Title: The fission yeast gamma-tubulin complex is required in G1 phase and is a component of the spindle assembly checkpoint
Abstract: Article15 November 2000free access The fission yeast γ-tubulin complex is required in G1 phase and is a component of the spindle assembly checkpoint Leah Vardy Leah Vardy Laboratory of Cell Regulation, Imperial Cancer Research Fund, PO Box 123, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Takashi Toda Corresponding Author Takashi Toda Laboratory of Cell Regulation, Imperial Cancer Research Fund, PO Box 123, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Leah Vardy Leah Vardy Laboratory of Cell Regulation, Imperial Cancer Research Fund, PO Box 123, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Takashi Toda Corresponding Author Takashi Toda Laboratory of Cell Regulation, Imperial Cancer Research Fund, PO Box 123, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Author Information Leah Vardy1 and Takashi Toda 1 1Laboratory of Cell Regulation, Imperial Cancer Research Fund, PO Box 123, 44 Lincoln's Inn Fields, London, WC2A 3PX UK *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:6098-6111https://doi.org/10.1093/emboj/19.22.6098 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Microtubule polymerization is initiated from the microtubule organizing centre (MTOC), which contains the γ-tubulin complex. We have identified fission yeast Alp4 and Alp6, which are homologues of the γ-tubulin-interacting proteins Sc.Spc97/Hs.Gcp2 and Sc.Spc98/Hs.Gcp3, respectively. The size of the fission yeast γ-tubulin complex is large (>2000 kDa), comparable to that in metazoans. Both Alp4 and Alp6 localize to the spindle pole body (SPB) and also to the equatorial MTOC. Temperature-sensitive (ts) alp4 and alp6 mutants show two types of microtubular defects. First, monopolar mitotic spindles form. Secondly, abnormally long cytoplasmic microtubules appear that do not stop at the cell tips and are still associated with the SPB. Alp4 function is required in G1 phase and ts mutants become lethal before S-phase. alp4 and alp6 mutants are hypersensitive to the microtubule- destabilizing drug thiabendazole (TBZ) and show a lethal 'cut' phenotype in its presence. Furthermore, alp4mad2 double mutants show an exaggerated multiple septation phenotype in TBZ. These results indicate that Alp4 and Alp6 may play a crucial role in the spindle pole-mediated checkpoint pathway. Introduction In many eukaryotic cells, microtubule nucleation occurs at a specific structure called the microtubule organizing centre (MTOC; Pickett-Heaps, 1969). The MTOC plays a vital role not only in nucleating microtubules, but also in determining their polarity. The slow growing minus end is embedded within the MTOC, while the fast growing plus end radiates out into either the cytoplasm or the nucleus (Heidemann and McIntosh, 1980). MTOC structure varies considerably between different species. In animal cells, the centrosome, in particular the pericentriolar material (PCM), functions as the MTOC (Gould and Borisy, 1978; Kellogg et al., 1994). In yeast, the equivalent structure is the spindle pole body (SPB). Central to MTOC function is the γ-tubulin complex. γ-tubulin, a member of the tubulin superfamily, was originally identified genetically as an intergenic suppressor of a β-tubulin mutation in Aspergillus nidulans (Oakley and Oakley, 1989). Subsequent work in various systems both in vivo and in vitro has established that γ-tubulin is a universal component of the MTOC, and plays a central role in microtubule nucleation (Oakley et al., 1990; Horio et al., 1991; Stearns et al., 1991; Zheng et al., 1991; Joshi et al., 1992, 1993; Oakley, 1992; Felix et al., 1994; Stearns and Kirschner, 1994; Schnackenberg et al., 1998). γ-tubulin does not appear to exist in the cell on its own; instead it is part of a large complex (Stearns and Kirschner, 1994). This γ-tubulin complex in animal cells comprises an open ring structure of 25 nm diameter, called the γ-tubulin ring complex (γTuRC), which exists in both the cytoplasm and the PCM (Moritz et al., 1995, 1998; Zheng et al., 1995) and functions as a minus end capping factor for microtubule nucleation (Keating and Borisy, 