In fission yeast, -tubulin (encoded by the gtb1⁺ gene), Alp4 (Spc97/GCP2), and Alp6 (Spc98/GCP3) are essential components of the -tubulin complex. We isolated gtb1 mutants as allele-specific suppressors of temperature-sensitive alp4 mutations. Mutation sites in gtb1 mutants and in several alp4 alleles were determined. The majority of substituted amino acids were mapped to a small area on the predicted surface of the -tubulin molecule that might directly interact with the Alp4 protein. The cold sensitivity of -tubulin mutants was almost completely suppressed by an -tubulin mutation and partially suppressed by a low concentration of thiabendazole, a microtubule assembly inhibitor. Other gtb1 mutants had increased resistance to this drug. Gel-filtration and immunoprecipitation analyses suggested that the mutant -tubulin formed an altered -tubulin complex with increased stability compared to wild-type -tubulin. In most gtb1 mutants, sexual development was impaired, and aberrant asci that contained an irregular spore shape and number were produced. In contrast, spore formation was not appreciably damaged in some alp4 and alp6 mutants, even at temperatures where vegetative proliferation was substantially defective. These results suggested that the function of the -tubulin complex or the requirement of each component of the complex is differentially regulated between the vegetative and sexual phases of the life cycle in fission yeast. In addition, genetic data indicated intimate functional connections of -tubulin with several kinesin-like proteins.

---

UBIQUITOUS in eukaryotes, -tubulin is a key component of the microtubule-organizing center (MTOC), such as the centrosome in animal cells. -Tubulin has a crucial role in microtubule nucleation and in the anchoring of the minus end of microtubules to the MTOC and thereby contributes to the proper formation of mitotic spindles and cytoplasmic microtubular arrays (OAKLEY 2000; SCHIEBEL 2000; JOB et al. 2003). In the budding yeast Saccharomyces cerevisiae, -tubulin interacts with Spc97 and Spc98 to form a functional complex, which localizes at the spindle pole body (SPB, centrosome-equivalent organelle in yeast; KNOP et al. 1997; KNOP and SCHIEBEL 1997, 1998). Proteins with sequence similarity to Spc97 and Spc98 have been identified from a number of species, including human, frog, fly, nematode, plant, and fungi (MURPHY et al. 1998; OEGEMA et al. 1999; GUNAWARDANE et al. 2000; VARDY and TODA 2000; ERHARDT et al. 2002; HANNAK et al. 2002). These proteins contain conserved sequence motifs and are called gamma-ring proteins (GRIPs)/gamma-tubulin complex proteins (GCPs; MURPHY et al. 1998, 2001; GUNAWARDANE et al. 2000). A large form of -tubulin ring complex (-TuRC) has been identified in several organisms, including fly, frog, and human (MORITZ et al. 1995, 2000: ZHENG et al. 1995; MURPHY et al. 1998). Upon incubation under high-salt conditions, a smaller complex is released from the Drosophila -TuRC. The -tubulin small complex (-TuSC) consists of Dgrip83, Dgrip91, and -tubulin and is thought to be structurally equivalent to the yeast Spc97/Spc98/-tubulin complex (OEGEMA et al. 1999). In vitro, -TuSC is capable of nucleating microtubules, but much less efficiently than -TuRC (OEGEMA et al. 1999). Genetic studies in several lower eukaryotes revealed that the nucleation of microtubules is not the sole function of -tubulin in vivo but that it is likely involved in other aspects of microtubule dynamics, including the regulation of spindle elongation, mitotic checkpoints, and cytoplasmic microtubule stability (PALUH et al. 2000; VOGEL and SNYDER 2000; HENDRICKSON et al. 2001; JUNG et al. 2001). How -tubulin protein achieves these functions and how the -tubulin complex is regulated remain to be determined.

-Tubulin in the fission yeast Schizosaccharomyces pombe is encoded by gtb1⁺ (also known as tug1⁺) and is essential for viability (HORIO et al. 1991; STEARNS et al. 1991). Fission yeast carries apparent homologs of Spc97 and Spc98 in S. cerevisiae, respectively, encoded by alp4⁺ and alp6⁺ (VARDY and TODA 2000). As anticipated from the sequence similarity, biochemical as well as genetic analyses demonstrated that Alp4/Alp6 interacts with -tubulin and localizes at the MTOC in fission yeast (VARDY and TODA 2000). Unlike the -tubulin complex in budding yeast, the -tubulin-Alp4/Alp6 complex in fission yeast appears to exist in larger complexes (VARDY and TODA 2000; FUJITA et al. 2002), analogous to the -tubulin complexes in higher eukaryotes.

We are interested in the function of the SPB in fission yeast, particularly during the sexual phase of the life cycle, where the SPB, as a nuclear membrane-associated organelle, performs various tasks that are required for spindle formation, nuclear migration, spore formation, and also probably chromosome arrangement (HIRATA and SHIMODA 1994; HAGAN and YANAGIDA 1995; CHIKASHIGE et al. 1997; DING et al. 1998; NIWA et al. 2000). Because -tubulin is a key element of the SPB, we examined how the requirements of -tubulin are differentially regulated for the varied SPB functions in fission yeast. Here we report the isolation of several -tubulin mutants that suppress temperature-sensitive (ts) alp4 mutations in an allele-specific manner. Sporulation, rather than vegetative proliferation, was highly susceptible to many of the -tubulin mutations and mutants in the components of the -tubulin complex differentially affected sporulation, suggesting that the function of each component of the -tubulin complex is differentiated at least between the vegetative and sexual phases of the life cycle in fission yeast. Also, -tubulin and kinesin-like proteins were functionally intimately related.

Strains and media:

Standard genetic methods for the fission yeast S. pombe were used (MORENO et al. 1991; ALFA et al. 1993). Yeast extract agar (YEA), malt extract agar (MEA), and Edinburgh minimal medium 2 (EMM2) were used as a complete rich medium, conjugation/sporulation medium, and minimal medium, respectively. The strains used in this study are listed in Table 1. The alp4-1891 and alp6-719 mutants were described by VARDY and TODA (2000). The other ts alp4 mutants used, alp4-B7, alp4-S5, and alp4-S7, were created by polymerase chain reaction (PCR)-based, gene-specific mutagenesis. Briefly, a C-terminal tagging module containing green fluorescent protein (GFP; BAHLER et al. 1998) was fused to the alp4⁺ gene and PCR fragments amplified under mutagenic conditions were generated and used for substitutive transformation. The ts growth defect of the alp4-B7 mutant, but not alp4-S5 and alp4-S7, was rescued by a multicopy plasmid carrying the gtb1⁺ gene. The hemagglutinin epitope (HA) sequence was fused to the C terminus of the alp4⁺ gene according to BAHLER et al. (1998). We attempted to fuse the same sequence at the C terminus of the alp4-1891 mutant gene that was chromosomally integrated. We also attempted to fuse the tag sequence at the N terminus in a plasmid-borne form, but failed to obtain a useful construct.

