The yeast microtubule organizing centre (MTOC), known as the spindle pole body (SPB), organizes the nuclear and cytoplasmic microtubules which are functionally and spatially distinct. Microtubule organization requires the yeast γ‐tubulin complex (Tub4p complex) which binds to the nuclear side of the SPB at the N‐terminal domain of Spc110p. Here, we describe the identification of the essential SPB component Spc72p whose N‐terminal domain interacts with the Tub4p complex on the cytoplasmic side of the SPB. We further report that this Tub4p complex‐binding domain of Spc72p is essential and that temperature‐sensitive alleles of SPC72 or overexpression of a binding domain‐deleted variant of SPC72 (ΔN‐SPC72) impair cytoplasmic microtubule formation. Consequently, polynucleated and anucleated cells accumulated in these cultures. In contrast, overexpression of the entire SPC72 results in more cytoplasmic microtubules compared with wild‐type. Finally, exchange of the Tub4p complex‐binding domains of Spc110p and Spc72p established that the Spc110p domain, when attached to ΔN‐Spc72p, was functional at the cytoplasmic site of the SPB, while the corresponding domain of Spc72p fused to ΔN‐Spc110p led to a dominant‐negative effect. These results suggest that different components of MTOCs act as receptors for γ‐tubulin complexes and that they are essential for the function of MTOCs.
Microtubules are part of the cytoskeleton of eukaryotic cells with essential functions in chromosome segregation in mitosis and meiosis, cell polarity, organelle positioning, secretion and cellular movement (reviewed in Huffaker et al., 1987; Hyman and Karsenti, 1996). Microtubules are hollow cylinders, and the wall of the cylinder consists of tubulin, a heterodimer of α‐ and β‐tubulin (reviewed by Mandelkow and Mandelkow, 1993). Microtubules form by the self‐assembly of tubulin, a process that starts in vivo at so‐called microtubule organizing centres (MTOCs) (reviewed in Brinkley, 1985; Kellogg et al., 1994; Pereira and Schiebel, 1997). The generic term MTOCs groups morphologically distinct structures with a common microtubule organization activity such as centrosomes, spindle pole bodies (SPBs) and basal bodies (Pickett‐Heaps, 1969).
The microtubule organization capability of MTOCs frequently is cell‐cycle regulated. For example, the number of microtubules at mammalian centrosomes increases 5‐fold at the onset of mitosis (Kuriyama and Borisy, 1981). Another example is the SPB from Schizosaccharomyces pombe which organizes nuclear microtubules only in mitosis (Hagan and Hyams, 1988; Masuda et al., 1992). Furthermore, one cell may have multiple MTOCs which are morphologically, spatially and functionally distinct. In this respect, plant cells are particularly interesting, since a number of different microtubule arrays exist: these include the cortical nuclear‐associated microtubules in interphase, the pre‐prophase band in G2 phase, and the mitotic spindle and the phragmoplast microtubules during mitosis (reviewed in Smirnova and Bajer, 1992; Marc, 1997). There is increasing evidence that the nuclear surface of higher plants serves as a MTOC during interphase and during telophase (Stoppin et al., 1994). Other MTOCs may exist, including the phragmoplast (Cleary et al., 1992). In the fission yeast S.pombe, interphase microtubules are organized mainly by a not very well defined MTOC that is localized at the cell equator (Hagan and Hyams, 1988). However, at the onset of mitosis, microtubules are organized from the two SPBs into a typical spindle (Ding et al., 1997).
The SPB of Saccharomyces cerevisiae offers an example of one MTOC that can organize functionally and spatially distinct classes of microtubules (Byers and Goetsch, 1975). The S.cerevisiae SPB is embedded in the nuclear envelope during the entire cell cycle. Substructures named the outer, central and inner plaques have been described by electron microscopy (Byers and Goetsch, 1975; Byers, 1981) (Figure 8). The outer and inner plaques organize the cytoplasmic and nuclear microtubules, respectively. The cytoplasmic microtubules have functions in nuclear positioning and nuclear movement (Palmer et al., 1992; Sullivan and Huffaker, 1992), while the nuclear microtubules are involved in spindle formation and chromosome segregation in mitosis and meiosis (Jacobs et al., 1988).
A universal component of MTOCs is γ‐tubulin which was first discovered in the fungus Aspergillus nidulans (Weil et al., 1986; Oakley and Oakley, 1989). Since then, the function of γ‐tubulin in microtubule formation has been established by antibody microinjection experiments (Joshi et al., 1992), genetic studies (Oakley et al., 1990; Horio et al., 1991; Sobel and Snyder, 1995; Marschall et al., 1996; Spang et al., 1996a) and biochemical approaches (Li and Joshi, 1995; Zheng et al., 1995). Biochemical studies using extracts of frog eggs (Zheng et al., 1995), mammalian cells (Stearns and Kirschner, 1994; Moudjou et al., 1996), S.cerevisiae (Knop and Schiebel, 1997) and A.nidulans (Akashi et al., 1997) cells revealed that γ‐tubulin is part of larger complexes. Purification of such a 25S complex from Xenopus laevis eggs identified α‐, β‐ and γ‐tubulin and at least four additional proteins (Zheng et al., 1995). The S.cerevisiae γ‐tubulin, Tub4p, forms a stable complex with two other proteins, Spc98p and Spc97p (Geissler et al., 1996; Knop and Schiebel, 1997), and this Tub4p complex is localized at the outer and inner plaques of the SPB (Rout and Kilmartin, 1990; Knop et al., 1997) (Figure 8). Conditional lethal mutants in SPC98 and SPC97 revealed a function of the encoded proteins in microtubule organization by the SPB (Geissler et al., 1996; Knop et al., 1997).