2000; Leguy et al., 2000; Moritz et al., 2000; Wiese and Zheng, 2000). Genetic studies in budding yeast have been instrumental in identifying other components of the γTuRC. Spc97 and Spc98 were identified as γ-tubulin-interacting proteins and shown to constitute the major components of the Saccharomyces cerevisiae γ-tubulin complex (Geissler et al., 1996; Knop and Schiebel, 1997; Knop et al., 1997). Homologues of Spc97 and Spc98 have subsequently been found in humans and are called Gcp2 and Gcp3, respectively (Martin et al., 1998; Murphy et al., 1998; Tassin et al., 1998). The budding yeast counterpart of γ-tubulin, Tub4, which also plays an important role in microtubule organization in this organism, is more divergent from γ-tubulins in other organisms (<40% amino acid identity with metazoan γ-tubulins as opposed to 70% identity between them), and metazoan γ-tubulin is not capable of substituting for Tub4 function (Burns, 1995; Sobel and Snyder, 1995; Marschall et al., 1996). Furthermore, the Tub4-containing complex is much smaller than the mammalian γTuRC (200–250 kDa versus >2000 kDa; Stearns and Kirschner, 1994; Zheng et al., 1995; Knop et al., 1997). The fission yeast γ-tubulin homologue Gtb1/Tug1 shares >70% identity with the vertebrate protein (Horio et al., 1991; Stearns et al., 1991) and, more importantly, human γ-tubulin can rescue the lethal gtb1 deletion (Horio and Oakley, 1994). Gtb1 localizes to the SPB throughout the cell cycle and to the equatorial MTOC in post-anaphase (Horio et al., 1991; Masuda et al., 1992). Like most other eukaryotes, fission yeast microtubule organization alters dynamically during cell cycle progression, where interphase cytoplasmic arrays give way for the mitotic bipolar spindle (Hagan and Hyams, 1988). Despite the attractions of this system, no conditional mutants that are defective in the components of the γ-tubulin complex have been available until recently (Paluh et al., 2000). The spindle assembly checkpoint is a surveillance mechanism that ensures that paired chromatids do not segregate until the chromosomes are aligned properly along the mitotic spindle, and it plays a pivotal role in the maintenance of genome integrity and fidelity of chromosome separation (Hoyt et al., 1991; Li and Murray, 1991; Lengauer et al., 1998). Recent analysis has shed more light on the complexities of this regulatory mechanism. In yeast, two distinct pathways are operational, one is Mad2 dependent whilst the other is Bub2 (Cdc16 in fission yeast) dependent (Burke 2000; Cerutti and Simanis, 2000). The Mad2 pathway is believed to be involved in the regulation of kinetochore function, such that its structural component plays a role in the checkpoint system (e.g. budding yeast Ndc10; Tavormina and Burke, 1998). In contrast, how microtubule/spindle integrity is monitored by the Bub2 pathway remains elusive, although recent analysis indicates that it regulates mitotic exit via nuclear positioning in budding yeast (Bardin et al., 2000; Bloecher et al., 2000; Pereira et al., 2000) and septation/cytokinesis in fission yeast (Cerutti and Simanis, 2000). We previously isolated a number of mutants that display temperature-sensitive (ts) defects in the maintenance of growth polarity control (alp loci, altered polarity; Radcliffe et al., 1998). In line with the fact that fission yeast microtubules play a crucial role in the determination of growth polarity, many of the alp+ genes encode conserved proteins that are required for microtubule function, including tubulins and cofactor homologues (Hirata et al., 1998; Radcliffe et al., 1998, 1999). In this study, we describe the characterization of Alp4 and Alp6, which are the fission yeast homologues of Spc97/Gcp2 and Spc98/Gcp3, respectively. We show that Alp4 and Alp6 are required for the regulation of both interphase microtubules and mitotic bipolar spindles. Importantly, these components execute their essential role in G1 phase. Furthermore, we show that Alp4 and Alp6 are essential components of the spindle assembly checkpoint. Results ts alp4 and alp6 mutants are defective in microtubule organization and show growth polarity defects ts alp4 and alp6 mutants were isolated from a visual screen for