View this table: In this window In a new window   TABLE 1 Strains used in this study

 

Mutant selection:

Mutagenesis with nitrosoguanidine (150 µg/ml) was performed according to UEMURA and YANAGIDA (1984). Revertants of the alp4-1891 or alp4-B7 ts mutants were crossed with a wild-type strain and random spore analysis was performed. Thirty descendant colonies from each cross were tested for the ts phenotype and, if ts, the revertant was judged to carry an extragenic suppressor of the alp4 mutant. Gene disruption using the G418-resistant marker replacement method was described previously (BAHLER et al. 1998). Correct disruption was verified by sequencing both of the gene replacement boundaries.

DNA sequencing:

A 2.4-kb DNA fragment from 270 bp upstream of the start codon through 540 bp downstream of the termination codon of the gtb1 gene was amplified by PCR and sequenced. Likewise, fragments covering the coding region of the alp4 gene were sequenced.

Reagents for immunochemical analyses:

A synthetic oligopeptide corresponding to the carboxyl terminus of fission yeast -tubulin was used to raise rabbit anti--tubulin antiserum, which was affinity purified before use. Mouse monoclonal anti-HA antibody (16B12) was purchased from Convance (Berkeley, CA). Electrochemiluminescence Western blotting detection kits (Amersham, Buckinghamshire, UK) were used for Western blot analysis.

Gel-filtration chromatography:

The experimental protocol described in VARDY and TODA (2000) was basically followed for gel filtration of the -tubulin complexes. Briefly, cell cultures were grown in minimal medium at 28° and shifted to 36° or 20°, followed by 6 and 7 hr of incubation, respectively. Cells suspended in buffer A (20 mM Tris-HCl, pH 7.5, 20% glycerol, 0.1 mM EDTA, 1 mM mercaptoethanol) containing 5 mM ATP, 1% protease inhibitor cocktail (Sigma Chemical, St. Louis), and 2 mM Pefabloc SC (Roche, Indianapolis) was disrupted with glass beads using a Multi-Beads shocker (Yasui Kikai, Osaka, Japan). Soluble cell extracts were obtained after repeated centrifugations at 18,000 x g for 10 min. Immediately after the preparation, 200–250 µl of the extract (approximate protein concentration was 30 mg/ml) was loaded on the Superose 6 10/300 GL column (Amersham), and proteins were eluted with buffer A containing 100 mM NaCl. Thyroglobulin (669 kD), apoferritin (443 kD), ß-amylase (200 kD), and bovine serum albumin (66 kD; Sigma Chemical) were used as size markers. According to the suggestion of Amersham, we did not use blue dextran, a 2000-kD marker, but rather, on the basis of the specification provided by the manufacturer, we assumed the void volume to be >2000 kD.

Immunoprecipitation:

Whole-cell extracts were prepared in TEG buffer (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 15% glycerol, 1% NP-40) containing 1% protease inhibitor cocktail and 2 mM Pefabloc SC by the glass beads disruption method. The soluble extracts, each containing 2 mg of protein, were brought to the indicated concentrations of NaCl and incubated at 0° for 30 min. Approximately 10 µg of the anti-HA antibody was added and incubated at 4° for 1 hr. Prewashed Affi-Prep Protein A support (Bio-Rad, Hercules, CA) was then added, followed by 1 hr of incubation at 4°. In some experiments (see Figure 4), the antibody was preincubated with the protein A beads at 4° for 1 hr in the absence of NaCl, mixed with the cell extracts, and incubated for 1 hr at 4° in the presence of the desired concentration of NaCl. The protein A beads were washed with TEG buffer at 4° using Spin Filters (CytoSignal, Irvine, CA) and proteins that remained bound to the beads were examined by Western blot analysis.

View larger version (46K): In this window In a new window Download PPT slide   FIGURE 4.— Immunoprecipitation analysis of Gtb1-Alp4 interaction. Cell extracts were prepared from strains W896 (wild type) and W897 (gtb1-93) grown at the indicated temperature and used for immunoprecipitation with anti-HA antibody. Proteins bound in the presence of the indicated concentrations of NaCl were detected with anti-HA (Alp4-HA) and anti--tubulin antibodies. The precipitation of both Alp4-HA and -tubulin was dependent on anti-HA antibody and protein A beads (data not shown).

 

Cytologic analysis:

Indirect immunostaining was performed with anti--tubulin monoclonal antibody (TAT1; WOODS et al. 1989) and with rabbit anti-Sad1 polyclonal antibody as described previously (GOTO et al. 2001; TANGE et al. 2002). The chromosomally integrated GFP-fused -tubulin gene expressed under the control of the nda2 promoter was a kind gift from H. Masuda (Kansai Advanced Research Center, Kobe, Japan). The yeast strain expressing GFP-fused Sid4 protein was used as an SPB marker (TOMLIN et al. 2002). The plasmid pDQ105 (DING et al. 1998) carrying a GFP-fused -tubulin gene was also used to visualize microtubules. The meioses shown in Figure 6B were induced in malt extract broth and galactose and mannose synthetic medium at 30° as described in TANGE et al. (1998). To examine the effect of suppressor gtb1 mutations on cytoplasmic microtubules in interphase cells of the alp4 mutant, synchronous cultures were made by the hydroxyurea arrest and release method as described in VARDY and TODA (2000).

View larger version (63K): In this window In a new window Download PPT slide   FIGURE 6.— Aberrant sporulation and meiosis in the gtb1 mutant. (A) Spores were produced by crossing HM123 and HM487 (wild type) or W604 and W609 (gtb1-93) on an MEA plate. Incubation was at 30° for 2 days. (B) Meioses were induced in YT374 (wild type; a, meiosis I; b, meiosis II) and YT375 (gtb1-93; c and d, meiosis I; f, meiosis II) at 30°. Sad1 (red), Sid4-GFP and Atb2-GFP (green), DAPI (blue). Bars, 5 µm.

 

Modeling of the -tubulin structure:

A 3-D structure model of fission yeast -tubulin was created based upon that for the /ß-tubulin heterodimer (NOGALES et al. 1998) using 3D Jigsaw (Version 2) software. RASMAC software was used to graphically view the 3-D structure.

Determination of mutation sites in alp4 mutant genes:

Mutation sites were determined in four different ts alp4 mutants. One of these, alp4-1891, was originally isolated from a collection of fission yeast mutants that displayed altered growth polarity (VARDY and TODA 2000). The others were created by PCR-based gene-specific mutagenesis (MATERIALS AND METHODS). Among them, alp4-B7 was suppressed by multiple copies of the gtb1⁺ gene, which encodes -tubulin in fission yeast, while alp4-S5, alp4-S7, and alp4-1891 mutants were not suppressed. alp4-1891 had an amino acid substitution: Ser at 194 was changed to Pro (S194P). The other mutants contained multiple substitutions: I499M, Q567K, and R619H in alp4-B7, P390H and L427M in alp4-S5, and R698Q and D735G in alp4-S7.