γ‐Tubulin complexes assemble in the cytoplasm of cells (Stearns and Kirschner, 1994; Moudjou et al., 1996; Pereira et al., 1998). From S.cerevisiae we know that Spc98p and Spc97p of the assembled γ‐tubulin complex (Tub4p complex) bind to the N‐terminal domain of the SPB component Spc110p (Knop and Schiebel, 1997). Analogously, other γ‐tubulin complexes may bind to MTOCs via such γ‐tubulin complex‐binding proteins (GTBPs). A GTPB may localize to only one MTOC within a cell and thereby contribute to the characteristic microtubule organization properties of an MTOC.
Spc110p is only associated with the inner plaque, while the Tub4p complex is located with the outer and inner plaques. Therefore, a protein other then Spc110p has to function as a GTBP at the outer plaque. The goal of this study was to identify the yeast GTBP at the outer plaque. Using the yeast two‐hybrid system, we identified a new, essential SPB component of the outer plaque, named Spc72p, whose N‐terminal domain interacts with Spc98p and Spc97p of the Tub4p complex. We further established that Spc72p fulfils similar functions in microtubule organization at the outer plaque as Spc110p at the inner plaque.
Cloning of SPC72 and its interaction with the Tub4p complex in the two‐hybrid system
Previously, we have shown that the N‐terminal domain of Spc110p interacts with Spc98p and Spc97p, but not with Tub4p (Knop and Schiebel, 1997) in the yeast two‐hybrid system (Fields and Song, 1989). However, an interaction with Tub4p was observed after co‐overexpression of SPC98 and SPC97. We used these criteria to search for further SPB components that interact with the Tub4p complex. Our screen resulted in a prey plasmid containing ∼270 codons of the 5′ end of the open reading frame (ORF) YAL047c. Since further experiments showed that ORF YAL047c encodes a SPB component, we renamed YAL047c as SPC72, for SPB component with a molecular weight of 72 kDa. SPC72 is located on chromosome I and it encodes a protein of 622 amino acids. The analysis of the amino acid sequence revealed that stretches of Spc72p have a high probability of forming coiled‐coil structures (Figure 1A), a structural motif that has been found in other SPB components such as Spc42p (Donaldson and Kilmartin, 1996) and Spc110p (Kilmartin et al., 1993). Spc72p does not show significant homology to any protein in the database.
Subdomains and the entire coding region of Spc72p were tested for their interactions with Tub4p, Spc98p and Spc97p using the yeast two‐hybrid system (Figure 1B). In agreement with our screening criteria, the N‐terminal domain of Spc72p (Spc72p1–271; the numbers denote amino acids) showed two‐hybrid interactions with Spc97p (Figure 1B, lane 4) and Spc98p (lane 5) but not with Tub4p (lane 3). We noticed that the strength of these interactions was dependent on whether Spc72p1ndash;271 was fused to Gal4p or lexA (compare lanes 4 and 8, and lanes 5 and 11). However, an indication that the two‐hybrid binding of Spc72p1–271 to Spc97p is specific came from the observation that the temperature‐sensitive spc97‐20 (lane 16) and spc97‐14 alleles (lane 14) showed no or a strongly reduced interaction with Spc72p1–271. In contrast to Spc72p1–271, no binding of the C‐terminal domain (lanes 6; data not shown) or the entire Spc72p (lane 7; data not shown) to Spc98p or Spc97p was observed.
Similarly to the N‐terminal domain of Spc110p (Spc110p1–176) (Knop et al., 1997; Figure 1C, lane 7), a strong interaction of Spc72p1–271 with lexA–Tub4p was only observed when SPC98 and SPC97 were co‐overexpressed simultaneously (Figure 1C, compare lanes 3 and 4 with 5), suggesting that the N‐terminal domain of Spc72p interacted indirectly with lexA–Tub4p via binding to Spc98p and Spc97p in the Tub4p complex. It is noteworthy that the first 176 amino acids of Spc72p (Spc72p1–176) were sufficient for this interaction and behaved as Spc72p1–271 in all these experiments (lane 6; data not shown). In conclusion, the two‐hybrid interactions of the subdomains of Spc72p with components of the Tub4p complex is remarkably reminiscent of Spc110p (Knop and Schiebel, 1997). In addition, the failure of Gal4–Spc72p to interact with Spc98p and Spc97p explains why the entire SPC72 was not obtained in the initial two‐hybrid screen.
Biochemical and genetic interactions of Spc72p with the Tub4p complex
We looked for biochemical evidence for an interaction of the Tub4p complex with Spc72p. A yeast strain harbouring a functional chromosomal gene fusion of SPC72 with three copies of the haemagglutinin epitope (3HA) was constructed. SPC72‐3HA cells were lysed under conditions which extracted ∼30% of SPC72p‐3HA, hardly any of Spc110p and most of Spc98p, Spc97p and Tub4p from the cells (data not shown). Spc72p‐3HA was then precipitated with anti‐HA (12CA5) antibodies. Besides Spc72p‐3HA, also Spc98p, Spc97p and Tub4p (Figure 2A), but not α‐ and β‐tubulin (data not shown), were detected in the immunoprecipitate, indicating a physical interaction between the Tub4p complex and Spc72p. Analysis of the immunoprecipitation supernatants revealed that only a small percentage of the Tub4p complex co‐precipitated with Spc72p‐3HA, while >90% of the extracted Spc72p‐3HA was precipitated. This suggests that only a minor fraction of the Tub4p complex is associated with Spc72p. We established that the co‐precipitation of Tub4p, Spc98p and Spc97p with Spc72p‐3HA was specific: no Tub4p complex was precipitated by the anti‐HA antibodies from an extract of SPC72 cells (Figure 2A, lanes 1). To confirm that Tub4p, Spc98p, Spc97p and Spc72p interact, we precipitated Spc97p‐3HA by anti‐HA antibodies. Spc98p, Tub4p and Spc72p were detected in the precipitate and this co‐precipitation was not observed from an extract containing Spc97p (Figure 2B).
