (Fig. 2 B). We conclude that YJL019W/YJL018W is a single gene that we call MPS3 (monopolar spindle). The 682–amino acid protein encoded by MPS3 has no clear homologues in other organisms but does contain several motifs found in other SPB proteins, including two predicted coiled-coil domains and a potential transmembrane domain (Fig. 2 B).
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At the permissive temperature, the spindle structure of mps3-1 mutants differs from that observed in wild-type cells in two aspects. First, the significant number of monopolar spindles seen in the mutant suggests that mps3-1 is compromised for function at the permissive temperature (Fig. 3 C), likely contributing to the increase in ploidy phenotype in mps3-1 strains. Second, 73% of the mitotic spindles in mps3-1 mutants grown at 23°C are short metaphase spindles, which we rarely observe in wild-type cells (17%; unpublished data). As a result, we frequently observe a mitotic delay in mps3-1 cells grown under permissive conditions.
Serial thin sections of entire nuclei from 19 large budded mps3-1 cells grown at 36°C were examined by EM to verify that cells contain a monopolar spindle. Consistent with immunofluorescence analysis, 14 of 19 nuclei (74%) contained a single SPB and a monopolar spindle (Fig. 3 D). The SPB structure observed in mps3-1 mutants is similar to that in cdc31 and kar1 mutants (Byers, 1981; Rose and Fink, 1987): the core SPB is intact, but the half-bridge is small and indistinct, and no satellite structure could be detected. Based on these observations, we conclude that MPS3 is required for SPB duplication.
MPS3 is required early in G1 for SPB duplication
The absence of a distinguishable half-bridge in mps3-1 suggests that Mps3p, like Cdc31p and Kar1p, is needed for the earliest step of SPB duplication. We performed an execution point experiment to determine which stage of SPB duplication requires MPS3 function. Wild-type and mps3-1 cells were arrested at an intermediate step of SPB duplication at 23°C using 
-factor (the satellite has formed at the distal end of the half-bridge but further SPB assembly is inhibited) and then were released into fresh media at 23°C or 36°C. 1 h after release from -factor arrest, the majority of cells had entered the first mitosis, as judged by bud formation and DNA replication (Fig. 4 A). Virtually all large budded wild-type cells examined at this time point contained bipolar mitotic spindles (Fig. 4 B). Although monopolar spindles were seen in mps3-1 mutants grown at 23°C or 36°C, greater than 50% of large budded mps3-1 mutants were able to form a bipolar mitotic spindle during the first mitosis (Fig. 4, B and C), indicating that MPS3 function is not required for later steps of SPB duplication. Instead, mps3-1 mutants appear to fail in SPB duplication during the second cell cycle. At 3 h after -factor release, 88% of large budded mps3-1 cells grown at 36°C now contained a monopolar spindle (Fig. 4, B and C).
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-factor is unlikely the result of incomplete inactivation of mps3-1p because preincubation of cells at 36°C for 1 h immediately before -factor release gave virtually identical results (unpublished data). Furthermore, mps3-1 is capable of a first cycle arrest. Cells released from S-phase (hydroxyurea) arrest in mitosis with monopolar spindles after undergoing a single failed round of SPB duplication in the absence of Mps3p function (unpublished data). These data indicate that MPS3 is required before the satellite-bearing stage of SPB duplication, although we cannot exclude the possibility that other alleles will show requirements for MPS3 in additional steps.
Mps3p localizes to the SPB half-bridge
To understand its role in SPB duplication, we examined the Mps3p protein. MPS3 is predicted to encode a 79-kD protein. To verify this prediction and demonstrate that YJL018W and YJL019W are one gene, we constructed a yeast strain in which the endogenous copy of MPS3 is fused at its COOH terminus to GFP (MPS3–GFP). Cells expressing Mps3p–GFP, but not an untagged control, contained a single protein of 120 kD that was recognized by anti-GFP antibodies (Fig. 5 A). Because GFP is 27 kD, this indicates that Mps3p migrates as a 90–95-kD protein on our gel system.
