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A novel protein complex promoting formation of functional alpha - and gamma -tubulin

1998; Springer Nature; Volume: 17; Issue: 4 Linguagem: Inglês

10.1093/emboj/17.4.952

1460-2075

Silke Geissler, Katja Siegers, Elmar Schiebel,

Plant nutrient uptake and metabolism

Article15 February 1998free access A novel protein complex promoting formation of functional α- and γ-tubulin Silke Geissler Silke Geissler Max-Planck Institut für Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany Search for more papers by this author Katja Siegers Katja Siegers Present address: The Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Bearsden, Glasgow, G61 1BD UK Max-Planck Institut für Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany Search for more papers by this author Elmar Schiebel Corresponding Author Elmar Schiebel Present address: The Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Bearsden, Glasgow, G61 1BD UK Search for more papers by this author Silke Geissler Silke Geissler Max-Planck Institut für Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany Search for more papers by this author Katja Siegers Katja Siegers Present address: The Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Bearsden, Glasgow, G61 1BD UK Max-Planck Institut für Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany Search for more papers by this author Elmar Schiebel Corresponding Author Elmar Schiebel Present address: The Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Bearsden, Glasgow, G61 1BD UK Search for more papers by this author Author Information Silke Geissler1, Katja Siegers2,1 and Elmar Schiebel 2 1Max-Planck Institut für Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany 2Present address: The Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Bearsden, Glasgow, G61 1BD UK ‡S.Geissler and K.Siegers contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:952-966https://doi.org/10.1093/emboj/17.4.952 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We describe the identification of GIM1/YKE2, GIM2/PAC10, GIM3, GIM4 and GIM5 in a screen for mutants that are synthetically lethal with tub4-1, encoding a mutated yeast γ-tubulin. The cytoplasmic Gim proteins encoded by these GIM genes are present in common complexes as judged by co-immunoprecipitation and gel filtration experiments. The disruption of any of these genes results in similar phenotypes: the gim null mutants are synthetically lethal with tub4-1 and super-sensitive towards the microtubule-depolymerizing drug benomyl. All except Δgim4 are cold-sensitive and their microtubules disassemble at 14°C. The Gim proteins have one function related to α-tubulin and another to Tub4p, supported by the finding that the benomyl super-sensitivity is caused by a reduced level of α-tubulin while the synthetic lethality with tub4-1 is not. In addition, GIM1/YKE2 genetically interacts with two distinct classes of genes, one of which is involved in tubulin folding and the other in microtubule nucleation. We show that the Gim proteins are important for Tub4p function and bind to overproduced Tub4p. The mammalian homologues of GIM1/YKE2 and GIM2/PAC10 rescue the synthetically lethal phenotype with tub4-1 as well as the cold-sensitivity and benomyl super-sensitivity of the yeast deletion mutants. We suggest that the Gim proteins form a protein complex that promotes formation of functional α- and γ-tubulin. Introduction Microtubules are hollow cylinders with a diameter of 25 nm. They are part of the cytoskeleton of eukaryotic cells and play an important role in numerous cellular processes, including chromosome segregation in mitosis and meiosis, organelle positioning and intracellular transport. The wall of the microtubule cylinder consists of 13 protofilaments which are strings of alternating α- and β-tubulin subunits pointing in the same direction (reviewed by Mandelkow and Mandelkow, 1993). Therefore, microtubules are polar structures with biochemically distinct ends. Microtubule formation is a complex process that includes the post-translational folding of α- and β-tubulin, their assembly into the heterodimer