We then tested whether the Tub4p complex binds to GST–Spc72p1–271 purified from Escherichia coli, as is the case for GST–Spc110p1–204 (Knop and Schiebel, 1997). Yeast extract containing Tub4p complex was incubated with recombinant GST, GST–Spc42p1–214 or GST–Spc72p1–271 bound to a glutathione resin. Binding of Tub4p, Spc98p and Spc97p to the resins was analysed by immunoblotting, because these species are minor components in total yeast cell lysate. A fraction of the Tub4p complex present in the crude lysate bound to GST–Spc72p1–271, but not to GST or GST–Spc42p1–214, indicating specific binding (Figure 2C).
Genetic evidence for an interaction of Spc72p with the Tub4p complex came from overexpression studies of SPC72. We found that moderate overexpression of SPC72 from the centromere‐based GalS promoter caused a slight growth defect (Figure 2D, compare lanes 1 and 2, galactose). This defect was increased further by expressing SPC72 from the stronger Gal1 promoter (lane 3, galactose) and was finally lethal when the Gal1–SPC72 promoter fusion was on a 2 μm multicopy plasmid (lane 4, galactose). In contrast, all cells grew equally on the repressing glucose plates. Interestingly, the toxic effects of SPC72 overexpression were much weaker in strain ESM387‐3 which carries the chromosomal Gal1–TUB4, Gal1–SPC98 and Gal1–SPC97 derivatives (Figure 2D, lanes 5–8). This result is explained most easily by a binding of Spc72p to the assembled Tub4p complex. Taken together, our biochemical and genetic analyses confirmed that the N‐terminal domain of Spc72p interacts with the Tub4p complex.
Spc72p is an essential component of the outer plaque of the SPB
Spc72p may be a cytoplasmic Tub4p complex‐binding protein, it could represent an additional subunit of the Tub4p complex, or it may function as a GTBP at the outer plaque. Only in the latter case we would expect to find Spc72p exclusively at the outer plaque of the SPB. To address these possibilities, we investigated the localization of Spc72p by indirect immunofluorescence and immunoelectron microscopy. We used affinity‐purified anti‐Spc72p1–271 antibodies for these experiments which were specific for Spc72p. This is indicated by the fact that predominantly one protein band with ∼85 kDa was detected in a cell lysate of SPC72 cells (Figure 3A, lane 3) and this band was shifted towards a higher molecular weight in an SPC72‐3HA cell extract (compare lanes 3 and 4). A comparison with the anti‐HA antibody (lanes 1 and 2) showed that the anti‐Spc72p antibodies (lanes 3 and 4) were more specific. On immunoblots, some Spc72p was shifted to higher molecular weights, and sometimes even multipe bands were resolved. One reason for this behaviour could be phosphorylation of Spc72p. By indirect immunofluorescence, Spc72p was detected as one or two dots at the nuclear periphery of all cells of an unsynchronized culture by the anti‐Spc72p antibodies (Figure 3B). Double labelling experiments with anti‐tubulin antibodies established that Spc72p is associated with the spindle poles, exactly where the SPBs are situated. An identical cellular distribution was observed with a functional Spc72p–green fluorescent protein (Spc72p–GFP) fusion (data not shown).
The substructural localization of Spc72p at the SPB was investigated by immunoelectron microscopy. Isolated SPBs were incubated with the anti‐Spc72p antibodies, followed by secondary antibodies coupled to colloid gold (15 nm). One to six gold particles were associated with the outer plaque region of the SPBs (n = 30; Figure 3C), suggesting that Spc72p is a component of the outer plaque. It is noteworthy that the inner plaque which is recognizable by the attached microtubules was never stained by the anti‐Spc72p antibodies.
To determine whether SPC72 is an essential gene, the entire coding region of SPC72 was disrupted in the diploid yeast strain YPH501 using the kanMX4 gene as disruption marker (ESM418). Analysis of tetrads from ESM418 suggested that SPC72 is indeed essential (data not shown). The essential function of SPC72 was confirmed by a plasmid shuffle experiment (Figure 7B). In summary, SPC72 encodes an essential SPB component that is associated with the outer plaque of the SPB.
Temperature‐sensitive SPC72 mutants are defective in spindle elongation, cytoplasmic microtubule organization and nuclear migration
The phenotype of mutants provides information about the function of the wild‐type gene. We were interested especially in the phenotype of spc72 mutants with a defect in the N‐terminal Tub4p complex‐binding domain. Therefore, we constructed an N‐terminal variant of SPC72 (ΔN‐SPC72), that carried a deletion of amino acids 2–176. However, ΔN‐SPC72 was unable to keep cells alive in the absence of the wild‐type SPC72 (Figure 7B). To overcome this problem, we constructed and analysed temperature‐sensitive spc72(ts) mutants with defects in the Tub4p complex‐binding region of Spc72p (Figure 4A). Synchronized cultures of SPC72 and spc72‐7 were shifted from 23 to 37°C. The DNA content, microtubule phenotypes and the distribution of the DNA were determined over time. SPC72 and spc72‐7 cells replicated their DNA with similar kinetics (Figure 4B). However, while SPC72 cells continued in the cell cycle, as indicated by the appearance of cells with 1N DNA content after 3 h, a spc72‐7 culture also showed cells with a DNA content >2N after 3 h.
When spc72‐7 cells were analysed using immunofluorescence microscopy, it was clear that most cells had strongly reduced cytoplasmic microtubule arrays (Figure 4C; for comparison, see Figures 3B and 5D, first column). We also noticed cells with an anaphase spindle in one cell body that still exhibited cytoplasmic microtubule remnants associated with the SPBs (Figure 4C, arrow). Figure 4D shows the distribution of the various microtubule phenotypes observed in wild‐type, spc72‐7 and spc72‐14 cells 3 h after release fom the α‐factor arrest at 37°C. As expected from this cytoplasmic microtubule deficiency, 29% of spc72‐7 cells were anucleated and lacked any microtubules, and about the same percentage of spc72‐7 cells had two separate 4,6′‐diamidino‐2‐phenylindole (DAPI)‐staining regions in the schmoo‐containing mother cell (Figure 4C and D). These two DAPI regions were still connected by long misaligned nuclear microtubules, indicating that nuclear division was not complete. The residual cells (∼46%) had one DAPI‐staining region with a short spindle of random orientation. The nucleus was either in the mother cell or in a cell without a bud. Nearly identical results were obtained with spc72‐14 cells which differ from spc72‐7 cells in their temperature sensitivity (data not shown and see Figure 4D). Taken together, our spc72(ts) cells show a clear defect in cytoplasmic microtubule functions, as indicated by the nuclear migration and spindle orientation defects. However, they show additional defects in spindle elongation or nuclear division that are not easily explainable.