growth polarity mutants (Radcliffe et al., 1998). Morphological characterization showed that these three strains showed similar, if not identical, phenotypes at the restrictive temperature, i.e. a bent shape associated with 'cut' phenotypes (9–10%). Furthermore, immunofluorescence microscopy using anti-α-tubulin antibody showed that mutant cells incubated for 6 h at 36°C displayed abnormal microtubules, and nuclear DNA often became displaced from the centre of the cell and segregated aberrantly into two or three masses (Figure 1A). These observations indicate that Alp4 and Alp6 are required for microtubule organization and proper chromosome separation. Figure 1.Defective phenotypes of ts alp4 and alp6 mutants and the cellular localization of Alp4 and Alp6 at the MTOC. (A) Wild-type (left, HM123, Table II), alp4-1891 (middle, DH1891) or alp6-719 (right, DH719) cells were incubated at 36°C for 6 h and processed for immunofluorescence microscopy. Merged images of anti-tubulin staining (TAT-1, red) and nuclear staining (DAPI, blue) are shown. Abnormally segregated mitotic chromosomes are marked with arrows. (B) Localization of Alp4. Fluorescence from GFP (two left panels; Alp4–GFP, LV11) or immunofluorescence microscopy using anti-HA antibody (two right panels; Alp4-3HA, LV15) are shown. (C) Localization of Alp4–GFP during the cell cycle. Triple staining using DAPI (left), anti-Sad1 (the second panels), GFP (the third panels) and merged pictures (right) during the cell cycle are shown. Representative images from interphase (row 1), metaphase (row 2), anaphase (row 3), post-anaphase (row 4) and septated cells (row 5) are presented. In the rightmost corner, combined images are depicted, in which blue corresponds to chromosomal DNA, orange shows the merged images between Sad1 and Alp4, and green presents Alp4 at the equatorial MTOC (marked with an arrow in row 4). (D) 'Ring' structures of the equatorial MTOC (shown by arrows, Alp4-3HA). Anti-HA signals from a post-anaphase cell (corresponding to row 4) have been rotating around a vertical axis after observation under a confocal microscope. The bar indicates 10 μm. Download figure Download PowerPoint alp4+ and alp6+ encode conserved components of the γ-tubulin complex A fission yeast genomic library was used to isolate genes that complemented the ts alp4 and alp6 mutations. Two different plasmids (pLV4-1 and pLV6-1) were isolated that complemented alp4-1891 and alp6-719, respectively. Genetic linkage analysis indicated that the gene in pLV4-1 is alp4+, whilst that in pLV6-1 is alp6+ (see Materials and methods). Nucleotide sequencing showed that alp4+ and alp6+ encode fission yeast homologues of universal components of the γ-tubulin complex, Gcp2 (human)/Spc97 (budding yeast) and Gcp3/Spc98, respectively (Schiebel, 2000). The identity between Alp4 and Gcp2 or Spc97 was 22% (32% if conservative changes are considered) and 11% (22%), whilst that between Alp6 and Gcp3 or Spc98 was 25% (39%) and 18% (32%), respectively, indicating that the fission yeast proteins are evolutionarily closer to vertebrates than budding yeast (Table I). In a similar manner to the vertebrate proteins (Martin et al., 1998; Murphy et al., 1998; Tassin et al., 1998), Alp4 and Alp6 show a distant evolutionary relatedness to each other (data not shown), and probably evolved from a common ancestor. Table 1. Homology of γ-tubulin-interacting proteins between fission yeast and other eukaryotes Gcp2 Dgrip84 Spc97 Alp4 22 (32) 18 (32) 11 (22) Gcp2 27 (42) 11 (24) Dgrip84 10 (22) Gcp3 Dgrip91 Spc98 Alp6 25 (39) 19 (32) 18 (32) Gcp3 29 (44) 15 (28) Dgrip91 15 (28) The percentage identity or similarity (in parentheses) in amino acid sequence between γ-tubulin-interacting proteins from fission yeast (Alp4 and Alp6), human (Gcp2 and Gcp3), fly (Dgrip84 and Dgrip91) and budding yeast (Spc97 and Spc98) is shown. The cellular localization of Alp4 and Alp6 at the MTOC In order to examine the cellular localization of Alp4 and Alp6, the green fluorescent protein (GFP) gene was fused to the C-terminus of the chromosomal alp4+ and alp6+ genes. GFP tagging did not interfere with protein function as strains containing Alp4–GFP or Alp6–GFP grew as well as wild-type