tubulin and finally the formation of microtubules from tubulin subunits (Solomon, 1991). All of these steps are assisted by proteins in vivo. The first post-translational step in the pathway leading to the formation of the tubulin heterodimer is the folding of α- and β-tubulin. Genetic (Ursic and Culbertson, 1991; Chen et al., 1994; Vinh and Drubin, 1994; Archer et al., 1995) and biochemical (Frydman et al., 1992; Yaffe et al., 1992; Melki et al., 1993; Sternlicht et al., 1993; Tian et al., 1996) evidence suggests that tubulins undergo facilitated folding via their interaction with cytoplasmic chaperonins. Conditional lethal mutations in TCP1, BIN2, BIN3 and ANC2, coding for subunits of cytoplasmic chaperonin (TRiC), affect microtubule as well as actin functions (Ursic and Culbertson, 1991; Chen et al., 1994; Li et al., 1994; Miklos et al., 1994; Vinh and Drubin, 1994). For example, tcp1-1 cells are cold-sensitive for growth, and accumulate multi-nucleated and anucleated cells with abnormal microtubule structures. This mutant is also sensitive towards the microtubule-depolymerizing drug benomyl (Ursic and Culbertson, 1991). Remarkably, bin3-1 cells display a normal distribution of unbudded, small-budded and large-budded cells, although the microtubules of bin3-1 cells disassemble at 14°C (Chen et al., 1994). While the cytoplasmic chaperonin is sufficient for the production of native actin in an ATP-dependent manner (Gao et al., 1992), folding of α- and β-tubulin requires the participation of additional cofactors. Such proteins, named cofactors A, B, C, D and E, have been identified by biochemical approaches (Tian et al., 1996, 1997). Potential yeast homologues of cofactors A, B, D and E are Rbl2p, Alf1p, Cin1p and Pac2p (Archer et al., 1995; Tian et al., 1996; Geiser et al., 1997). Surprisingly, ALF1, RBL2, CIN1 and PAC2 are not essential for growth of yeast cells (Hoyt et al., 1990; Stearns et al., 1990; Archer et al., 1995). However, their deletion causes sensitivity towards benomyl and they show multiple genetic interactions with genes coding for components of the microtubule system. In addition, Rbl2p interacts directly with yeast β-tubulin (Archer et al., 1995). In many cell types, microtubule assembly is initiated at centrosomes, basal bodies, spindle pole bodies (SPBs) or nucleus-associated bodies, structures for which Pickett-Heaps (1969) coined the generic term microtubule-organizing centre (MTOC). MTOCs assist the assembly of the first tubulin monomers into oligomers, a step known as microtubule nucleation (Mitchison and Kirschner, 1984). A universal component of MTOCs involved in microtubule nucleation is γ-tubulin (Oakley et al., 1990; Horio et al., 1991; Stearns et al., 1991). γ-Tubulin forms a complex with additional proteins (Stearns and Kirschner, 1994; Moudjou et al., 1996; Akashi et al., 1997), and purification of such a complex from Xenopus laevis eggs identified α-, β- and γ-tubulin and proteins with mol. wts of 195, 133, 109 and 75 kDa (Zheng et al., 1995). In the yeast Saccharomyces cerevisiae, the MTOC is known as the SPB. It is a multi-layered structure which is embedded in the nuclear envelope (Byers and Goetsch, 1975; Byers, 1981). The yeast γ-tubulin is encoded by the essential TUB4 gene (Sobel and Snyder, 1995; Marschall et al., 1996; Spang et al., 1996). Tub4p forms a 6S complex with the SPB components Spc98p and Spc97p (Geissler et al., 1996; Spang et al., 1996; Knop et al., 1997). Purification of this complex suggests that it contains only one molecule of Spc98p and Spc97p, but two or more molecules of Tub4p (Knop and Schiebel, 1997). Fractionation experiments suggest that the Tub4p complex assembles in the cytoplasm followed by its nuclear import via an essential nuclear localization sequence in Spc98p (G. Pereira et al., 1998). Finally, Spc98p and Spc97p of the Tub4p complex interact in the nucleus with the amino-terminal domain of the SPB component Spc110p (Knop and Schiebel, 1997). The binding site for the Tub4p complex at the outer plaque is still unknown. To identify genes that are involved in yeast γ-tubulin functions, we performed a genetic screen for mutants that are