Overexpression of ΔN‐SPC72 is lethal and results in multi‐nucleated and anucleated cells, while more cytoplasmic microtubules are observed after SPC72 overexpression
Since the nuclear phenotypes of spc72‐7 and spc72‐14 cells are not fully understood, we looked for additional experiments that could lead to a better understanding of Spc72p's function. We reasoned that overproduction of ΔN‐Spc72p should displace Spc72p from the outer plaque. A ΔN‐Spc72p‐containing outer plaque may then be unable to bind Tub4p complex and thereby would fail to organize cytoplasmic microtubules. On the contrary, overproduced Spc72p may increase the number of Tub4p complex‐binding sites at the outer plaque and thereby elevate the number of cytoplasmic microtubules.
Mild overexpression of ΔN‐SPC72 from the galactose‐inducible GalS promoter was lethal for the cells, as indicated by the failure of GalS–ΔN‐SPC72 cells to grow on galactose plates, while GalS–SPC72 and GalS control cells grew (Figure 5A). Analysis of cell extracts confirmed GalS‐dependent expression of ΔN‐Spc72p and Spc72p (Figure 5B).
To gain an understanding of why overexpression of ΔN‐SPC72 is toxic, we investigated whether overproduced ΔN‐Spc72p and Spc72p are capable of binding to the SPB and whether they replace endogeneous Spc72p from the SPB. We established that GFP–ΔN‐Spc72p associated with the SPB after mild overproduction (data not shown). In addition, we found that 64% of the nuclei of SPC72–GFP cells did not show a Spc72p–GFP signal at the SPB after induction of GalS–ΔN‐SPC72 for 9 h (corresponding to two doubling times of the GalS cells), while the rest of the SPC72–GFP nuclei showed one SPB signal. In contrast, all nuclei of cells, in which the central plaque of the SPB was marked by GFP fused to Spc42p (Spc42p–GFP), revealed one or two SPB signals after induction of GalS–ΔN‐SPC72 (Figure 5C). Most probably, Spc72p–GFP that was already assembled into the outer plaque at the time of the GalS–ΔN‐SPC72 induction was not replaced by ΔN‐Spc72p, while ΔN‐Spc72p competed with Spc72p for the incorporation into the newly formed SPB. Similarly, the SPB of GalS–SPC72 cells was stained more strongly by the anti‐Spc72p antibodies compared with the GalS control (data not shown), and overproduced Spc72p replaced Spc72p–GFP from a newly formed SPB (Figure 5C), suggesting that more Spc72p bound to the outer plaque of GalS–SPC72 cells than in wild‐type.
The positions of the nucleus and microtubule structures in GalS, GalS–SPC72 and GalS–ΔN‐SPC72 cells were analysed after growth in the inducing galactose‐containing medium. Most interestingly, many GalS–ΔN‐SPC72 cells were unbudded and contained no, two or four nuclei (Figure 5D). We noticed cells which appear to have only one nucleus. As reported for the tub2‐401 mutant (Sullivan and Huffaker, 1992), we assume that these cells probably have more than one nuclei situated on top of or next to each other where they cannot be resolved by fluorescence microscopy. This is also suggested by the large number of anucleated cells in the GalS–ΔN‐SPC72 culture (Table I). They resulted from cells that did complete anaphase nuclear division in the mother cell body, giving birth to one anucleated and one binucleated cell. It is noteworthy that multi‐ and anucleated cells were a specific phenotype of ΔN‐SPC72 overexpression. Such cell types were not found in the GalS or GalS–SPC72 cultures (Table I). Furthermore, hardly any cytoplasmic microtubules were observed in GalS–ΔN‐SPC72 cells, while the nuclear microtubules were of normal appearance: depending on the cell‐cycle stage of the cell, a monopolar spindle organized by one SPB (cell with four nuclei in Figure 5D), a parallel array of microtubules organized by two SPBs, or even two nuclei with an anaphase B spindle within one cell body were observed. We also noticed anucleated cells with one microtubule and nucleated cells with microtubules that were not connected with the SPB (Figure 5D, arrows), raising the possibility that microtubules detached from the SPB in GalS–ΔN‐SPC72 cells or that microtubule formation took place independently of the SPB.
Mild overexpression of GalS–SPC72 dramatically increased the tubulin signal in the cytoplasm, suggesting that more cytoplasmic microtubules were organized by the outer plaque (Figure 5D). In addition, ∼60% of the GalS–SPC72 cells were large budded, with a single DAPI‐staining region in the mother cell body, positioned close to the bud neck (Figure 5D). About 80% of the nuclei contained two SPBs (GFP–Spc42p; Figure 5C) and most probably a short spindle which was masked by the strong cytoplasmic microtubule staining. Taken together, the nuclear phenotype of SPC72 overexpression is consistent with the notion that these cells pause in mitosis due to a defect in mitotic spindle assembly.