Isolation of suppressors of ts alp4 mutants:

To examine whether the ts alp4 mutants can be suppressed by altering the -tubulin protein sequence, we first sought extragenic suppressors of the alp4-1891 mutant. Mutated alp4 mutant cells were plated and incubated at 36°, the restrictive temperature for the mutant. Of 96 colonies examined, 7 had a severe growth defect at 20°. Each of these 7 revertants was crossed with a wild-type strain and 3 of them produced ts progeny, indicating that they had lost the ts phenotype due to extragenic suppressors. Sequencing of genomic DNA indicated that one of them had a substitution mutation (P302L) in the gtb1 gene. This mutation was identical to that previously isolated by PALUH et al. (2000) as a synthetic lethal mutation with a complete loss-of-function mutation in the pkl1⁺ gene that encodes a member of the Kar3-family kinesin-like protein (PALUH et al. 2000 described that the substitution occurred at position 301, but we followed the numbering of the Sanger Center Fission Yeast Database and designated the mutation to be at 302). We named the mutant allele gtb1-93. Further genetic analysis by tetrad dissection confirmed that the mutation was responsible for suppression of the alp4 mutation. Consistent with the previous result (PALUH et al. 2000), the gtb1-93 mutation alone conferred a cold-sensitive (cs) phenotype, which was more severe than that of the gtb1 alp4 double mutant (Figure 1, bottom). This indicated that the alp4-1891 mutation functioned, although weakly, to suppress gtb1-93.

View larger version (83K): In this window In a new window Download PPT slide   FIGURE 1.— Suppression of alp4-1891 by gtb1-93 mutation. Strains with the indicated mutations were plated on YEA plates and incubated at the indicated temperatures for 3 days (35° and 30°) or for 6 days (20°). HM123 (wild type), W604 (gtb1-93), W705 (alp4-1891), and W733 (alp4-1891 gtb1-93) were used.

 
Another screening was performed to isolate more gtb1 mutants. In the second screening, the cs phenotype was not taken into account. From 20 revertants due to extragenic suppressors, we isolated a new gtb1 mutant, gtb1-85, which had an L301S substitution. Like gtb1-93, the gtb1-85 mutant was cs, although the growth defect was slightly leaky. The cs phenotype of gtb1-85 was partially relieved by the alp4-1891 mutation (data not shown). Thus, two cs gtb1 mutants and an alp4 mutant were mutually suppressing, consistent with direct in vivo interactions between these two gene products (VARDY and TODA 2000).

Both the gtb1-93 and the gtb1-85 mutant, when crossed with themselves or with each other, produced abnormal asci containing an aberrant number and shape of spores even at high temperatures (see below). Thus, we further screened for gtb1 mutants from revertants of alp4-1891, as those produced aberrant asci when crossed with the gtb1-93 mutant. This screening revealed eight independent mutants that produced abnormal asci. All eight were verified to be gtb1 mutants by nucleotide sequencing. As summarized in Table 2, gtb1 mutants isolated as suppressors of alp4-1891 were classified into four groups with respect to the mutation sites. The majority of the mutants had mutations at position 301 or 302, while the remaining gtb1-29 carried a substitution at position 87.

View this table: In this window In a new window   TABLE 2 -Tubulin mutants isolated as suppressors of the alp4-1891 mutation

 

Allele-specific suppression of alp4 by the gtb1 mutants:

The clustered mutation sites in a class of suppressor gtb1 mutants and the fact that two cs mutants of this class mutually suppressed the alp4-1891 mutation suggested that the mutated region interacts directly with Alp4 and -tubulin proteins. If this is the case, it might be that the suppression is specific to particular alleles of the alp4 gene. To test this, we combined the gtb1 mutations with different alp4 alleles and examined their colony-forming ability (Table 3). The gtb1-93, gtb1-85, and gtb1-22 mutants did not suppress alp4 alleles other than alp4-1891. The gtb1-93 mutation was lethal at any temperature examined (33°, 30°, and 26°) when it was combined with alp4-B7 or alp4-S5. gtb1-85 had a similar but weaker synergistic effect with these alp4 mutants. In contrast, gtb1-29 suppressed the alp4-B7 and alp4-1891 mutations. Both gtb1-22 and -29 mutants became cs in the background of the alp4-B7 mutation.

View this table: In this window In a new window   TABLE 3 Allele-specific suppression of alp4 by gtb1 mutants

 
Another screening was performed for gtb1 mutants that suppress the alp4-B7 mutant. From eight independent batches of mutated alp4-B7 cells, 23 revertants due to extragenic suppressors were isolated and sequenced for the gtb1 gene. We obtained five independent mutants (six mutants in total) that had a substitution mutation in the coding region of the gene. All of them had an identical mutation: Ser at 263 was changed to Asn (named gtb1-101). The gtb1-101 mutation suppressed not only the alp4-B7 but also the alp4-S5 mutant, although the suppression was weak for the latter mutation, while it did not suppress the alp4-1891 mutation at all (Table 3). The gtb1-29 allele was not isolated from the new screening, indicating that the screening was not exhaustive. Nevertheless, two different alp4 mutants, alp4-1891 and alp4-B7, were suppressed by a different range of -tubulin mutants. In particular, gtb1 alleles with a substitution at 301 or 302 seemed to have very specific suppressor activity for alp4-1891, consistent with the idea that these sites are in a region that is involved in direct protein interactions (PALUH et al. 2000 and see DISCUSSION).

The gtb1-101 mutant cells were examined to determine if they contained more -tubulin protein, because the alp4-B7 mutant was suppressed by an increased dosage of the gtb1⁺ gene (data not shown). -Tubulin protein was not increased in the gtb1-101 mutant as determined by measuring the band intensity in a Western immunoblot analysis (data not shown). The amount of -tubulin was also not appreciably changed in the gtb1-93, gtb1-22, or gtb1-29 mutants.

Suppression of the gtb1-93 mutation:

We obtained a spontaneous revertant of gtb1-93 that lost the cs growth defect, producing normal-sized colonies at 22°. Subsequent genetic analysis revealed that the reversion was due to a mutation in the atb2 gene (atb2-607), which encoded altered 2-tubulin with a P173L substitution. This atb2 mutation, either alone or with gtb1-93, conferred hypersensitivity to the microtubule-destabilizing drug, thiabendazole (TBZ). It did not form colonies in the presence of 10 µg/ml TBZ at 33°. The atb2-607 mutation also suppressed another cs mutant, gtb1-85. The cs growth defect of the gtb1-93 mutant is suppressed by a TBZ-sensitive allele of nda2 (1-tubulin; PALUH et al. 2000). Thus, both 1- and 2-tubulin, when altered to presumed microtubule-destabilizing forms, suppressed the cs phenotype of the -tubulin mutants. Consistently, a low concentration of TBZ partially rescued the cs growth defect of the gtb1 mutants (Figure 2). Figure 2 also demonstrates that the non-cs gtb1 mutants, gtb1-22, gtb1-29, and gtb1-101, were more resistant than wild-type cells to TBZ.