synthetically lethal with tub4-1. We identified SPC98 and SPC97 coding for components of the yeast γ-tubulin complex and five additional genes, which we named GIM1–GIM5. Their products are not associated with the SPB, instead they form cytoplasmic multi-protein complexes which promote formation of functional α-tubulin and Tub4p. Most interestingly, the Gim proteins are phylogenetically conserved proteins, and mouse and human homologues function in yeast, indicating that the mammalian proteins fulfil a very similar role. Results Identification of GIM1/YKE2, GIM2/PAC10, GIM3, GIM4 and GIM5 in a screen for mutants that are synthetically lethal with a mutated yeast γ-tubulin In the yeast S.cerevisiae, the γ-tubulin Tub4p forms a complex with the SPB components Spc98p and Spc97p. Physical interaction between Tub4p, Spc98p and Spc97p is reflected by multiple genetic interactions, including synthetic lethality (Geissler et al., 1996; Knop et al., 1997). The latter is an indication of a functional relationship of two gene products. To identify further components that functionally interact with TUB4, we performed a genetic screen for mutants that are synthetically lethal with tub4-1, yielding 12 mutants with this phenotype. Subsequent analysis showed that two of these mutants were defective in SPC98 and two in SPC97. The other eight belong to five complementation groups which we named GIM1–GIM5 (genes involved in microtubule biogenesis). Plasmids containing GIM1–GIM5 were isolated by transforming the mutants with a yeast genomic library. Subcloning of DNA fragments and complementation analysis showed that GIM1 is identical to YKE2 (Shang et al., 1994), coding for a protein of 114 amino acids (Table I). GIM2 had already been identified as PAC10, a gene that becomes essential in the absence of the CIN8-encoded kinesin motor (Geiser et al., 1997). GIM3 corresponds to the open reading frame (ORF) YNL153c, encoding a protein of 129 amino acids. The DNA fragment complementing gim4 cells contained three overlapping ORFs, one of which was interrupted by an intron. The various coding regions were cloned directly behind the ADH promoter of plasmid p415-ADH. Only the ADH promoter fusion with the intron-containing ORF YEL003w complemented gim4 cells (data not shown). Gim4p is a protein of 132 amino acids. GIM5 (ORF YML094w) also contains a small intron and encodes a protein of 163 amino acids. Table 1. Properties of the GIM genes and the corresponding deletion mutants ORF name Amino acids in encoded protein Cold-sensitivea Benomyl-sensitiveb (2.5 μg/ml) Relative volumec Suppression of benomyl super-sensitivityd GIM1/YKE2 YLR200w 114 yes yes 2.4 yes GIM2/PAC10 YGR078c 199 yes yes 2.2 yes GIM3 YNL153c 129 yes yes 2.4 yes GIM4 YEL003w 132 no yes 2.4 yes GIM5 YML094w 163 yes yes 2.3 yes a Cold sensitivity was determined as described in the legend to Figure 2B. b Benomyl sensitivity was determined as in Figure 2C. c Determination of cell volumes (compared with wild-type) is described in Materials and methods. d Suppression of benomyl super-sensitivity by overexpression of TUB1 or RBL2 was determined as in Figure 5C. The Gim proteins are phylogenetically conserved coiled-coil proteins As reported for Gim2p/Pac10p (Geiser et al., 1997), all Gim proteins have a high probability of forming coiled-coils (Lupas, 1996). Sequence comparisons have shown that Gim1p/Yke2p, Gim2p/Pac10p, Gim3p, Gim4p and Gim5p are related proteins. They also have relatives in mammals, Caenorhabditis elegans and Schizosaccharomyces pombe, revealing that they are phylogenetically conserved (Figure 1). Remarkably, Gim1p/Yke2p and Gim5p are homologous to two proteins of the archaebacterium Methanococcus jannaschii, while no relatives were found in eubacteria. We also noticed that the Gim proteins are more closely related to their homologues from other species than to each other (Figure 1). Figure 1.GIM1/YKE2, GIM2/PAC10, GIM3, GIM4 and GIM5 encode phylogenetically conserved proteins. Dendrogram of the Gim proteins and their homologous proteins from S.pombe (Sp), C.elegans (Ce), mouse (Mm), human (Hs) and M.jannaschii (Mj) generated by the PROTDIST and FITCH modules of PHYLIP (Felsenstein, 1996). The dendrogram is based on an alignment by A.Lupas (personal communication) using MACAW (Schuler et al., 1991). Accession Nos: Sp-Gim1p, Z99260; Mm-KE2, I53651; Ce-KE2, P52554; Mj-Gim1p, C64423; Sp-Yas7p, Q10143; Ce-Gim2p, Z81587; Hs-VBP-1, U56833; Ce-Gim3p, Z73102; Hs-C1, U41816; Ce-Gim5p, U00036; Hs-MYCbp, Q99471; Mj-Gim5p, H64418. Download figure Download PowerPoint Deletion of the GIM genes causes microtubule defects To understand the function of the GIM gene products, we investigated whether they are essential for growth of yeast cells. We disrupted the entire coding regions of the GIM genes in the diploid yeast strain YPH501. As shown for Δgim5 (Figure 2A), spore analysis revealed that GIM1/YKE2, GIM3, GIM4 and GIM5 are not essential for growth. In agreement with a previous report (Geiser et al., 1997), we observed that Δpac10 cells were defective in spore germination. Such a defect was not apparent for the other GIM mutants, raising the possibility that GIM2/PAC10 has an additional specialized function in spore germination. The disruption of any of the GIM genes resulted in a slow growth phenotype at 30°C (Figure 2A and B). A more detailed analysis indicated that the doubling time of Δgim1/yke2, Δgim2/pac10, Δgim3 and Δgim5 cells grown in liquid medium at 30°C was increased by a factor of 1.4 compared with the wild-type, while the doubling time of Δgim4 cells was only affected 1.1-fold. It has been reported previously that a deletion mutant of GIM1/YKE2 shows normal growth over a wide range of temperatures (Shang et al., 1994). This discrepancy with our study is explained either by strain differences or by the fact that Shang et al. (1994) did not disrupt the entire coding region of YKE2. Figure 2.Δgim5 cells are cold- and benomyl-sensitive. (A) Tetrads of a diploid GIM5/Δgim5::kanMX4 strain were analysed for growth at 30°C on YPD plates. All spores from a tetrad germinated and formed colonies. These colonies were tested for growth on YPD plates containing the kanamycin derivative G418. Only cells which carry the kanMX4 gene grow on these plates. Two of the spores of each tetrad, which were G418 resistant (Δgim5::kanMX4), grew more slowly in comparison with the G418-sensitive GIM5 cells. (B) The growth defect of Δgim5::kanMX4 cells on YPD plates is more pronounced at lower temperatures. Serial dilutions of Δgim5 and GIM5 cells were grown on YPD plates. One plate was incubated for 3 days at 30°C. The plates at 23 and 14°C were incubated until the sizes of the GIM5 colonies were approximately the same as on the 30°C plate. (C) Δgim5::kanMX4 cells are super-sensitive towards the microtubule-depolymerizing drug benomyl. Serial dilutions of Δgim5::kanMX4 and GIM5 cells were grown on YPD plates containing 2.5 μg/ml benomyl at 30°C. Download figure Download PowerPoint Common phenotypes of mutants affecting the microtubule cytoskeleton are a cold-sensitive growth defect and an increased sensitivity towards the microtubule-depolymerizing drug benomyl (Neff et al., 1983; Huffaker et al., 1988). Therefore, we investigated whether the growth defects of the haploid gim null strains were intensified at lower temperatures. The gim null strains showed an enhanced growth defect at reduced temperatures in comparison with the wild-type (Figure 2B; compare 30°C with 23°C and 14°C plates). An exception was Δgim4 cells, which did not show an enhanced growth defect at lower temperatures (Table I). In contrast to the wild-type, all gim null strains were super-sensitive to 2.5 μg/ml benomyl (Figure 2C). Such a strong benomyl sensitivity has only been observed for mutants defective in microtubule biogenesis (Stearns et al., 1990; Chen et al., 1994; Archer et al., 1995; Tian et al., 1997). We investigated whether the Gim proteins function in parallel pathways. In this case double mutants should have more pronounced defects compared with the single mutants. However, the double deletion mutants of Δgim1/yke2 together with any one of Δgim2/pac10, Δgim3, Δgim4 or Δgim5 were viable and as benomyl-sensitive as the single mutants. We also tested whether overexpression of any of the GIM genes rescued the synthetically