GalS, GalS–ΔN‐SPC72 and GalS–SPC72 cells were inspected by thin section electron microscopy. The size and morphology of five SPBs examined from GalS–SPC72 (Figure 6B) and GalS–ΔN‐SPC72 (Figure 6E) cells were similar to those of the SPB from GalS cells (Figure 6A). Since the cytoplasmic microtubules are difficult to detect by electron microscopy in yeast, it was impossible to judge by this experiment whether their number was affected. However, the nuclear microtubules are easily detectable and their appearance in GalS–ΔN‐SPC72 (Figure 6C and D) and GalS–SPC72 cells (Figure 6B) was the same as in the GalS control (Figure 6A). In contrast to GalS cells (Figure 6A), GalS–ΔN‐SPC72 cells assembled the anaphase spindle in one cell body (Figure 6C). Furthermore, thin serial sections through several GalS–ΔN‐SPC72 cells confirmed that some cells contained two completely separated nuclei (for an example, see Figure 6D). In addition, cells were found that did not contain nuclear membrane structures (data not shown). In conclusion, the phenotypes of ΔN‐SPC72 and SPC72 overexpression are consistent with a function of the N‐terminal domain of Spc72p in cytoplasmic microtubule organization, most likely by recruiting the Tub4p complex to the outer plaque.
The Tub4p complex‐binding domain of Spc110p is functional at the outer plaque
A comparison of the amino acid sequences of the Tub4p complex‐binding sites of Spc72p and Spc110p did not indicate any homology. This raises the possibility that the two Tub4p complex‐binding domains evolved independently and that they may contain features which are important for their specific functions at the inner and outer plaques. To test this possibility, we constructed ΔN2–176‐SPC110 (ΔN‐SPC110), SPC722–176–SPC110177–944 (N‐SPC72–SPC110) and SPC1101–176–SPC72177–622 (N‐SPC110–SPC72) derivatives (Figure 7A) and tested whether they are expressed and functional.
We noticed that ΔN‐SPC110 does not provide SPC110 function (Figure 7B) and that its overexpression from the GalS promoter resulted in a lethal phenotype (Figure 7D), indicating that the Tub4p complex‐binding domain of Spc110p is essential for its function. In contrast to ΔN‐SPC110, hardly any transformants were obtained with a plasmid carrying N‐SPC72–SPC110 expressed from the SPC110 promoter, suggesting that N‐SPC72–SPC110 is dominant lethal. Consequently, GalS–N‐SPC72–SPC110 expression was lethal (Figure 7D), such that 95% of the cells were no longer viable 1 h after the induction of the GalS promoter (data not shown). Most interestingly, the N‐SPC110–SPC72 gene fusion rescued a SPC72 deletion (Figure 7B), raising the possibility that the Tub4p‐binding domain of Spc110p functions at the outer plaque. To exclude that the rescuing effect of N‐Spc110p was due to a simple stabilization of the C‐terminal domain of Spc72p, we constructed a KAR1–SPC72 gene fusion which did not function for SPC72 (data not shown). Finally, immunoblots established that N‐SPC110–SPC72 (Figure 7C), GalS–ΔN‐SPC110, GalS–SPC110 and GalS–N‐SPC72–SPC110 were expressed in yeast (Figure 7E).
We were interested in whether N‐Spc110–Spc72p fulfils Spc72p function at the SPB. This is indicated by the fact that N‐Spc110–Spc72p produced from the GalS promoter replaced endogenous GFP–Spc72p from the newly formed SPB (data not shown). For a more detailed study, N‐SPC110–SPC72 and SPC110 were integrated into the leu2 locus of Δspc72::kanMX4 cells to exclude artefacts caused by loss or an increased copy number of the plasmid. N‐SPC110–SPC72 cells had a slightly longer doubling time (2.8 h) at 30°C than SPC72 cells (2.4 h), and a small population of large budded N‐SPC110–SPC72 cells had two separate DAPI‐staining regions in the mother cell body (Figure 7F, arrow), while such cells were not observed in the SPC72 culture (Table II). The cytoplasmic microtubules were often longer in N‐SPC110–SPC72 cells undergoing anaphase as compared with SPC72 cells. Summarizing, our results demonstrate that the N‐terminal domain of Spc110p functions for the corresponding domain of Spc72p, suggesting that the Tub4p‐binding domain of Spc110p is functional at the outer plaque.
A universal component of MTOCs involved in microtubule organization is γ‐tubulin (Horio et al., 1991; Liu et al., 1993; Spang et al., 1996a; Ding et al., 1997) and possibly other subunits of γ‐tubulin complexes (Rout and Kilmartin, 1990; Spang et al., 1996a; Knop et al., 1997). γ‐Tubulin complexes assemble in the cytoplasm of cells (Stearns and Kirschner, 1994; Moudjou et al., 1996; Akashi et al., 1997; Pereira et al., 1998), followed by their binding to MTOCs. This suggests that MTOCs must have GTBPs which dock the γ‐tubulin complex to the scaffold of the MTOC. Using yeast as a model system, we have started to investigate the nature, specificity and regulation of GTBPs.
Spc72p is the GTBP of the outer plaque
We recently identified the SPB component Spc110p (Kilmartin et al., 1993; Kilmartin and Goh, 1996) as a GTBP of the inner plaque (Knop and Schiebel, 1997) (Figure 8). Here, we describe the identification of the essential SPB component Spc72p as a GTBP of the outer plaque. This notion is supported by the findings that Spc72p is an outer plaque component whose N‐terminal domain interacts with Spc98p and Spc97p, but not with Tub4p, in the yeast two‐hybrid system. Further proof for an interaction of Spc72p with the Tub4p complex came from the co‐immunoprecipitation of the Tub4p complex with Spc72p‐3HA and from the in vitro binding of the Tub4p complex to purified GST‐Spc72p1–271. In the immunoprecipitation experiment, we noticed that the phosphorylated and the unphosphorylated forms of Spc98p were precipitated by Spc72p‐3HA with about equal efficiency, which was unexpected since previous experiments indicated that Spc98p at the outer plaque is in its unphosphorylated form (Pereira et al., 1998). We assume that phosphorylated Tub4p complex from the inner plaque bound to Spc72p‐3HA after its extraction from the SPB. This assumption is in agreement with the finding that both forms of Spc98p bound to GST‐Spc72p1–271 in vitro.