View larger version (59K): In this window In a new window Download PPT slide   FIGURE 2.— Effect of TBZ on gtb1 mutants. Strains with the indicated gtb1 allele were plated on YEA plates containing the indicated concentration (in micrograms per milliliter) of TBZ and incubated at the indicated temperatures for 3 days (30° –TBZ), 4 days (30° +TBZ and 26°), 5 days (22°), or 6 days (20°). Strains used were HM123, W604, W650, W665, W671, and W867.

 

Gel-filtration analysis of the -tubulin complex in gtb1 mutants:

We performed a gel-filtration analysis to examine whether the gtb1-93 mutation affects the formation of the -tubulin complex. Protein extracts were prepared from cells with or without gtb1-93 and alp4-1891 mutations that were grown at 36° or at 20°. Consistent with previous reports (VARDY and TODA 2000; FUJITA et al. 2002), a major part of -tubulin in the alp4 mutant as well as in wild-type cells was eluted near the void volume, which corresponded to >2000 kD, and this profile was not appreciably changed by the gtb1 mutation irrespective of the growth temperature (Figure 3). When extracts of cells grown at 36° were compared, however, there was a discrete peak centering around 550 kD in the gtb1 single mutant that was barely detectable in other strains, including the gtb1 alp4 double mutant. Alternatively, in these strains, there was a broad distribution of -tubulin in fractions ranging from 400 to 150 kD. The alp4-ts mutant contained a reduced amount of -tubulin in this range of fractions compared with wild type, while the gel-filtration pattern of the alp4 gtb1 double mutant was very similar to that of the wild type. Thus, the effect of a single mutation of the gtb1 and alp4 genes on the chromatographic properties of protein complexes appeared to negate each other. This result was consistent with the mutual suppression of these mutations described above. The 550-kD peak in the gtb1 mutant became less prominent when extract was prepared from cells grown at 20° (Figure 3), the restrictive temperature for the gtb1 mutant, suggesting that the complex formation was somehow impaired at this temperature.

View larger version (92K): In this window In a new window Download PPT slide   FIGURE 3.— Gel-filtration analysis of the -tubulin complex. Soluble cell extracts were prepared from HM123 (wild type), W705 (alp4-1891), W604 (gtb1-93), and W733 (alp4-1891 gtb1-93), which were incubated at either 36° or 20°. Each fraction was analyzed by Western blotting with anti--tubulin antibody. Positions of size markers (669, 443, 200, and 66 kD) were determined by a parallel control chromatography (see MATERIALS AND METHODS).

 
We examined whether the apparent peak shift observed in the gtb1-93 mutation was specific to this mutation. In both gtb1-22 and gtb1-29 mutants grown at 36°, there was a shift to higher-molecular-weight fractions, although the shift was not as evident as in gtb1-93. Between these two mutants, the gtb1-29 mutant had a clearer shift, and interestingly, the gel-filtration pattern was affected only slightly by the alp4-1891 mutation (data not shown).

Altered stability of the -tubulin complex in gtb1 mutants:

Because the gel filtration was performed in the presence of 100 mM NaCl to avoid nonspecific protein interaction, the observed difference in the protein distribution could have been due to differential stability of the protein complex at various salt concentrations. Thus, we made a tagged version of the Alp4 protein in which the HA epitope sequence was fused to the C terminus of Alp4 (see MATERIALS AND METHODS). Previous studies indicated that this fused protein retained its function (VARDY and TODA 2000). Cell extracts were prepared from two strains that carried either the gtb1⁺ or the gtb1-93 allele together with the alp4⁺-HA gene. Immunoprecipitation with an anti-HA antibody revealed that the amount of wild-type -tubulin that co-immunoprecipitated with Alp4-HA decreased sharply with increased concentrations of NaCl, while mutant -tubulin remained largely associated with Alp4-HA (Figure 4). Similar experiments were performed with other gtb1 alleles, gtb1-85, gtb1-29, and gtb1-22. Gtb1-85 was stably associated with Alp4-HA, while Gtb1-29 had slightly reduced stability and Gtb1-22 had almost wild-type stability with Alp4-HA (data not shown). Thus, the salt stability of each mutant -tubulin appeared to be generally correlated with the chromatographic property. Therefore, the peak shift in the gel-filtration chromatography that occurred in the gtb1 mutants might have been partially due to altered stability of the -tubulin complex from the salt. Although further biochemical analyses are needed to elucidate the nature of the protein complexes separated in the gel chromatography, it is probable that the gtb1 mutation affects the stability of the -tubulin complex.

The salt-resistant property of the mutant -tubulin complex was only slightly changed by the growth temperature (Figure 4), yet the chromatographic pattern was different; that is, the prominent peak around 550 kD was apparently shifted to a lower-molecular-weight form in the extract of cells grown at a low temperature (Figure 3). Therefore, the reduction of the 550-kD peak in this extract was unlikely due to altered salt sensitivity, suggesting that the gtb1 mutant cells grown at low temperatures contained a reduced amount of a particular form of the -tubulin complex.

Aberrant cytoplasmic microtubule arrays in gtb1 mutants:

Wild-type fission yeast cells in the interphase contain six to eight microtubule bundles that run mostly parallel to the long axis of the cell (HAGAN 1998). PALUH et al. (2000) reported that the gtb1-93 mutant cells at a permissive temperature contained aberrant cytoplasmic microtubule bundles that were fewer in number and longer than wild-type microtubules. This observation was verified by immunofluorescent staining with an anti--tubulin antibody (Figure 5A). Similar aberrant cytoplasmic microtubules were observed by use of GFP-tagged 2-tubulin (DING et al. 1998; Figure 5B). The suppressor atb2 for the cs gtb1-93 mutant negated the detrimental effect of the gtb1 mutation on microtubule formation (Figure 5A). In gtb1-85 and gtb1-22 mutants, cytoplasmic microtubule arrays visualized with GFP-fused tubulin were similar to those in gtb1-93. gtb1-29 mutant cells contained two types of abnormal microtubular arrays. One type contained microtubules similar to those in the gtb1-93 mutant (Figure 5B, bottom left), but the other type had thinner microtubules (Figure 5B, bottom right). On the other hand, cytoplasmic microtubules formed in the gtb1-101 cells were only marginally different from wild type (data not shown).

View larger version (34K): In this window In a new window Download PPT slide   FIGURE 5.— Abnormal cytoplasmic microtubule bundles formed in gtb1 mutants. Strains YT106 (wild type), W607 (gtb1-93), W650 (gtb1-85), W870 (gtb1-93 atb2-607), W871 (gtb1-85 atb2-607), and W869 (atb2-607) were used. (A) Effect of atb2 mutation on microtubule formation. Cells with the indicated mutations were incubated at 33° in YE medium and stained with anti--tubulin antibody (TAT-1, green) and 4',6-diamidino-2-phenylindole (DAPI; red). (B) Interphase cells carrying the indicated gtb1 alleles were transformed with the plasmid pDQ105 to express GFP-fused -tubulin at 30° in EMM2 containing 10 µM of thiamine. Bars, 5 µm.