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We performed immuno-EM on asynchronously growing MPS3–GFP cells to further refine the subcellular localization of Mps3p. We were able to detect Mps3p–GFP at 37 of 40 SPBs examined using anti-GFP antibodies (Fig. 5 E). Within the SPB, we rarely detected gold particles at the inner plaque (IP) or outer plaque (OP) regions of the SPB. Like the examples shown in Fig. 5 E, virtually all the gold particles at the SPB (96%) were found at the half-bridge (B) or at the interface between the half-bridge and central plaque (CP), suggesting that Mps3p is a component of the SPB half-bridge. These gold particles were most frequently observed on the nuclear side of the half-bridge (Fig. 5 E), however, some were clearly on its cytoplasmic face (unpublished data). We also observed that a fraction of the gold particles (13%) was distributed randomly in the nuclear envelope (NE), not at the SPB, consistent with observations in living cells of an additional perinuclear localization for Mps3p–GFP (Fig. 5, C and D).
Mps3p is an integral membrane protein
Our immuno-EM analysis suggests that Mps3p is a half-bridge protein. Because Mps3p contains a putative transmembrane domain, we might expect that it is a membrane protein of the half-bridge like Kar1p (Vallen et al., 1992b). To determine if Mps3p associates with membranes, spheroplasts were prepared from cells containing a COOH-terminal fusion of MPS3 to three copies of the HA epitope (MPS3–HA3). After a mild lysis, extracts were fractionated by differential centrifugation, and the distribution of Mps3p–HA3 to the supernatant and pellet fractions was determined by Western blotting. Mps3p–HA3 was detected almost exclusively in the pellet fraction (P) that contains the cellular membranes (Fig. 6). The cytoplasmic protein Zwf1p was a minor contaminant in our membrane preparations, whereas the membrane protein Vam3p was greatly enriched in the pellet fraction (Fig. 6) (Srivastava and Jones, 1998; Kumar et al., 2002). The fact that Mps3p–HA3 could only be extracted from the pellet in the presence of the detergent Triton X-100 indicates that Mps3p, like Vam3p, is an integral membrane protein (Fig. 6).
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We examined the localization of Cdc31p in mps3-1 to determine if Mps3p is involved in its recruitment to the SPB. Using affinity-purified anti-Cdc31p antibodies, we were able to detect Cdc31p at SPBs in nearly all wild-type cells examined (Fig. 7 D). Cdc31p was also found at SPBs in mps3-1 cells grown at 23°C (unpublished data). However, when mps3-1 mutants were shifted to the nonpermissive temperature (36°C), Cdc31p staining at the SPBs was diminished and was visible in only 54% of cells (Fig. 7 D), suggesting that Mps3p is required for efficient recruitment of Cdc31p to SPBs. For comparison, we also examined Cdc31p localization in cells containing a mutation in the other Cdc31p binding partner, Kar1p. In contrast to previous studies (Biggins and Rose, 1994; Spang et al., 1995), we were able to detect Cdc31p at the SPB in 17% of kar1-17 mutants grown at 36°C (Fig. 7 D). We suspect that superior detection using a digital camera allowed us to detect Cdc31p at the SPB in kar1 mutants, especially because the Cdc31p signal in mps3-1 and kar1-17 mutants was often reduced compared with wild type (Fig. 7 D). The fact that we were unable to detect Cdc31p at the SPB in mps3-1 kar1-17 (Fig. 7 D) indicates that residual Cdc31p at the SPB in mps3-1 is due to Kar1p and suggests that Mps3p and Kar1p are the two Cdc31p binding partners at the SPB.