Conditional lethal spc72 mutants showed defects in cytoplasmic microtubule functions
We studied the function of Spc72p in yeast cells using temperature‐sensitive alleles of SPC72. If Spc72p is the GTBP at the outer plaque, we expected to find that the cytoplasmic microtubules are defective in spc72(ts) cells, resulting in nuclear migration and nuclear positioning defects (Palmer et al., 1992; Sullivan and Huffaker, 1992). This was indeed the case: a high proportion of anucleated spc72(ts) cells and large budded spc72(ts) cells with one or two DAPI‐staining regions randomly positioned in the mother cell body were observed. In addition, the mitotic spindle of large budded spc72(ts) cells was not aligned along the mother–bud axis as is the case in wild‐type cells (Palmer et al., 1992). A direct proof of a cytoplasmic microtubule defect in spc72(ts) cells is the reduction or complete loss of cytoplasmic microtubule staining in indirect immunofluorescence experiments. However, compared with other mutants which affect cytoplasmic microtubule functions (Sullivan and Huffaker, 1992; Eshel et al., 1993; Li et al., 1993; Cottingham and Hoyt, 1997), spc72(ts) cells did not complete anaphase nuclear division in the mother cell body and therefore failed to give rise to multi‐nucleated cells. Instead, ∼50% of spc72(ts) cells formed a short spindle, or 24% of the cells formed an anaphase spindle randomly positioned in the mother cell, but failed to divide the nucleus. The reason for these nuclear phenotypes of spc72(ts) cells is unknown. Possible explanations are that Spc72p may have additional functions in cell‐cycle regulation, nuclear microtubule organization or nuclear division. Alternatively, aggregates of Spc72‐7p, which we observed occasionally by indirect immunofluorescence in the cytoplasm of spc72‐7 cells (data not shown), may bind other components of the SPB and thereby influence SPB functions. This would then result in the activation of the mitotic checkpoint (Hoyt et al., 1991; Li and Murray, 1991) in addition to the nuclear migration phenotype.
In contrast to spc72‐7 cells, overexpression of ΔN‐SPC72 resulted in polynucleated cells. Hardly any cytoplasmic microtubules were detectable in GalS–ΔN‐SPC72 cells, while the nuclear microtubules were of normal appearance and they were probably functional, indicated by the segregation of sister chromatids and the formation of anaphase spindles. Remarkably, the phenotype of GalS–ΔN‐SPC72 overexpression is nearly identical to that of tub2‐401 cells which are selectively defective in cytoplasmic microtubule functions (Sullivan and Huffaker, 1992). This similarity supports the notion that Spc72p has a specific function in cytoplasmic microtubule organization. The specific cytoplasmic microtubule defect of ΔN‐SPC72 is explained by the finding that ΔN‐Spc72p competes with GFP–Spc72p for the incorporation into the outer plaque of newly formed SPBs. Such SPBs are then unable to recruit Tub4p complex to the outer plaque, which as a consequence fails to organize cytoplasmic microtubules.
Further support for Spc72p's function as a GTBP comes from the observation that the moderate overexpression of SPC72 gives rise to more Spc72p signal at the SPB, accompanied by an increase in cytoplasmic microtubule staining. The elevated level of Spc72p at the outer plaque may recruit more Tub4p complex to this location of the SPB, with the consequence that more cytoplasmic microtubules are formed. Overexpression of SPC72 showed toxic effects, but these cells were not defective in cytoplasmic microtubule functions, since the nucleus was positioned in the bud neck. Instead, GalS–SPC72 cells were delayed in the formation of an anaphase spindle. Based on the observation that overproduced Spc72p did not accumulate inside the nucleus (data not shown), we favour the idea that the cytoplasmic Spc72p binds Tub4p complex and may prevent its nuclear import via Spc98p (Pereira et al., 1998). The depletion of Tub4p complex in the nucleus then interferes with nuclear microtubule functions which then leads to the activation of the mitotic spindle checkpoint (Hoyt et al., 1991; Li and Murray, 1991). This model is supported by the observation that co‐overexpression of TUB4, SPC98 and SPC97, which gives rise to large amounts of Tub4p complex (Pereira et al., 1998), suppresses the lethal effect of multicopy Gal1–SPC72 overexpression.
The Tub4p‐binding domain of Spc110p functions at the outer plaque
Sequence analysis of the Tub4p complex‐binding domains of Spc110p and Spc72p revealed no homology, raising the possibility that these domains evolved independently and did not arise by gene duplication. Whether GTBPs contribute to the specific microtubule properties of an MTOC is an important question. Therefore, we tested whether hybrid proteins between the N‐terminal domain of Spc72p and the C‐terminal Spc110p, and vice versa, are functional. Our results show that N‐SPC110–SPC72 rescues a SPC72 null mutant, indicating that N‐SPC110–SPC72 either fulfils SPC72 function or by‐passes its requirement. That N‐Spc110p–Spc72p functions directly for Spc72p at the outer plaque is suggested by the association of the hybrid with the SPB upon its overexpression, thereby displacing Spc72p–GFP from the outer plaque. Does N‐Spc110–Spc72p fully substitute for Spc72p? Analysis of a chromosomal integrated N‐SPC110–SPC72 allele identified a weak growth defect and a small percentage of anucleated cells and cells with two DAPI‐staining regions connected by a spindle in one cell body. These phenotypes indicate that nuclear migration is delayed in at least some N‐SPC110–SPC72 cells. Our conclusion is supported further by the observation that more N‐SPC110–SPC72 cells of a logarithmically growing culture have a 2N DNA content compared with the SPC72 control (data not shown). The nature and extent of these defects are comparable with the weak cytoplasmic microtubule defects of DYN1 (Eshel et al., 1993; Li et al., 1993) and KAR9 (Miller and Rose, 1998) deletion mutants incubated at 30°C. However, in contrast to Δdyn1 cells, the nuclear migration defect of N‐SPC110–SPC72 cells was not increased by reducing the growth temperature to 14°C (data not shown).