 
The alp4 and alp6 mutants contain aberrant cytoplasmic microtubules that resemble those in the gtb1 mutants (VARDY and TODA 2000). We examined whether the combination of the alp4-1891 mutation with its suppressor gtb1-22 could reverse the aberrant cytoplasmic microtubules to the wild-type configuration. The double mutant, however, contained mutant cytoplasmic microtubules (data not shown). Similar results were obtained with the gtb1-93 mutation. Thus, although the gtb1 mutants suppressed lethal spindle dysfunction caused by the alp4 mutation (data not shown), they appeared not to affect the cytoplasmic microtubules.

Impaired sporulation and meiosis in gtb1 mutants:

As mentioned above, gtb1 mutants isolated as suppressors of the alp4-1891 mutation produced aberrant asci containing an abnormal number and shape of spores (Figure 6A). We examined the sporulation defect quantitatively by counting the number of mature spores per ascus and compared this number with that of other gtb1-related mutants. The incubation temperature was either 30° or 33° where gtb1 mutants had no or only a slight defect in vegetative growth on the basis of the colony size on a rich medium plate (Figure 2) and from the doubling time of the logarithmic phase of liquid cultures (Figure 7). Four gtb1 mutants that suppressed the alp4-1891 mutation produced aberrant asci and two cs mutants, gtb1-93 and gtb1-85, were more severely impaired (Table 4). Another mutant, gtb1-101, which suppressed the alp4-B7 but not the alp4-1891 mutation, had no sporulation defect. The gtb1-101 allele, therefore, had no discernible phenotype in either the vegetative or the sporulation phase, except for its suppressor activity to alp4-B7 and alp4-S5 mutants as well as increased resistance to TBZ. We also examined the effect of alp4-1891 and alp6-719 mutations on sporulation. These mutants had only marginal sporulation defects at both 30° and 33°. At 33°, vegetative growth of the alp4-1891 mutant was very poor (Figure 7). Although the alp4-1891 mutant itself conferred little sporulation defect, it had marked synergy with the gtb1-93 and gtb1-22 mutants (Table 4). Note that the alp4-1891 mutation suppressed the vegetative growth of the cs gtb1-93 mutant, but had an adverse effect on sporulation in the same gtb1 mutant. The other alp4 mutant, alp4-B7, had defective sporulation, although the defect was not as severe as in the gtb1 mutants. Nevertheless, the ts growth defect of alp4-B7 was not as severe as that of alp4-1891 (data not shown). These results demonstrated that mutations in the genes that encode the components of the -tubulin complex exhibit differential effects on vegetative growth and on sporulation. The findings also indicated that sporulation in fission yeast was generally more susceptible to gtb1 mutation than to alp4 and alp6 mutations.

View larger version (54K): In this window In a new window Download PPT slide   FIGURE 7.— The effect of temperature on vegetative growth of gtb1 and alp4/6 mutants. (Left) Logarithmic phase of cultures in YE liquid medium at 26° were shifted to the indicated temperature at time 0 hr and aliquots of cultures were taken for microscopically counting the number of cells. HM123 (wild type: circle), W705 (alp4-1891: triangle), and W549 (alp6-719: square) were used. (Right) Liquid cultures at 33° were shifted to the indicated temperatures and the increase in cell number was plotted. HM123 (wild type: circle), W604 (gtb1-93: triangle), W715 (gtb1-29: square), and W716 (gtb1-22: diamond) were used. For all cultures, cell concentrations at time 0 were between 1.5 x 106 and 2.2 x 106 cells/ml. In the graphs, relative increase in cell number was plotted.

 

View this table: In this window In a new window   TABLE 4 Abnormal ascus formation in gtb1 and related mutants

 
We further examined meiotic divisions in the gtb1-93 mutant that occurred prior to sporulation. Two defective phenotypes were observed in meiosis I; one was impaired chromosome segregation with an apparently normal spindle (Figure 6B, c and e) and the other was a monopolar spindle (d). Figure 6B(f) shows a defective phenotype, probably in meiosis II. How these defects are related to the sporulation phenotype is not clear. Detailed analyses of the sexual phase of -tubulin mutants are in progress.

Microtubule-destabilizing agents rescued the sporulation defect in gtb1 mutants:

Because the cs gtb1 mutants were suppressed by the atb2-607 mutation and also partially suppressed by a low concentration of TBZ, both of which are presumed microtubule-destabilizing conditions, we examined whether the sporulation defect of gtb1 mutants was affected by the same agents. The results summarized in Table 5 demonstrated the following. The atb2-607 mutation suppressed defective sporulation in the gtb1-93, gtb1-22, and gtb1-29 mutants. TBZ (10 µg/ml) was as effective as the atb2 mutant in rescuing sporulation in all of these gtb1 mutants. Thus, aberrant sporulation occurring in gtb1 mutants might be correlated with stabilized forms of microtubules, and thus microtubule-destabilizing agents could abrogate the deleterious effect of microtubules with altered stability. The gtb1-101 mutant had an elevated resistance to TBZ, yet it produced almost normal asci (Table 5). The gtb1-101 mutant was less resistant to TBZ than the other two gtb1 mutants, gtb1-22 and gtb1-29 (see Figure 2), and also did not contain abnormal cytoplasmic microtubules, like the other -tubulin mutants.

View this table: In this window In a new window   TABLE 5 Effect of thiabendazole and the atb2 mutation on sporulation

 

Genetic interactions of -tubulin with kinesin-like proteins:

Previous studies demonstrated that a null allele of the pkl1 gene (encoding a Kar3 family of kinesin-like protein) created by a gene-disruption method (hereafter called pkl1) was synthetically lethal with the gtb1-93 mutant (PALUH et al. 2000). To confirm the previous observation, we made a genetic cross to produce double-mutant strains at the gtb1 and pkl1 loci. The strains were viable but produced smaller colonies at 33° compared to the respective single mutants. Also, the cold sensitivity of the gtb1-93 mutant was enhanced in the pkl1 background. Thus, although the pkl1-null mutation was not synthetically lethal with the gtb1 mutant at high temperatures, at least in the genetic background of our strains, there was a clear genetic interaction between these genes. A similar genetic interaction was reported in Aspergillus nidulans using a klpA (a homolog of pkl1) deletion mutant and a -tubulin mutant (PRIGOZHINA et al. 2001). Another Kar3-related protein in fission yeast is encoded by klp2⁺ (TROXELL et al. 2001). Unlike pkl1, the klp2 mutant had very little synergy with the gtb1 mutation in vegetative growth. This finding, consistent with the previous result (TROXELL et al. 2001), demonstrated that pkl1⁺ and klp2⁺ genes have different roles in fission yeast. In the sexual life cycle, however, there was a severe defective phenotype in the klp2 gtb1 double mutant (Y. TANGE and O. NIWA, unpublished results).