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-factor arrest point strongly suggest that Mps3p functions at the same step in SPB duplication as Cdc31p and Kar1p (Byers, 1981; Rose and Fink, 1987; Winey et al., 1991). All three proteins are essential for half-bridge elongation and satellite formation in wild-type yeast cells (Fig. 8 A). We have shown that Mps3p not only localizes to the SPB half-bridge, but also associates with Cdc31p. Binding between Cdc31p and the COOH terminus of Mps3p suggests a specific function for Mps3p in SPB duplication, and it allows us to predict the topology of Mps3p in the nuclear membrane (Fig. 8 B). MPS3 is an essential gene, and our observations that mps3-1 mutants arrest with monopolar spindles at the nonpermissive temperature are consistent with the possibility that its essential function is in SPB duplication. However, this does not exclude other Mps3p functions. The mps3-1 mutant exhibits several phenotypes at the permissive temperature, including a delay in metaphase. Although a partial SPB defect at the permissive temperature could explain the mitotic delay, the fact that it is not alleviated by deletion of spindle checkpoint genes (MAD1, MAD2, or MAD3; unpublished data) suggests that mps3-1 is defective in a second process. Localization of a fraction of Mps3p to the nuclear periphery suggests that Mps3p could be involved in some aspect of nuclear membrane function. Cdc31p and Kar1p have multiple functions in the cell (Vallen et al., 1992a; Sullivan et al., 1998), and Mps3p might also participate in these processes.
MPS3 encodes a novel SPB component, but MPS3 shares features common to other genes encoding SPB proteins. First, the predicted protein has two potential coiled-coil domains between residues 242–260 and 366–390 (Wigge et al., 1998). Second, the promoter of MPS3 contains two partial matches to the MluI cell cycle box (ACGCGTNA) at positions -209 to -202 (ACGgGTAA) and -193 to -186 (ACGCGaAA) (McIntosh et al., 1991). This promoter element leads to G1-specific gene transcription and is found in the promoters of SPC110 and SPC42 (Kilmartin et al., 1993; Donaldson and Kilmartin, 1996). mRNA levels of MPS3 (both YJL019W and YJL018W) are highest in G1 (Spellman et al., 1998), suggesting that expression of Mps3p is cell cycle regulated.
MPS3 was not found using other protein-based techniques used to analyze SPB composition. Two-hybrid analysis, including a CDC31 two-hybrid screen, has been used extensively to analyze protein–protein interactions within the SPB and to identify new SPB components (Sullivan et al., 1998; Schramm et al., 2001). In addition, SPBs have been enriched from yeast and the protein composition of the SPB analyzed by mass spectrometry (Wigge et al., 1998). Mps3p was not detected by these methods. However, most also failed to identify the other membrane protein of the half-bridge, Kar1p.
In addition to Mps3p, Cdc31p also binds to Kar1p (Biggins and Rose, 1994; Spang et al., 1995). In fact, the essential function of Kar1p in SPB duplication is to recruit Cdc31p to the SPB. Kar1p is thought to be the primary binding partner for Cdc31p at the SPB, but a second Cdc31p binding partner had been postulated (Vallen et al., 1994). The most compelling evidence for a second Cdc31p binding partner comes from observations that a dominant allele of CDC31, CDC31-16, suppresses a complete deletion of KAR1 and that the protein encoded by this mutant, Cdc31-16p, localizes to the SPB in the complete absence of Kar1p (Biggins and Rose, 1994). Our findings that a mps3-1 CDC31-16 strain is unable to grow at any temperature, that overexpression of wild-type CDC31 rescues the growth defect of mps3-1, and that Cdc31p binds Mps3p in vitro suggest that Mps3p might be the other protein that binds Cdc31p at the half-bridge. We confirmed this possibility by examining the localization of Cdc31p in mps3-1 mutants. The fact that Cdc31p localization to the SPB is reduced in mps3-1 or kar1-17 strains at 36°C and is abolished in the mps3-1 kar1-17 double mutant indicates that Kar1p and Mps3p are the half-bridge proteins required to recruit Cdc31p to the SPB (Fig. 8 B).