N‐SPC72–SPC110 expressed from the SPC110 promoter on a centromere‐based plasmid resulted in a dominant lethal phenotype which was dependent on the N‐SPC72 portion, since the non‐functional ΔN‐SPC110 version did not affect cell viability. The only way to analyse N‐SPC72–SPC110 was the expression of the hybrid gene from a regulated promoter, e.g. the GalS promoter. Although this promoter is weaker than Gal1 (Mumberg et al., 1995), it still caused overexpression of N‐SPC72–SPC110. Analysis of GalS–SPC72–SPC110 cells clearly showed more nuclear microtubules than in wild‐type cells. However, these cells also failed to duplicate the SPB (M.Knop, unpublished). Although our observations are consistent with the view that N‐Spc72–Spc110p functions as a GTBP at the inner plaque, it remains unclear why even low levels of N‐Spc72–Spc110p are lethal for cells. Either N‐SPC72–SPC110 expressed from the SPC110 promoter also affects SPB duplication as does GalS–SPC72–SPC110, or the nuclear microtubules organized by N‐Spc72–Spc110p are not functional, resulting in a defective spindle followed by cell death. Taken together, our results are consistent with the notion that the N‐terminal domains of Spc110p and Spc72p fulfil an essential role by the organization of the nuclear spindle and the cytoplasmic microtubules respectively, and that these functions are connected to the ability of these domains to bind to the Tub4p complex.
GTBPs represent MTOC‐specific components of microtubule attachment sites
The components of the yeast Tub4p complex have been localized to the outer and inner plaques (Rout and Kilmartin, 1990; Spang et al., 1996a; Knop et al., 1997), suggesting that they represent universal components of the microtubule organization machinery. In contrast, Spc110p and Spc72p are the first side‐specific proteins of the SPB involved in microtubule organization. Our data suggest that Spc110p and Spc72p have at least two functionally distinct domains: an N‐terminal domain that interacts with the Tub4p complex and a C‐terminal domain which binds to at least another SPB component. Cell‐cycle‐dependent modification of the N‐terminal domain of Spc110p and Spc72p could in fact modify the microtubule organization properties of the inner and outer plaques. In this respect, it is interesting that Spc110p is a phosphoprotein (Friedmann et al., 1996; Stirling and Stark, 1996) and that Spc72p was resolved by SDS–PAGE into multiple bands (Figure 2B), suggesting that it is modified. The C‐terminal domains of Spc110p and Spc72p carry the information as to which side of the SPB the proteins bind. Spc110p has been purified in complex with Spc42p, calmodulin and an SPB component with an apparent mol. wt of 35 kDa (Knop and Schiebel, 1997), indicating a physical interaction between the four SPB components (Figure 8). How Spc72p is bound to the SPB is still an open question.
In conclusion, based on the analysis of the yeast SPB, we favour the idea that each MTOC has a specific set of GTBPs which interact with components of the γ‐tubulin complex as well as with proteins of the MTOC. In some cases, GTBPs may already bind to cytoplasmic γ‐tubulin complexes, explaining their variation in size and complexity. The binding properties, post‐transitional modification and abundance of GTBPs may contribute to the specific microtubule‐organizing properties of an MTOC.
Materials and methods
Growth media and general methods
Basic yeast methods and growth media were as described (Sherman, 1991). Yeast strains were grown in yeast extract, peptone, dextrose (YPD) medium containing 100 mg/l adenine. For Gal1‐ or GalS‐controlled gene expression, yeast strains were grown in synthetic complete (SC) medium containing raffinose (2%) as carbon source. Galactose (2%) or glucose (2%) were added to induce or repress the Gal1 or GalS promoters, respectively. Yeast strains were transformed by the lithium acetate method (Schiestl and Gietz, 1989). The E.coli strains were transformed by electroporation (Dower et al., 1988). PCR was performed with a mixture of 0.4 U of Vent polymerase (New England Biolabs) and 2 U of Taq polymerase (Gibco‐BRL) per 100 μl reaction. Recombinant DNA methodology was as described by Sambrook et al. (1989).
A two‐hybrid screen was performed using the entire coding sequence of SPC98 fused to the DNA‐binding domain of the GAL4 gene in the TRP1‐based plasmid pGBT9 (Fields and Song, 1989). Strain Y190 (Bai and Elledge, 1996) carrying pGBT9‐SPC98 was then transformed with a yeast cDNA library fused to the GAL4 activation domain. Among the 20 000 Leu+ Trp+ transformants, 25 grew on plates containing 50 mM aminotriazole and showed β‐galactosidase activity. The prey plasmids were transformed into SGY37 with plasmid pMK16 (pEG202‐SPC97) or pEG202. For three prey plasmids, expression of β‐galactosidase was dependent on plasmid pMK16. These prey plasmids were transformed into strain SGY37 together with pSG21 (pEG202‐TUB4) and with or without plasmid pMK155 (p414‐Gal1‐SPC97 Gal1‐SPC98). One prey plasmid resulted in expression of β‐galactosidase when SPC98 and SPC97 were co‐expressed. Sequence analysis revealed that the positive prey plasmid contained an in‐frame fusion of GAL4 to the first 270 codons of SPC72 (YAL047c).
Plasmids and yeast strains
Plasmid and yeast strains used during this study are listed in Table III. Where PCR products were used for cloning, the resulting plasmids were either sequenced (pSM447, pSM572, pMK230–257) or PCR products from two independent reactions were cloned (two‐hybrid plasmids). In all cases, the independent constructs behaved identically. Chromosomal encoded terminal protein fusions with GFP or 3HA were constructed using a PCR targeting strategy with GFP–KanMX (Wach et al., 1997) or 3HA‐KanMX (M.Knop, B.Windsor, K.Siegers and C.Schiebel, in preparation) modules.
Construction of temperature‐sensitive alleles of SPC72
Codons 1–176 of SPC72 were mutagenized by PCR (Cadwell and Joyce, 1992) using Primer 1 that is homologous to the 63 bp upstream of the start codon of SPC72 and Primer 2 that binds to codons 177–198 of SPC72 as shown in Figure 4A. The mutagenized 5′ region of SPC72 was combined with the not mutagenized 3′ region of SPC72 as outlined in Figure 4A. Conditional lethal alleles of SPC72 were selected as described (Muhlrad et al., 1992).