lethal phenotype of gim tub4-1 cells. This was the case for GIM1 which weakly suppressed the defect of gim4 tub4-1 cells (data not shown). The strong benomyl super-sensitivity of the haploid gim null strains suggests that the gene products are needed for either tubulin formation or microtubule stability. To analyse these possibilities, we investigated, by indirect immunofluorescence, the microtubule arrays of Δgim1/yke2, Δgim2/pac10 and Δgim3 cells incubated at 14°C for 20 h. The phenotype is shown for Δgim1/yke2 cells as a representative, since it is very similar to that of the other mutants. Δgim cells lost most of their cytoplasmic and nuclear microtubules, leaving only a small spot near the nucleus, most likely at the SPB (Figure 3A). This observation suggests that microtubule attachment to the SPB via the Tub4p complex is still taking place in Δgim cells. Instead microtubule stability seems to be impaired. Furthermore, cells with a large bud (Figure 3A, arrow) had only one 4′,6'-diamidino-2-phenylindole (DAPI) staining region in one of the two cell bodies, a phenotype that is consistent with the observed defect in microtubule organization. Despite these defects, gim null cells incubated for 20 h at 14°C were still as viable as wild-type cells when then grown at 30°C (data not shown). Based on the distributions of the DNA content (Figure 3B) and of the cell morphologies (Figure 3A), Δgim cells did not arrest at a defined stage of the cell cycle. This observation was surprising, since many mutants with a defect in spindle formation arrest in mitosis due to a mitotic check point (Hoyt et al., 1990; Li and Murray, 1991). The non-arrest phenotype could point to a role for the Gim proteins in mitotic check point control. However, all GIM deletion mutants arrested in the cell cycle like wild-type cells in response to the microtubule-depolymerizing drug nocodazole, while the mitotic check point control mutant bub2 did not (Hoyt et al., 1990; Li and Murray, 1991; Geiser et al., 1997; data not shown). This led us to conclude that the GIM gene products are not part of a mitotic check point. Taken together, the strong benomyl sensitivity and the microtubule defects of the gim null mutants at reduced temperature are most consistent with a role for the Gim proteins in tubulin biogenesis. Figure 3.Microtubule defects of Δgim1/yke2 cells. (A) Microtubule staining of the gim1/yke2 null mutant. Cells of Δgim1/yke2::kanMX4 and GIM1/YKE2 were grown in YPD medium at 30°C. The cells were diluted to 5×106 cells/ml using pre-cooled YPD medium. The cultures were then incubated at 14°C for two doubling times (20 h). Microtubules were detected by indirect immunofluorescence with anti-tubulin antibodies. DNA was stained with DAPI. Cells were also inspected by phase contrast microscopy (PC). The arrows point to a Δgim1/yke2 cell with a large bud that contains no mitotic spindle and only one DAPI-staining region. Bar: 5 μm. (B) DNA content of Δgim1/yke2 cells. Δgim1/yke2 cells were incubated in YPD at 14°C. Samples were taken at the indicated time points. The DNA content of these cells was analysed by flow cytometry. (C) Sensitivity of the indicated strains towards latrunculin-A. Relative apparent sensitivity was determined as described (Ayscough et al., 1997). (D) Sensitivity of gim null strains towards high osmolarity. Wild-type cells, Δgim1/yke2, Δgim2/pac10, Δgim3, Δgim4 and Δgim5 cells were grown for 3 days at 30°C on YPD plates containing 0, 1.4 or 1.8 M sorbitol. Download figure Download PowerPoint gim null mutants are sensitive towards the actin inhibitor latrunculin-A and are osmotically sensitive We noticed that the volume of cells of the GIM deletion mutants incubated at 14°C was ∼2.2- to 2.4-fold increased compared with the wild-type (Table I). Such a phenotype has been reported, among others, for mutants affecting the actin cytoskeleton (reviewed by Drubin, 1990). Many mutants with actin defects are sensitive towards the actin-binding drug latrunculin-A (Ayscough et al., 1997) and are osmotically sensitive (Drubin, 1990). We found that the gim null strains were more sensitive towards latrunculin-A than the wild-type, which