strains, and no morphological or mitotic defects were apparent. Signals from Alp4–GFP and Alp6–GFP were essentially the same (although the signal from Alp4–GFP was stronger than that from Alp6–GFP). Signals were seen either as a single or double spot around the nuclear periphery and/or as dot-like structures in the cell centre in exponentially growing Alp4–GFP cells (the left panels in Figure 1B). As this localization appeared similar to that of the SPB, double staining with both Alp4–GFP and anti-Sad1 antibodies was performed (Sad1 is an SPB component; Hagan and Yanagida, 1995). As shown in Figure 1C, in both interphase (rows 1) and mitotic cells (rows 2 and 3), Alp4–GFP co-localized precisely with Sad1 either as a single (row 1) or double spot (rows 2 and 3). Of particular interest were post-anaphase cells (row 4), in which the localization of Alp4–GFP differed from that of Sad1. At this stage, in addition to the SPBs, which are located on the side of each nucleus, an equatorial dot(s) of Alp4–GFP but not anti-Sad1 was clearly visible (arrow in row 4). After the completion of septum formation, this central dot(s) disappeared (row 5). These dots appeared to be identical to those seen with anti-Gtb1, which have been termed the equatorial MTOC and which generate the post-anaphase array during cytokinesis (Hagan and Hyams, 1988; Horio et al., 1991; Hagan, 1998). In order to observe the three-dimensional structure of the equatorial MTOC, confocal images were rotated around a vertical axis. It was found that these central signals are in fact not 'dots', but instead formed a 'ring' structure during the early stages of cytokinesis (arrow, Figure 1D). It has been reported previously that cytoplasmic microtubules also exist as 'equatorial rings' in the centre of the cell at the end of anaphase and during telophase (Pichová et al., 1995). These results show that Alp4 and Alp6 are not only homologous to components of the MTOC in their amino acid sequence, but are also an integral part of the two fission yeast MTOCs that are functional during the mitotic cell cycle. Physical and genetic interactions between Alp4, Alp6 and γ-tubulin Immunoprecipitation experiments were performed to explore the physical interactions between Alp4, Alp6 and Gtb1. Haemagglutinin (HA) epitope-tagged strains (Alp4-3HA and Alp6-3HA) were constructed in a manner similar to GFP tagging. Immunoblotting using anti-HA antibody identified Alp4-3HA and Alp6-3HA on an SDS–polyacrylamide gel (Figure 2A). These gene fusions were also used to confirm that Alp4 and Alp6 localize to the MTOC. Immunofluorescence microscopy using anti-HA antibody showed the same localization patterns as those of GFP-tagged proteins (see the right two panels in Figure 1B). Immunoprecipitation experiments showed that Alp4-3HA and Alp6-3HA co-precipitated with Gtb1 (Figure 2B, lanes 4 and 5). It appeared that the amount of Gtb1 that was co-precipitated was not as much as the total protein (compare lanes 1 and 2 with lanes 3 and 4). It is possible that Alp4-3HA and Alp6-3HA, which form a complex with Gtb1, are less efficient than free proteins for precipitation with anti-HA antibody, or that a subpopulation of Gtb1 may exist independently of Alp4 and Alp6. Figure 2.Physical and genetic interactions between Alp4/Alp6 and γ-tubulin. (A) Identification of the alp4+ and alp6+ gene products. Immunoblotting was performed with anti-HA antibody against cell extracts prepared from an Alp4-3HA (lane 1, LV15) or Alp6-3HA strain (lane 2, LV16). Gtb1 (γ-tubulin) was used as a loading control. (B) Physical interaction between Alp4/Alp6 and γ-tubulin. Cell extracts were prepared from an Alp4-3HA strain (lanes 1 and 4, LV15), an Alp6-3HA strain (lanes 2 and 5, LV16) or an untagged wild type (lanes 3 and 6), and immunoprecipitation performed with anti-HA antibody (lanes 4–6). Precipitated proteins were detected with anti-HA or anti-Gtb1 antibody. Total cell extracts (corresponding to 1/50 amount used for immunoprecipitation) were also run (lanes 1–3). (C) Gel filtration chromatography. Soluble cell extracts were analysed by immunoblotting with anti-HA or anti-Gtb1 antibody. Total extract (10 μg) was run in the far-left panel. Protein extracts