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¹ Department of Molecular Biology, and ² Department of Developmental Molecular Anatomy, Graduate School of Medical Science, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
³ Max-Planck-Institut für molekulare Physiologie, Abteilung Strukturelle Biologie, Otto-Hahn-Strasse 11, D-44227 Dortmund, Germany

	Abstract

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RCC1, a conserved chromosomal protein with a seven-bladed propeller is a GDP/GTP nucleotide exchange factor for RanGTPase that mediates various cellular events. We isolated 16 temperature-sensitive (ts) mutants of S. pombeRCC1-homolog, pim1⁺, by error-prone PCR. Five pim1^ts mutants had a single mutation. The obtained pim1^ts mutations and previously reported mutations were localized on similar sites in seven RCC1 repeats. Those mutations resulted in a reduced binding of Pim1 with Spi1. All pim1^ts mutants showed a defect in nucleocytoplasmic protein transports, whereas the majority of them showed a normal mRNA export. In all pim1^ts examined, chromosomal DNA replication was completed. However, mitotic spindle formation was abrogated, the septum was formed being uncoupled with nuclear division and abnormally widened, thus resulting in chromosomal DNA mis-segregation and the accumulation of enucleated cells. As a result, a defect of RanGEF/Pim1 abolished the orchestration of sequential mitotic events, spindle formation, septation and cytokinesis that are essential to produce two identical daughter cells.

	Introduction

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Ran is an abundant Ras-like small GTPase, which is mainly localized in the nucleus (Bischoff & Ponstingl 1991a). The RanGEF: Ran-GDP/GTP exchange factor, RCC1 (Kai et al. 1986; Bischoff & Ponstingl 1991b), is localized on chromatin (Ohtsubo et al. 1989) and RanGAP: RanGTPase activating protein, Rna1 (Bischoff et al. 1995), is in the cytoplasm (Feng et al. 1999). Therefore, a gradient of Ran-GTP concentration exists from the nucleus to the cytoplasm (Kalab et al. 2002). This gradient is important for such Ran-mediated biological functions as nucleocytoplasmic transport, spindle microtubule organization and nuclear membrane-fusion (Kahana & Cleveland 1999; Clarke 2001; Gruss et al. 2001; Dasso 2001, 2002; Hetzer et al. 2002). The Ran-mediated cellular events should be coordinated with the cell cycle time events (Pines 1999). In this regard, it is noticeable that RCC1 is localized on chromatin (Ohtsubo et al. 1989; Frasch 1991; Li et al. 2003), and the replication of chromatin is an important time event of the cell cycle. We have proposed the idea that in order to couple cellular space and time events, chromosomal RCC1 senses the status of chromatin regarding such processes as replication, DNA damage and transcription, and then transfers these data to cellular spatial machines through Ran (Nishimoto 1999, 2000). In this context, RanGEF; RCC1, should interact not only with Ran, but also with several unknown proteins that may be localized on either chromatin or mitotic chromosome. Indeed, Drosophila RCC1 homolog, BJ1, is contained in a large protein complex (Frasch 1991) and mammalian RCC1 has been reported to interact with histone H2A and H2B (Nemergut et al. 2001). The two-hybrid method has been a main avenue to identify an interacting protein with a target protein. However, we could not identify RCC1 interacting protein(s) thus far with this method, while we did obtain a lot of Ran-interacting proteins using Ran, as a bait (Yokoyama et al. 1995; Noguchi et al. 1996, 1997). To identify RCC1-interacting proteins, we then chose a genetic method, namely, the isolation of gene(s) that suppresses the pim1^ts defects when it is over-expressed. As the first step towards this purpose, we presently isolated a series of temperature-sensitive (ts) mutants of S. pombe RCC1-homolog, pim1⁺, using the error-prone PCR as previously described (Oki et al. 1998). Thus far, pim1^ts has been isolated as a cell cycle mutant (Matsumoto & Beach 1991; Sazer & Nurse 1994), sns (septated, not in S phase) (Matynia et al. 1998). The presently identified pim1^ts mutations did not overlap with the previously isolated ones, except for pim1-46^ts (Matsumoto & Beach 1991).