Fission yeast contains two kinesin-like proteins, encoded by the klp5⁺ and klp6⁺ genes, which belong to the Kin I family (WEST et al. 2001). This family of proteins has microtubule-destabilizing activity (DESAI et al. 1999). Klp5 and Klp6 share overlapping functions in both mitosis and meiosis (WEST et al. 2001, 2002; GARCIA et al. 2002a,b). The klp5/6 mutants produce aberrant asci and abnormally elongated cytoplasmic microtubules that resemble those observed in the gtb1 mutants in the present study (WEST et al. 2001; GARCIA et al. 2002a). Hence, we examined whether there were any genetic interactions between the gtb1⁺ and klp5⁺/klp6⁺ genes. The gtb1-22 and gtb1-29 mutants had very little synergy with either of the null mutants of the klp5/6 genes, but combining the gtb1-93 mutation with the klp5 gene was lethal at any temperature examined (33°, 30°, and 26°). Similarly, cells carrying gtb1-93 and klp6 did not form colonies at 33°. These findings indicated an intimate functional relationship between -tubulin and the kinesin-family protein. Both Klp5/Klp6 and -tubulin might thus be required for proper microtubule dynamics in fission yeast cells. The Kin I family of proteins share essential mitotic functions with Dis1 and Alp14/Myc1 kinetochore-binding proteins (NAKASEKO et al. 2001; GARCIA et al. 2002b). Consistently, the gtb1-93 dis1 double mutant was extremely sick, producing very tiny colonies that were barely viable at 33° (data not shown). As anticipated, when a plasmid carrying the dis1⁺ gene was introduced into the double mutant, the synergistic effect completely disappeared.

We examined whether the mutations of kinesin-related genes had a synergistic effect on alp4 and alp6 mutations. The temperature sensitivity of alp4-1891 as well as of alp6-719 was greatly enhanced in the pkl1 background, while the other null mutations, klp2, klp5, and klp6, did not have notable synergistic effects.

The 3-D structures of polymerized - and ß-tubulin have been determined (NOGALES et al. 1998). Both tubulins have basically identical structures in which a core of two ß-sheets is surrounded by 12 helices. We used the structure of -tubulin as a template for modeling the 3-D structure of -tubulin. The predicted structure of -tubulin in the form of an /-tubulin heterodimer is shown in Figure 8, together with mutated amino acid residues in the -tubulin mutants. Figure 8 illustrates that all of the mutations occur in loops between the ß-strand and the helix, which are located on the predicted surface of the -tubulin protein. This is consistent with the interaction of the mutated regions in -tubulin with other cellular factors. Of particular interest were positions 301 and 302, at which the gtb1-22, gtb1-85, and gtb1-93 mutation sites are mapped. These -tubulin mutants suppressed alp4-1891, but not other alp4 mutants, and two cs mutants among the gtb1 mutants were partially suppressed by the alp4-1891 mutation. The allele-specific suppression as well as the mutual suppression suggests that Gtb1 and Alp4 interact directly via the protein regions containing altered amino acids in the mutants. The results of the gel-filtration analysis (Figure 3) are consistent with mutual suppression via direct protein interaction.

View larger version (53K): In this window In a new window Download PPT slide   FIGURE 8.— A 3-D structure model of an /-tubulin heterodimer. The positions of the altered amino acid residues in -tubulin mutants isolated in the present study are indicated.

 
Another notable feature of the suppressor -tubulin mutants is that, although the ts alp4 alleles were suppressed, they produced microtubules with altered dynamics so that they were more resistant to the microtubule-destabilizing drug, TBZ, or their defective phenotype was suppressed by TBZ-sensitive -tubulin and partially by TBZ. The effect of these microtubule-destabilizing agents was more pronounced in sexual development in the mutants. In addition, one of the gtb1 mutants was lethal when cells lacked one of the microtubule-depolymerizing enzymes, encoded by the klp5⁺ and klp6⁺ genes, suggesting that microtubules formed under this condition are too stable to support cell viability. Thus, an "inappropriately stabilized microtubule" might be a common characteristic of gtb1 mutants isolated as suppressors of the alp4 mutants. Such "stabilized microtubules" in -tubulin mutants have also been reported in other organisms, but are not a general property of all -tubulin mutants (HENDRICKSON et al. 2001; JUNG et al. 2001). Our suppressor screenings were not exhaustive and might be biased, particularly because in the second screening we selected only those with a sporulation-defective phenotype. Therefore, it is possible that there are other types of suppressor gtb1 mutations. Nevertheless, it seems likely that the suppression of tsalp4 is closely related to the microtubule stability phenotype. These arguments suggest that any gtb1 mutant could suppress the ts alp4 mutation if the gtb1 mutant produces microtubules with adequately altered stability for the alp4 mutation. It is not clear how this idea can be reconciled with the notion of allele-specific suppression through the postulated direct interaction of -tubulin and Alp4 protein as discussed above, although these ideas are not mutually exclusive.

An intriguing finding in this study was that all of the gtb1 mutations that suppressed the temperature sensitivity of the alp4-1891 mutant had highly defective sporulation, while the alp4 mutant itself did not, even at temperatures at which vegetative growth was substantially compromised. Even more striking was that the alp4-1891 mutation was able to rescue, albeit weakly, the cs growth defect of gtb1-93 and gtb1-85 mutants in the vegetative growth phase, yet it enhanced the sporulation defect of the same gtb1 mutants in the sexual phase. These and other results strongly suggest that the functional requirements of the -tubulin complex are different in the vegetative and sexual phases. Because the sporulation defect was effectively rescued by either TBZ or a TBZ-sensitive form of -tubulin, stabilized forms of microtubules might be deleterious to sporulation. This is consistent with the fact that reduced expression of the Kin I family of kinesin-like proteins gave rise to the sporulation defect (WEST et al. 2001), which was very similar to that observed in the gtb1 mutants. In the normal sporulation process, a characteristic modification of the SPB occurs during meiosis II, which is required for forespore membrane deposition and subsequent spore wall formation (HIRATA and SHIMODA 1994; IKEMOTO et al. 2000). The fusion of two SPBs and nuclear membranes, which occurs during conjugation prior to meiosis, is also required for normal sporulation and meiosis (TANGE et al. 1998). We demonstrated that at least one of the gtb1 mutants was highly defective in meiosis. This defect is likely to be closely related to the impaired sporulation. However, detailed cytologic analyses are needed to determine the stage of meiosis and/or sporulation that is impaired in the -tubulin mutants and how such defects are related to the postulated altered microtubule stability. Such analyses might also elucidate further functions of -tubulin, particularly in the sexual development of fission yeast.

We thank Aki Minoda (Cancer Research UK) for modeling of the protein 3-D structure and Jan Paluh for discussion. We also thank Hirohisa Masuda (Kansai Advanced Research Center) and Kathy Gould (Vanderbilt University) for supplying the yeast strains, and A. Kurabayashi (Kazusa DNA Research Institute) for help with the nucleotide sequencing. Special thanks are due to Manabu Nakayama (Kazusa DNA Research Institute) for his kind assistance in gel-filtration chromatography. This study was supported by the Kazusa DNA Research Institute Foundation and by Cancer Research UK.