So how are Cdc31p, Kar1p, and Mps3p arranged at the half-bridge? We detect binding between Mps3p and Cdc31p, and genetic experiments also support the idea that Mps3p and Cdc31p interact. The weak signal seen in the Cdc31p gel overlay with Mps3p could arise from any number of reasons, including the use of a truncated form of Mps3p (full-length Mps3p did not express in bacteria; unpublished data), the use of unmodified, recombinant Cdc31p and Mps3p, inherent low affinity between Cdc31p and Mps3p, or the fact that Mps3p is denatured in this assay. Preliminary results from fluorescence resonance energy transfer experiments between Mps3p and other SPB components suggest that Mps3p and Kar1p interact in vivo (Muller, E., and T. Davis, personal communication). Perhaps Mps3p forms a trimeric complex with Cdc31p and Kar1p in vivo, and its primary function is to regulate the Kar1p–Cdc31p interaction rather than directly bind Cdc31p. The Cdc31p binding site in Kar1p has been identified by Kar1p deletion analysis and is a 19–amino acid acidic -helix in the central part of Kar1p (Spang et al., 1995). We have yet to conclusively identify a similar site in the primary sequence of Mps3p, but Mps3p is predicted to contain multiple -helical domains. Mps3p also contains several other protein–protein interaction domains that might mediate the formation of a complex with Cdc31p and Kar1p.
Mps3p, Kar1p, and Cdc31p all localize to the SPB throughout the cell cycle (Spang et al., 1993, 1995; Biggins and Rose, 1994), and we speculate that these are the structural proteins of the half-bridge. This raises an interesting question of how initiation of satellite formation is restricted to only once during early G1, given that the key half-bridge components are present at the SPB during the entire cell cycle. Although an exact equivalent of the half-bridge is not found in higher eukaryotic centrosomes, it does seem that an analogous template structure exists and is important for centrosome duplication (Adams and Kilmartin, 2000; Hinchcliffe and Sluder, 2001). Centrosomes are composed of two orthogonally oriented centrioles. During centrosome duplication, the centrioles split apart and a new centriole is built adjacent to the two preexisting centrioles. Assembly of the centriole precursor, the procentriole, occurs at a set distance from the existing centriole just as assembly of the SPB precursor, the satellite, occurs at a set distance from the existing SPB. The length of the half-bridge is what seems to determine the SPB/satellite distance, and recruitment of Cdc31p to the half-bridge is likely a key step in SPB duplication. Similarly, a fraction of centrin localizes to the region between the new and the old centriole, and it appears to play some role in controlling centrosome duplication (Paoletti et al., 1996; Salisbury et al., 2002). Therefore, understanding the mechanisms that regulate Mps3p and Kar1p function and their interaction with Cdc31p is relevant not only to yeast SPB duplication but also to centrosome duplication in other eukaryotes.
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Materials and methods |
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SPB assembly screen
A ts bank of mutants was created by mutagenizing five pools of SLJ764 with ethylmethane sulfonate to 60–70% killing as previously described (Rose et al., 1990). Strains unable to induce protein expression from the GAL promoter were eliminated from the collection using a ß-galactosidase assay to monitor induction of H2B1–LacZp (Rose et al., 1990). Approximately 150,000 mutagenized cells yielded 755 ts strains that were microscopically screened for SPB assembly defects as described in the text. The gene responsible for the SPB assembly defect in each mutant was identified by complementation of the ts phenotype using a centromere-based library (Rose and Fink, 1987), linkage analysis, and intragenic complementation testing (Table I; Fig. 2). Two mutants were not cloned.
Lysate preparation and Western blotting
Yeast lysates were prepared by resuspending 2 OD600 U/ml of cells in 0.2 ml SDS sample buffer (125 mM Tris-HCl, pH 6.8, 5% SDS, 1.4 M ß-mercaptoethanol, 20% glycerol, 0.3% bromophenol blue, 2 mM PMSF) and lysing by mechanical disruption. Proteins were fractionated by differential centrifugation as previously described (Srivastava and Jones, 1998). Samples equal to 0.5 OD600 U/ml of cells were loaded on SDS-PAGE gels, and proteins were detected by immunoblotting with the following dilutions of antibodies: 1:2,000 anti-GFP mAb B34 (Covance); 1:1,000 anti-HA mAb 16B12 (Covance); 1:1,000 affinity-purified anti-Vam3p antibody (a gift of G. Odorizzi, University of Colorado); 1:20,000 affinity-purified glucose-6-phosphate dehydrogenase antibody (Zwf1p; Sigma-Aldrich).