indicates an influence of the Gim proteins on the actin cytoskeleton (Figure 3C). This sensitivity was stronger compared with that of the cold-sensitive mutant tcp1-1, but was, however, clearly weaker compared with the actin mutant act1-2. TCP1 encodes a component of TRiC which is required for actin folding (Ursic and Culbertson, 1991). Furthermore, similarly to actin mutants, Δgim1/yke2, Δgim2/pac10 and Δgim5 cells did not grow on plates containing 1.4 M sorbitol. In contrast, Δgim3 cells grew slowly, while Δgim4 grew as wild-type cells (Figure 3D). At 1.8 M sorbitol, Δgim3 cells failed to grow and a weak growth defect of Δgim4 cells was observed (Figure 3D). We sought additional evidence for a role for the Gim proteins in actin function. However, deletion of GIM1/YKE2 was synthetically lethal with neither act1-2 nor act1-3. In addition, Δgim1/yke2, Δgim2/pac10, Δgim3 and Δgim4 cells did not show an obvious defect in actin organization as judged by indirect immunofluorescence using anti-actin antibodies (data not shown), suggesting that the gim null mutants have only subtle actin defects. In conclusion, the GIM genes may have multiple functions, since their deletion causes microtubule defects as well as an increased sensitivity towards the actin-specific drug latrunculin-A and towards high osmolarity. The GIM genes interact genetically with genes involved in microtubule biogenesis To gain further insight into the role of the GIM genes, we investigated their genetic interactions with other genes involved in microtubule function. We first addressed the question of whether total loss of GIM gene function is synthetically lethal with mutants of the Tub4p complex. Using the approach described in Figure 4A, we could show that this is indeed the case for tub4-1. This result was confirmed by tetrad analysis using a GIM4/Δgim4::HIS3MX6 TUB4/tub4-1 strain. No viable temperature-sensitive HIS3 spores were obtained, confirming that Δgim4::HIS3MX6 combined with tub4-1 is lethal. Microscopic inspection of the non-colony-forming spores identified several hundred cells. Therefore, Δgim4 tub4-1 cells died only after multiple duplications. A similar result was obtained with GIM3 (data not shown). Figure 4.Genetic interactions of GIM1/YKE2 with TUB4, SPC98 and SPC97. (A) GIM1/YKE2 of strain ESM183 (Δtub4::HIS3 pRS316-TUB4) was disrupted using the kanMX4 gene. The resulting strain SGY119 was unable to grow on 5-FOA plates which selects against the URA3-based plasmid (sector 1), confirming that TUB4 is an essential gene (Spang et al., 1996). However, strain SGY119 was able to grow on 5-FOA when it contained a LEU2-based plasmid carrying TUB4 (sector 3), but not when it contained tub4-1 (sector 2), indicating that Δgim1/yke2 is synthetically lethal with tub4-1. Controls established that tub4-1 (sector 4) and Δgim1/yke2 cells (sector 5) grow on 5-FOA at 30°C. (B) In a similar manner as in (A), using strain ESM243 (Δspc98::HIS3 pRS316-SPC98), we tested for genetic interactions of GIM1/YKE2 with SPC98. Strain SGY120 (ESM243 Δgim1/yke2) was unable to grow on 5-FOA (sector 1), as SPC98 is essential for growth (Geissler et al., 1996). As expected, growth was observed in the presence of a LEU2-based plasmid containing SPC98 (sector 4). In contrast, spc98-2 (sector 2) and spc98-1 (sector 3) did not or hardly support growth, indicating synthetic lethality and synthetic toxicity of Δgim1/yke2 spc98-2 (sector 2) and Δgim1/yke2 spc98-1 (sector 3). We established that spc98-2 (sector 5) and spc98-1 (sector 6) cells grow well on 5-FOA at 30°C. (C) GIM1/YKE2 of strain YMK10 (Δspc97::HIS3 pRS316-SPC97) was disrupted. The resulting strain SGY121 was unable to grow on 5-FOA (sector 1), unless the plasmid pRS315-SPC97 was present (sector 4). Since spc97-20 Δgim1/yke2 cells barely grow at 23°C (sector 2), while cells of spc97-20 (sector 5) grow well, we conclude that Δgim1/yke2 is synthetically toxic with spc97-20. In contrast, spc97-14 combined with Δgim1/yke2 (sector 3) grow as spc97-14 cells at 23 or 30°C (sector 6). Download figure Download PowerPoint In a similar manner, we tested whether GIM1/YKE2, GIM2/PAC10 and GIM4 showed interactions with