from two strains (Alp4-3HA and Alp6-3HA) were loaded on separate columns, and separation profiles were superimposed according to a control pattern using anti-Gtb1 antibody. The positions of size markers (2000, 669 and 232 kDa) are also shown. (D) Suppression analysis by multicopy plasmids. ts alp4 or alp6 mutants were transformed with an empty vector or multicopy plasmids containing alp4+ or alp6+ (pUR-Alp4 or pUR-Alp6, respectively), and transformants were streaked on rich plates and incubated at 36°C for 3 days. Download figure Download PowerPoint In order to address the size of the γ-tubulin complex in fission yeast, gel filtration analysis was performed. As shown in Figure 2C, Alp4, Alp6 and Gtb1 co-fractionate predominantly in a large complex (>2000 kDa). It should be noted that this large size is comparable to that in higher eukaryotes such as Drosophila and mammalian cells (Martin et al., 1998; Murphy et al., 1998). We have observed that a small amount of Gtb1 is also found in the smaller fractions (fractions 13–22), some of which still overlap with Alp4 and Alp6 (13–16). It is possible that, as in other eukaryotes, multiple forms of the complex may exist in fission yeast, which vary in size (Akashi et al., 1997; Moritz et al., 1998). We sought genetic interactions to substantiate the biochemical data that Alp4, Alp6 and Gtb1 physically interact. As shown in Figure 2D, multicopy plasmids containing the alp4+ gene suppressed the defects of the ts alp6-719 mutant. Taken together, these results show that Alp4 and Alp6 localize to the MTOC and constitute part of the γ-tubulin complex, and that the alp4+ and alp6+ genes interact genetically with gtb1+ and also with each other. alp4 mutants exhibit defects in the formation of bipolar mitotic spindles and maintenance of the length of cytoplasmic microtubules In order to characterize the ts alp4 phenotypes more carefully, a culture of alp4 cells was synchronized with respect to cell cycle progression by centrifugal elutriation and shifted to the restrictive temperature. Small early G2 alp4-1891 cells grown at 26°C were collected by elutriation and the culture was divided into two halves. One half was incubated at 26°C to monitor the degree of synchrony of cell cycle progression, whilst the other half was shifted up to 36°C to examine the phenotypes arising from incubation under the restrictive conditions. Samples were collected at 20 min intervals in order to measure viability and to use for immunofluorescence analysis of microtubules and the SPB. Synchrony was high as the septation index was <0.5% at 0 min, reached 62% at 120 min (the first mitosis), dropped to 1% at 200 min and peaked again up to 47% at 300 min (the second mitosis) at 26°C (Figure 3A). At the restrictive temperature, defective phenotypes, including abnormal mitotic DNA (Figure 1A), became evident only during the second mitosis; the first mitosis appeared to occur normally (Figure 3B; note the normal mitotic pattern until 200 min). Cell viability remained high during the first 100 min when the septation index (equivalent to the population of cells that have passed mitosis) peaked, and then started to drop as the second cycle proceeded. It is of note that in fission yeast, G1 phase is very short and S phase occurs almost simultaneously with nuclear division and septation (Moreno et al., 1991). This result, therefore, suggested that alp4 mutants are committed to death before the second M phase. Figure 3.Alp4 is required for both the formation of mitotic bipolar spindles and the integrity of interphase cytoplasmic microtubules. (A and B) Centrifugal elutriation. Small early G2 cells of an alp4-1891 strain (DH1891) were collected by centrifugal elutriation and the cultures were divided into two parts, one part incubated at 26°C (A) and the other at 36°C (B). Samples were collected at 20 min intervals, and cell number (open diamonds in dark blue), septation index (open squares in black) and viability (open diamonds in red, the percentage of viable colonies) were measured. Immunofluorescence microscopy with anti-tubulin, anti-Sad1 antibody and DAPI was performed to examine the percentage of cells containing anaphase B nuclei (open circles