Cdc31p antibodies
Antibodies against purified 6His–Cdc31p (see below) were generated in rabbits (Animal Pharm) and purified according to the manufacturer's instructions on a Cdc31p column created using the Sulfo-link kit (Pierce Chemical Co.). Purified antibodies recognize a single major band the size of Cdc31p in yeast extracts (not depicted).
Cytological techniques
The DNA content of 20,000 mid-log phase cells stained with propidium iodide was determined by flow cytometry as previously described (Hunter and Eipel, 1979). Samples were analyzed on a Becton Dickinson FACScan® flow cytometer using CELL QUEST software. Cells were fixed with 4% paraformaldehyde in 0.1 M sucrose for 20 min at room temperature, spheroplasted, methanol/acetone fixed to glass sides, and processed for immunofluorescence as previously described (Rose et al., 1990). The following primary antibody dilutions were used: microtubules, 1:500 rat anti-tubulin antibody YOL 1/34 (Accurate Chemical & Scientific Corp.); SPBs, 1:500 affinity-purified anti-Tub4p antibodies (a gift of T. Stearns, Stanford University, Palo Alto, CA); Mps3–GFP, 1:5,000 affinity-purified anti-GFP polyclonal antibodies (CLONTECH Laboratories, Inc.); and Cdc31p, 1:500 affinity-purified anti-Cdc31p antibodies. Secondary antibodies included 1:10,000 CY3-conjugated goat anti–rabbit IgG (Chemicon) and 1:200 fluorescein-conjugated goat anti–rat IgG (Cappel). DNA was visualized by staining with 1 µg/ml DAPI for 5 min immediately before mounting with Citifluor (Ted Pella, Inc.). Cells were examined with a Leica fluorescence microscope using 63x magnification, and images were captured with a Cooke Sensicam and processed with Slidebook (3I). Cells were high pressure frozen, freeze substituted, sectioned, and stained as previously described (Giddings et al., 2001) to examine spindle structure by EM or to detect Mps3p–GFP by immuno-EM. Serial thin sections were viewed on a Philips CM10 electron microscope, and images were captured with a Gatan digital camera and viewed with Digital Micrograph Software.
Gel overlay assay
6His–Cdc31p was purified from M15 Escherichia coli transformed with pQE10-CDC31 and induced with 0.5 mM IPTG for 2 h at 25°C by metal affinity chromatography using Talon resin as described by the manufacturer (CLONTECH Laboratories, Inc.). 500 µg of purified Cdc31p was dialyzed overnight at 4°C against PBS and labeled with the infrared dye Alexa®680 according to the manufacturer's instructions (Molecular Probes).
Expression of GST, GST–Kar1p, and GST–Mps3p-Ct in the E. coli strain BL21(DE3) was induced by the addition of 0.5 mM IPTG to log-phase cells for 2 h at 25°C. 10 µl of GST–Kar1p, GST–Mps3p-Ct, and cell only and 2 µl of GST extract from 1 ml culture were separated by 10% SDS-PAGE, and proteins were electrophoretically transferred to nitrocellulose membranes (Scheiler & Schuell). Membranes were probed with 10 µg labeled 6His–Cdc31 as previously described (Biggins and Rose, 1994) and were imaged on an infrared scanner (LiCor) to detect Alexa®680–Cdc31p binding. Expression of bacterial fusion proteins was confirmed by Western blotting with a 1:1,000 dilution of anti-GST antibody (Amersham Biosciences).
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Footnotes |
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Acknowledgments |
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This work was supported by funding from the National Institutes of Health (GM51312 to M. Winey), the Keck Foundation (to S.L. Jaspersen), and the Helen Hay Whitney Foundation (to S.L. Jaspersen).
Submitted: 28 August 2002
Revised: 6 November 2002
Accepted: 6 November 2002
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References |
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