Binding of Tub4p complex to recombinant GST–N‐Spc72p
Binding of the Tub4p complex to recombinant GST fusion proteins was performed as described before (Knop and Schiebel, 1997). Plasmids were pGEX‐5X‐1 (GST), pSM363 (GST–Spc42p1–214) and pGP68 (GST–Spc721–271). An extract of cells from strain YMK47 (SPC97‐3ProA) was used. The samples were analysed by immunoblotting with affinity‐purified rabbit anti‐Tub4p and rabbit anti‐Spc98p antibodies. Spc97‐3ProA was always detected on the immunoblot due to the binding of the protein A part to IgGs.
Antibodies specific for Spc72p were produced against recombinant TrpE–Spc72p1–425 protein. In brief, a BamHI restriction site was introduced by PCR just upstream of the ATG start codon of SPC72. Using this BamHI site, a 1279 bp BamHI–ClaI fragment of SPC72 (codons 1–425) was cloned into the BamHI–ClaI sites of vector pATH11 (pSM430). The TrpE–Spc72p1–425 fusion protein was induced by indoleacrylic acid as described (Koerner et al., 1991). The 87 kDa TrpE–Spc72p1–425 fusion protein was purified from inclusion bodies. The protein was solubilized in SDS–PAGE buffer and separated by SDS–PAGE (Laemmli, 1970). Proteins were transferred onto a nitrocellulose membrane and stained by Ponceau S. The TrpE–Spc72p1–425‐containing membrane strip was cut out and solubilized with dimethylsulfoxide (DMSO). Antibodies were raised as described (Harlow and Lane, 1988). For affinity purification of the antibody, CNBr‐Sepharose (Pharmacia) with immobilized GST–Spc72p1–271 was used.
The polyclonal rabbit anti‐Tub4p (Spang et al., 1996a), anti‐Spc97p (Knop and Schiebel, 1997), anti‐Spc98p (Knop et al., 1997), anti‐Spc110p3–175 (Spang et al., 1996b) and anti‐Spc110p293–756 (Stirling et al., 1994) have been described before. Rabbit anti‐Tub1p and rabbit anti‐Tub2p were a kind gift from F.Solomon, and the mouse monoclonal anti‐β‐tubulin antibody (Wa3) was a gift from U.Euteneuer‐Schliwa. The mouse monoclonal anti‐HA antibodies (12CA5) were obtained from Hiss Diagnostics, and anti‐Myc antibodies (9E10) were purchased from Boehringer Ingelheim. Secondary antibodies used in immunofluorescence, immunoelectron microscopy and immunoblotting were goat anti‐mouse and goat anti‐rabbit antibodies coupled to Cy2 or Cy3, or goat anti‐rabbit antibodies coupled to colloid gold particles or to horseradish peroxidase (all from Jackson Immuno Research Laboratories), respectively.
Immunofluorescence of formaldehyde‐fixed yeast cells was performed as described (Knop et al., 1996) with 1 h fixation time. Microtubules were stained using the Wa3 antibodies. Spc72p or Spc110p were detected using affinity‐purified anti‐Spc72p or anti‐Spc110p3–175 antibodies. DNA was stained with DAPI. For double detection of GFP‐labelled proteins and DNA, cells carrying the indicated GFP constructs were fixed with 4% paraformaldehyde/0.1 M KPO4 pH 6.5 for 5 min. Cells were washed twice with phosphate‐buffered saline (PBS) and then incubated with 10 μg/ml DAPI in PBS for 10 min. Cells were harvested and resuspended in PBS containing 1 μg/ml DAPI.
Immunoprecipitation of Spc72‐3HA or Spc97‐3HA was performed as described (Knop et al., 1997; Pereira et al., 1998) using TBS‐T (20 mM Tris pH 7.5, 135 mM NaCl, 2.5 mM KCl, 1% Triton X‐100) as buffer. The anti‐HA antibodies were cross‐linked to protein A–Sepharose beads (Harlow and Lane, 1988). For immunoblotting, proteins were transferred onto nitrocellulose membranes. The blocked membranes were incubated with the indicated primary antibodies. Secondary antibodies were coupled to peroxidase. The immunoreaction was visualized by an ECL Kit from Amersham.
SPB isolation, electron microscopy and immunoelectron microscopy
SPBs were isolated from strain YPH499 as described previously (Rout and Kilmartin, 1990). Immunoelectron microscopy using anti‐Spc72p antibodies was performed as follows. Isolated SPBs were fixed with 4% paraformaldehyde/0.5% glutaraldehyde in Bt‐Mg (10 mM BisTris pH 6.5, 0.1 mM MgCl2) for 25 min at room temperature. Fixation reactions were diluted 5‐fold with cold Bt‐Mg and the SPBs were centrifuged onto round cover slides as described (Mitchison and Kirschner, 1984). After immunodecoration of the SPBs with the appropriate antibodies, the SPBs were post‐fixed in 2% glutaraldehyde (10 min), 2% osmium tetroxide (15 min). The samples were dehydrated followed by embedding in Spurr low viscosity resin (Polyscience). Yeast cells were prepared for thin section electron microscopy following a published protocol (Byers and Goetsch, 1991).
Yeast cells were prepared for flow cytometry as described (Hutter and Eipel, 1979). Samples were measured using a FACS‐calibur (Becton‐Dickson).
We thank Dr M.Stark for the anti‐Spc110p serum and Dr S.Elledge for the cDNA yeast two‐hybrid library. We would like to mention G.K.Pereira who constructed the plasmids pGP62‐68. We are also grateful to M.Matzner from the Max‐Planck Institute and to M.O'Prey from the Beatson Institute for thin serial sections of embedded samples for electron microscopy. P.McHardy is acknowledged for technical support with microscopy. Part of this work was performed at the Max‐Planck Institute for Biochemistry, Martinsried, Germany, with support from the BMBF. This work was carried out with support from the Cancer Research Campaign.
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