ALFA, C., P. FANTES, J. HYAMS, M. MCLEOD and E. WARBRICK, 1993 Experiments With Fission Yeast: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

BAHLER, J., J. Q. WU, M. S. LONGTINE, N. G. SHAH, A. MCKENZIE, III et al., 1998 Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast 14: 943–951.[CrossRef][Medline]

CHIKASHIGE, Y., D.-Q. DING, Y. IMAI, M. YAMAMOTO, T. HARAGUCHI et al., 1997 Meiotic nuclear reorganization: switching the position of centromeres and telomeres in the fission yeast Schizosaccharomyces pombe. EMBO J. 16: 193–202.[CrossRef][Medline]

DESAI, A., S. VERMA, T. J. MITCHISON and C. E. WALCZAK, 1999 Kin I kinesins are microtubule-destabilizing enzymes. Cell 96: 69–78.[CrossRef][Medline]

DING, D. Q., Y. CHIKASHIGE, T. HARAGUCHI and Y. HIRAOKA, 1998 Oscillatory nuclear movement in fission yeast meiotic prophase is driven by astral microtubules, as revealed by continuous observation of chromosomes and microtubules in living cells. J. Cell Sci. 111: 701–712.[Abstract]

ERHARDT, M., V. STOPPIN-MELLET, S. CAMPAGNE, J. CANADAY, J. MUTTERER et al., 2002 The plant Spc98p homologue colocalizes with -tubulin at microtubule nucleation sites and is required for microtubule nucleation. J. Cell Sci. 115: 2423–2431.[Abstract/Free Full Text]

FUJITA, A., L. VARDY, M. A. GARCIA and T. TODA, 2002 A fourth component of the fission yeast -tubulin complex, Alp16, is required for cytoplasmic microtubule integrity and becomes indispensable when g-tubulin function is compromised. Mol. Biol. Cell 13: 2360–2373.[Abstract/Free Full Text]

GARCIA, M. A., N. KOONRUGSA and T. TODA, 2002a Two kinesin-like Kin I family proteins in fission yeast regulate the establishment of metaphase and the onset of anaphase A. Curr. Biol. 12: 610–621.[CrossRef][Medline]

GARCIA, M. A., N. KOONRUGSA and T. TODA, 2002b Spindle-kinetochore attachment requires the combined action of Kin I-like Klp5/6 and Alp14/Dis1-MAPs in fission yeast. EMBO J. 21: 6015–6024.[CrossRef][Medline]

GOTO, B., K. OKAZAKI and O. NIWA, 2001 Cytoplasmic microtubular system implicated in de novo formation of a Rabl-like orientation of chromosomes I fission yeast. J. Cell Sci. 114: 2427–2435.[Abstract/Free Full Text]

GUNAWARDANE, R. N., O. C. MARTIN, K. CAO, L. ZHANG, K. DEJ et al., 2000 Characterization and reconstitution of Drosophila -tubulin ring complex subunits. J. Cell Biol. 151: 1513–1523.[Abstract/Free Full Text]

HAGAN, I., 1998 The fission yeast microtubule cytoskeleton. J. Cell Sci. 111: 1603–1612.[Abstract]

HAGAN, I., and M. YANAGIDA, 1995 The product of the spindle formation gene sad1⁺ associates with the fission yeast spindle pole body and essential for viability. J. Cell Biol. 129: 1033–1047.[Abstract/Free Full Text]

HANNAK, E., K. OEGEMA, M. KIRKHAM, P. GONCZY, B. HABERMANN et al., 2002 The kinetically dominant assembly pathway for centrosomal asters in Caenorhabditis elegans is -tubulin dependent. J. Cell Biol. 157: 591–602.[Abstract/Free Full Text]

HENDRICKSON, T. W., J. YAO, S. BHADURY, A. H. CORBETT and H. C. JOSHI, 2001 Conditional mutations in -tubulin reveal its involvement in chromosome segregation and cytokinesis. Mol. Biol. Cell 12: 2469–2481.[Abstract/Free Full Text]

HIRATA, A., and C. SHIMODA, 1994 Structural modification of spindle pole bodies during meiosis II is essential for the normal formation of ascospores in Schizosaccharomyces pombe: ultrastructural analysis of spo mutants. Yeast 10: 173–183.[CrossRef][Medline]

HORIO, T., S. UZAWA, M. K. JUNG, B. R. OAKLEY, K. TANAKA et al., 1991 The fission yeast -tubulin is essential for mitosis and is localized at microtubule organizing centers. J. Cell Sci. 99: 693–700.[Abstract]

IKEMOTO, S., T. NAKAMURA, M. KUBO and C. SHIMODA, 2000 S. pombe sporulation-specific coiled-coil protein Spo15p is localized to the spindle pole body and essential for its modification. J. Cell Sci. 113: 545–554.[Abstract]

JOB, D., O. VALIRON and B. OAKLEY, 2003 Microtubule nucleation. Curr. Opin. Cell Biol. 15: 111–117.[CrossRef][Medline]

JUNG, M. K., N. PRIGOZHINA, C. E. OAKLEY, E. NOGALES and B. R. OAKLEY, 2001 Alanine-scanning mutagenesis of Aspergillus -tubulin yields diverse and novel phenotypes. Mol. Biol. Cell 12: 2119–2136.[Abstract/Free Full Text]

KNOP, M., and E. SCHIEBEL, 1997 Spc98p and Spc97p of the yeast gamma-tubulin complex mediate binding to the spindle pole body via their interaction with Spc110p. EMBO J. 16: 6985–6995.[CrossRef][Medline]

KNOP, M., and E. SCHIEBEL, 1998 Receptors determine the cellular localization of a gamma-tubulin complex and thereby the site of microtubule formation. EMBO J. 17: 3952–3967.[CrossRef][Medline]

KNOP, M., G. PEREIRA, S. GEISSLER, K. GREIN and E. SCHIEBEL, 1997 The spindle pole body component Spc97p interacts with the -tubulin of Saccharomyces cerevisiae and functions in microtubule organization and spindle pole body duplication. EMBO J. 16: 1550–1564.[CrossRef][Medline]

MORENO, S., A. KLAR and P. NURSE, 1991 Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 194: 795–823.[Medline]

MORITZ, M., M. B. BRAUNFELD, J. W. SEDAT, B. ALBERTS and D. A. AGARD, 1995 Microtubule nucleation by gamma-tubulin-containing rings in the centrosome. Nature 378: 638–640.[CrossRef][Medline]

MORITZ, M., M. B. BRAUNFELD, V. GUENEBAUT, J. HEUSER and D. A. AGARD, 2000 Structure of the -tubulin ring complex: a template for microtubule nucleation. Nat. Cell Biol. 2: 365–370.[CrossRef][Medline]

MURPHY, S. M., L. URBANI and T. STEARNS, 1998 The mammalian gamma-tubulin complex contains homologues of the yeast spindle pole body components spc97p and spc98p. J. Cell Biol. 141: 663–674.[Abstract/Free Full Text]