SPC98 and SPC97, coding for components of the yeast γ-tubulin complex. Δgim1/yke2 was synthetically lethal with spc98-2 and synthetically toxic when combined with spc98-1 (Figure 4B). Allele-specific genetic interactions of GIM1/YKE2 were also observed with SPC97: deletion of GIM1/YKE2 was synthetically toxic in spc97-20 cells, while it did not affect growth of the spc97-14 mutant (Figure 4C). In contrast to Δgim1/yke2, Δgim2/pac10 or Δgim4 combined with spc98-1, spc98-2 or spc97-20 hardly affected growth (Table II). It is important to note that the spc98 and spc97 alleles have distinct phenotypes and are suppressed differently by TUB4 (Geissler et al., 1996; Knop et al., 1997). In particular, spc97-20 has an SPB duplication defect, while spc97-14 fails to form a mitotic spindle (Knop et al., 1997). Table 2. Genetic interactions of GIM1/YKE2, GIM2/PAC10, GIM3, GIM4 and GIM5 with genes involved in microtubule assembly tub4-1 spc98-1/2 spc97-20 tub1-4 tub2-403/-405 bin2-1 bin3-1 Δrbl2 gim1/yke2 SLa SL SL SL SL SL SL SL Δgim2/pac10 SL +b + n.d.c n.d. n.d. n.d. SL Δgim3 SL n.d. n.d. n.d. n.d. n.d. n.d. SL Δgim4 SL + + n.d. + SLd SL n.d. Δgim5 SL n.d. n.d. n.d. n.d. n.d. n.d. n.d. a SL, synthetic lethality. This was determined by plasmid shuffle unless otherwise indicated. For plasmid shuffle experiments, the mutant strains (see Table III) were transformed with a plasmid-encoded GIM gene (URA plasmid). The corresponding GIM gene on the chromosome was disrupted by use of the kanMX4 marker as described. The resulting strains were transformed with plasmids containing the GIM gene or the wild-type allele of the respective mutated gene and a control vector and were tested for growth on 5-FOA selecting against the URA3 plasmid. Non-growth of the transformants with the control plasmid on 5-FOA indicates synthetic lethality. However, the transformants with the additional GIM gene or the wild-type allele of the mutated gene grew. b +, not synthetically lethal. c n.d., not determined. d Tested for synthetic lethality by tetrad analysis. The bin2-1 mutant and the gim4 null mutant were crossed. The resulting strain was sporulated and the spores were analysed. Finally, GIM1/YKE2, GIM2/PAC10 and GIM4 were in part tested for their genetic interactions with TUB1, TUB2, BIN2, BIN3 and RBL2. TUB1 and TUB2 code for α- and β-tubulin in yeast (Neff et al., 1983; Schatz et al., 1986a), while RBL2 encodes a β-tubulin-binding protein, the homologue of mammalian cofactor A (Archer et al., 1995; Tian et al., 1996). Bin2p and Bin3p are subunits of TRiC (Chen et al., 1994). Δgim1 showed synthetic lethality with tub1-4, tub2-403, tub2-405, bin2-1, bin3-1 and Δrbl2 (Table II). In contrast, Δgim4 was only synthetically lethal in combination with bin2-1, bin3-1 and Δrbl2, but not with tub2-403 or tub2-405. In summary, the GIM genes show a broad range of genetic interactions with genes involved in tubulin biogenesis and microtubule nucleation. Deletion of the GIM genes results in reduced levels of α-tubulin, which explains the benomyl super-sensitivity, but not the synthetic lethality with tub4-1 A defect of the gim null mutants in tubulin biogenesis may result in reduced levels of α-, β- or γ-tubulin. The amount of these proteins was determined by immunoblotting using specific antibodies. While the α-tubulin content was about half to a third compared with wild-type, β-tubulin and Tub4p levels were approximately the same (Figure 5A and B). It is noteworthy that deletion of GIM4 affected α-tubulin less severely than deletion of the other GIM genes. We also noticed that the reduction of α-tubulin levels was independent of the incubation temperature of the cultures (data not shown). Figure 5.Haploid gim null mutants have reduced α-tubulin levels, resulting in benomyl super-sensitivity. (A) Wild-type cells (lane 1) and cells of Δgim1/yke2 (lane 2), Δgim2/pac10 (lane 3), Δgim3 (lane 4), Δgim4 (lane 5) and Δgim5 (lane 6) were grown in YPD medium at 30°C to mid-log phase. Equal amounts of protein (50 μg) from the six strains were separated by SDS–PAGE, followed by immunodetection of α-tubulin, β-tubulin and Tub4p, using specific antibodies. An identical res