in green) and abnormal mitotic DNA (open triangles in blue). (C) Abnormally long interphase microtubules in the alp4-1891 mutant. Confocal microscopy was performed using synchronous interphase samples (220 min at 36°C) with anti-tubulin (red) and anti-Sad1 (green) antibodies. Merged images are shown. (D) Aberrant mitotic spindles. Characteristic cells showing monopolar-like spindles (320 min) are presented. (E) Displaced long interphase microtubules. Interphase-like cells with longer cytoplasmic microtubules after an aberrant mitosis (420 min) are shown. (F) Tea1 localization in the alp4-1891 mutant. alp4 mutants were incubated at 26°C for 3 h in the presence of 10 mM HU and shifted up to 36°C after washout of HU. After 2.5 h incubation, cells were fixed and processed for immunofluorescence microscopy using anti-tubulin (left) and anti-Tea1 (middle) antibodies. Merged images are shown on the right. (G) Defects in microtubules by overexpression of alp4+. Cells containing integrated nmt1-alp4+ were grown in the absence of thiamine, and processed for immunofluorescence microscopy using anti-tubulin antibody. Images are from confocal microscopy. Cells from a wild-type control (left) and alp4+ overexpression after 16 h (right) are shown. The bar indicates 10 μm. Download figure Download PowerPoint Immunofluorescence microscopy at each time point enabled us to follow temporally the defective phenotypes of the alp4 mutant and delineate the kinetics of alterations in microtubules. The first phenotype to be seen was the appearance of abnormally long cytoplasmic microtubules. In wild-type cells, the interphase microtubule cytoskeleton consists of several filamentous forms that extend along the long axis of the cell with varied lengths but never curl around the cell tip (Hagan, 1998). In contrast, in the alp4 mutant, the majority of interphase cells had longer microtubules, which curved around the cell end (220 min, Figure 3C). Whilst most of the microtubules in wild-type cells appear to be independent of the SPB (Hagan, 1998), these longer microtubules generally associated with or passed through the SPB. Upon entry into the second mitosis, chromosome separation often occurred, albeit only partially, and the SPB separated into two bodies. However, bipolar spindles were rarely observed, and, instead, aberrant 'monopolar'-like spindles were formed (320 min, Figure 3D). In these cells, spindles generally emanated from only one of the two SPBs. The cell cycle continued despite these mitotic defects and cells exited mitosis as monopolar spindles disappeared and the septum cleaved the undivided nuclei to produce a 'cut' phenotype. At this stage, the interphase microtubules reappeared (420 min, Figure 3E). Again, like the earlier interphase time point (Figure 3C), the length of cytoplasmic microtubules was abnormal and they grew right around the end of the cell. These cytoplasmic microtubules were often formed only in one half of the cell, which corresponded to the side with the displaced nucleus (see Figure 1A). Taken together, this analysis established the notion that Alp4 is required for the formation of cell cycle-dependent microtubule structures. In interphase, it is required to maintain the length of cytoplasmic microtubules and, in mitosis, it is essential for the formation of bipolar spindles. Localization of the end marker Tea1 is not defective in alp4 mutants Abnormally long interphase microtubules have been reported previously in tea1 mutants, which are defective in the cell end marker for growth polarity control (Mata and Nurse, 1997). In the absence of Tea1, which usually localizes to the cell end, as well as along microtubules and at their tips, cytoplasmic microtubules do not terminate at the cell end; instead they curve around the cell cortex. In order to examine whether or not the long microtubules observed in alp4 mutants are attributable to a disfunction of Tea1, double immunofluorescence microscopy was performed with anti-Tea1 and anti-tubulin antibodies. A synchronous culture was prepared using a hydroxyurea (HU) block and release method. Upon washout of HU and release to the restrictive temperature, elongated and curved microtubules were observed (Figure 3F). Co-staining with anti-Tea1 antibody revealed that Tea1 localization is not disturbed. Tea1 localized to both the cell ends and the tips of microtubules. Therefore, the longer cytoplasmic microtubules are not ascribable to Tea1 mislocalization, rather they might be due to defects in SPB function. Longer interphase microtubules were also seen in cells in which alp4+ or alp6+ were overexpressed ectopically. The thiamine-repressible strong nmt1+ promoter was integrated into the chromosomal alp4+ and alp6+ loci just prior to the initiation codon. It was found that ectopic overexpression of alp4+ was toxic and inhibits colony formation on plates in the absence of thiamine (data not shown). Very few mitotic spindles (<0.5%, in contrast to 2–4% in exponentially growing wild-type culture) were observed after 24 h of overinduction. Instead, as in ts mutants, elongated interphase microtubules were seen (16 h after induction, Figure 3G). These results indicate that the protein levels or activities of Alp4 and Alp6 have to be regulated correctly during the cell cycle to ensure the formation of functional interphase microtubules and mitotic spindles. Alp4 function is executed prior to S phase regardless of the fact that the mutant shows mitotic abnormalities in the subsequent M phase We were interested in the cell cycle stage at which Alp4 executes its essential function. The results of the previous elutriation analysis were somewhat unexpected, as, although the alp4 mutant showed a variety of mitotic defects, these phenotypes appeared only in the second mitosis. In order to address the question of when Alp4 executes its essential role, centrifugal elutriation experiments were repeated with the following modifications; after elutriation, the cultures were kept at 26°C for various times (0, 40, 80, 100, 120, 140 and 160 min; Figure 4A), then shifted up to 36°C and the appearance of abnormal mitosis in each sample was followed. The results are summarized schematically in Figure 4B. It transpired from these manipulations that there is a critical time when Alp4 function is executed. Until 100 min when the mutant was finishing the first mitosis (Figure 4A), the defects appeared in the second mitosis, as in the case when the culture was shifted up at 0 min (Figure 4B). On the other hand, if the culture was shifted up at 140 min or later after the first mitosis was complete, the defective phenotypes started to appear upon entry into the third mitosis. Thus, it appeared that the crucial stage at which Alp4 function is executed occurs between 120 and 140 min, which is likely to correspond to G1 phase. These results suggest that an Alp4 function has already been accomplished before S, G2 and M phases, irrespective of the fact that alp4 mutants show defective phenotypes only in the subsequent mitosis. Figure 4.Essential Alp4 function is executed in G1 phase. (A) Elutriation centrifugation and shift-up at different times. Centrifugal elutriation was performed to collect small G2 cells from an exponentially growing alp4 mutant at 26°C, and the synchronized cells continued to grow at 26°C. After various incubation times at 26°C (shown by arrows, 0, 40, 80, 100, 120, 140 and 160 min), the cultures were shifted up to 36°C. The septation index at 26°C is shown (up to the third mitosis). (B) Appearance of cells showing the defective mitosis at different time points. The percentage of cells that show aberrant mitosis after the first (black column), second (grey) or third mitosis (hatched) is plotted. (C) HU block and release experiments. The cultures of alp4 mutant grown at 26°C in rich medium containing 10 mM HU for 3 h were shifted up to 36°C after washing out the HU. The septation index (open squares in black), viability (open diamonds in red), anaphase B nuclei (open circles in green) and abnormal mitotic DNA (open triangles in blue) at 36°C were examined as in Figure 3A and B. (D) Nitrogen deprivation and addition experiments. alp4(LV6) cells starved of nitrogen for 7 h at 26°C were divided into two parts: to one half, fresh rich medium was added and shifted up at 36°C, whilst the other half was kept in nitrogen-deprived medium and shifted up at 36°C. Symbols are the same