MURPHY, S. M., A. M. PREBLE, U. K. PATEL, K. L. O'CONNELL, D. P. DIAS et al., 2001 GCP5 and GCP6: two new members of the human -tubulin complex. Mol. Biol. Cell 12: 3340–3352.[Abstract/Free Full Text]

NAKASEKO, Y., G. GOSHIMA, J. MORISHITA and M. YANAGIDA, 2001 M phase-specific kinetochore proteins in fission yeast: microtubule-associating Dis1 and Mtc1 display rapid separation and segregation during anaphase. Curr. Biol. 11: 537–549.[CrossRef][Medline]

NIWA, O., M. SHIMANUKI and F. MIKI, 2000 Telomere-led bouquet formation facilitates homologous chromosome pairing and restricts ectopic interaction in fission yeast meiosis. EMBO J. 19: 3831–3840.[CrossRef][Medline]

NOGALES, E., S. G. WOLF and K. H. DOWNING, 1998 Structure of the ß-tubulin dimmer by electron crystallography. Nature 391: 199–203.[CrossRef][Medline]

OAKLEY, B. R., 2000 -Tubulin. Curr. Top. Dev. Biol. 49: 27–54.[Medline]

OEGEMA, K., C. WIESE, O. C. MARTIN, R. A. MILLIGAN, A. IWAMATSU et al., 1999 Characterization of two related Drosophila gamma-tubulin complexes that differ in their ability to nucleate microtubules. J. Cell Biol. 144: 721–733.[Abstract/Free Full Text]

PALUH, J. L., E. NOGALES, B. R. OAKLEY, K. MCDONALD, A. L. PIDOUX et al., 2000 A mutation in -tubulin alters microtubule dynamics and organization and is synthetically lethal with the kinesin-like protein Pkl1p. Mol. Biol. Cell 11: 1225–1239.[Abstract/Free Full Text]

PRIGOZHINA, N. L., R. A. WALKER, C. E. OAKLEY and B. R. OAKLEY, 2001 -Tubulin and the C-terminal motor domain kinesin-like protein, KLPA, function in the establishment of spindle bipolarity in Aspergillus nidulans. Mol. Biol. Cell 12: 3161–3174.[Abstract/Free Full Text]

SCHIEBEL, E., 2000 -Tubulin complexes: binding to the centrosome, regulation and microtubule nucleation. Curr. Opin. Cell Biol. 12: 113–118.[CrossRef][Medline]

STEARNS, T., L. EVANS and M. KIRSCHNER, 1991 Gamma-tubulin is a highly conserved component of the centrosome. Cell 65: 825–836.[CrossRef][Medline]

TANGE, Y., T. HORIO, M. SHIMANUKI, D.-Q. DING, Y. HIRAOKA et al., 1998 A novel fission yeast gene, tht1⁺, is required for the fusion of nuclear envelopes during karyogamy. J. Cell Biol. 140: 247–258.[Abstract/Free Full Text]

TANGE, Y., A. HIRATA and O. NIWA, 2002 An evolutionarily conserved fission yeast protein, Ned1, implicated in normal nuclear morphology and chromosome stability, interacts with Dis3, Pim1/RCC1 and an essential nucleoporin. J. Cell Sci. 115: 4375–4385.[Abstract/Free Full Text]

TOMLIN, G. C., J. L. MORRELL and K. L. GOULD, 2002 The spindle pole body protein Cdc11p links Sid4p to the fission yeast septation initiation network. Mol. Biol. Cell 13: 1203–1214.[Abstract/Free Full Text]

TROXELL, C. L., M. A. SWEEZY, R. R. WEST, K. D. REED, B. D. CARSON et al., 2001 pkl1⁺ and klp2⁺: two kinesins of the Kar3 subfamily in fission yeast perform different functions in both mitosis and meiosis. Mol. Biol. Cell 12: 3476–3488.[Abstract/Free Full Text]

UEMURA, T., and M. YANAGIDA, 1984 Isolation of type I and II DNA topoisomerase mutants from fission yeast: single and double mutants show different phenotypes in cell growth and chromatin organization. EMBO J. 3: 1737–1744.[Medline]

VARDY, L., and T. TODA, 2000 The fission yeast -tubulin complex is required in G1 phase and is a component of the spindle assembly checkpoint. EMBO J. 19: 6098–6111.[CrossRef][Medline]

VOGEL, J., and M. SNYDER, 2000 The carboxy terminus of Tub4p is required for -tubulin function in budding yeast. J. Cell Sci. 113: 3871–3882.[Abstract]

WEST, R. R., T. MALMSTROM, C. L. TROXELL and J. R. MCINTOSH, 2001 Two related kinesins, klp5⁺ and klp6⁺, foster microtubule disassembly and are required for meiosis in fission yeast. Mol. Biol. Cell 12: 3919–3932.[Abstract/Free Full Text]

WEST, R. R., T. MALMSTROM and J. R. MCINTOSH, 2002 Kinesins klp5⁺ and klp6⁺ are required for normal chromosome movement in mitosis. J. Cell Sci. 115: 931–940.[Abstract/Free Full Text]

WOODS, A., T. SHERWIN, R. SASSE, T. H. MACRAE, A. J. BAINES et al., 1989 Definition of individual components within the cytoskeleton of Trypanosoma brucei by a library of monoclonal antibodies. J. Cell Sci. 93: 491–500.[Abstract/Free Full Text]

ZHENG, Y., M. L. WONG, B. ALBERTS and T. MITCHISON, 1995 Nucleation of microtubule assembly by a gamma-tubulin-containing ring complex. Nature 378: 578–583.[CrossRef][Medline]

This article has been cited by other articles:

				Y. Tange and O. Niwa Novel mad2 Alleles Isolated in a Schizosaccharomyces pombe {gamma}-Tubulin Mutant Are Defective in Metaphase Arrest Activity, but Remain Functional for Chromosome Stability in Unperturbed Mitosis Genetics, April 1, 2007; 175(4): 1571 - 1584. [Abstract] [Full Text] [PDF]

				H. Masuda, T. Toda, R. Miyamoto, T. Haraguchi, and Y. Hiraoka Modulation of Alp4 function in Schizosaccharomyces pombe induces novel phenotypes that imply distinct functions for nuclear and cytoplasmic {gamma}-tubulin complexes Genes Cells, April 1, 2006; 11(4): 319 - 336. [Abstract] [Full Text] [PDF]

				H. Masuda, R. Miyamoto, T. Haraguchi, and Y. Hiraoka The carboxy-terminus of Alp4 alters microtubule dynamics to induce oscillatory nuclear movement led by the spindle pole body in Schizosaccharomyces pombe Genes Cells, April 1, 2006; 11(4): 337 - 352. [Abstract] [Full Text] [PDF]

				S. Zimmerman and F. Chang Effects of {gamma}-Tubulin Complex Proteins on Microtubule Nucleation and Catastrophe in Fission Yeast Mol. Biol. Cell, June 1, 2005; 16(6): 2719 - 2733. [Abstract] [Full Text] [PDF]