	Results

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Isolation of pim1^ts mutants

First, an appropriate pREP plasmid carrying pim1⁺ was determined based on its ability to rescue a temperature-sensitive lethality of pim1-46^ts (Matsumoto & Beach 1991), since an over-expression of pim1⁺ is lethal for S. pombe (data not shown). The plasmid carrying the weakest nmt promoter, designated as pREP81X-pim1, was found to efficiently rescue a temperature-sensitive lethality of pim1-46^ts. It was introduced into the strain, SP1054 (pim1:: ura4⁺/pim1⁺leu1-32/leu1-32, diploid) (Matsumoto & Beach 1991). Transfected cells were sporulated to obtain a haploid cell, pim1::ura4⁺ carrying pREP81X-pim1 designated as MM-1 (Fig. 1A) that was lethal in the presence, but not in the absence, of thiamine (data not shown). Second, 3.5 kb of DNA fragment containing pim1⁺ ORF was amplified by PCR in an error prone condition as described in the Experimental procedures. The resultant amplified DNA fragments were integrated into the genome of the strain, MM-1 by homologous recombination in a flanking region of pim1⁺ ORF as shown in Fig. 1B. Transfectants were incubated in the presence of 5-FOA (5-FOA is toxigenic for the Ura4 protein) and leucine at 26 °C, to isolate a haploid cell that had the mutagenized pim1 DNA fragment at the chromosomal pim1⁺site, and lost pREP81X-pim1. The colonies that formed were replicated on YE5S plates, containing phloxine B, and each half of plates was incubated at 26 °C and at 36 °C, respectively, to select colonies temperature-sensitive for growth. Finally, 37 pim1^ts mutants were isolated and their mutation sites were determined by sequencing, thus resulting in 16 independent alleles of pim1^ts as shown in Table 1. Their temperature-sensitive lethality was varied among pim1^ts alleles. A representative of results are shown in Fig. 1C.

View larger version (39K): [in this window] [in a new window]   Figure 1  Isolation of pim1ts mutants. (A) Isolation of S. pombe MM-1. From S. pombe diploid strain SP1054, a haploid S. pombe strain, MM-1 was constructed as described. (B) DNA fragment containing pim1+ gene was amplified by error-PCR using a genomic DNA clone from wild-type cells and then it was introduced into the MM-I strain. Transformants were cultivated in EMM2 containing leucine and plated on EMM2 plate containing 5-FOA (1.5 mg/mL) and leucine at 26 °C. Temperature-sensitive colonies were selected as described in the Experimental procedures. (C) Representative results of pim1ts mutants. The temperature sensitivity was checked on EMM plates supplemented with uracil at the indicated temperature.

The mutated amino acid residues of the presently isolated 16 pim1^ts alleles were localized on Pim1 according to the human RCC1 crystal structure model (Renault et al. 2001) (Table 1; Fig. 2), along with the yeast and mammalian temperature-sensitive mutation sites of Prp20/Pim1/RCC1 so far reported (Uchida et al. 1990; Matsumoto & Beach 1991; Kadowaki et al. 1992, 1993; Lee et al. 1994; Matynia et al. 1998; Ideue et al. 2004). Out of 16 presently isolated pim1^ts mutants, 5 pim1^ts mutants had a single amino acid change (Fig. 2, shown by red underlines) while another 5 pim1^ts mutants had two amino acid changes. The rest of the pim1^ts mutants had 3 or 4 amino acid changes. Although we did not determine whether all of the amino acid changes detected were responsible for the temperature-sensitivity in pim1^ts carrying multi hits mutations, the following points should be noted.

View larger version (50K): [in this window] [in a new window]   Figure 2  Localization of ts mutations on human RCC1 structure. Amino acid sequences of (A) human, (B) S. cerevisiae and (C) S. pombe RCC1 homologs aligned based on the crystal structure of human RCC1. Four ß-sheets of each blade are shown by big colored arrows. The squares show conserved amino acid residues. Green arrows are supposed to be the Ran-interacting sites (Renault et al. 2001). Dotted orange vertical lines indicate conserved amino acids among ß-sheets in each blade. The mutation sites of presently isolated pim1ts mutants are shown in red capital letters. The underlined amino acid changes indicate those found in the single point mutants. The previously isolated mutation sites of S. pombe Pim1, S. cerevisiae Prp20 and hamster RCC1 are shown by blue, purple, and brown capital letters, respectively.

8-29 RCC1

A single mutation, L113S, which corresponds to pim1-46^ts (Matsumoto & Beach 1991), is localized at the same upward ß-sheet of the 2nd RCC1-blade as another novel single point mutation, G285E of pim1-26^ts on the 5th RCC1-blade. The other mutation, Q230R of pim1-123^ts (Table 1), is also localized at the same site as pim1-46^ts on a different RCC1-blade (Fig. 2).

Both novel single point mutants, N135S of pim1-121^ts and F201S of pim1-7^ts, possess their amino acid changes at the same domain of the 2nd and 3rd RCC1-blades, respectively, which is confirmed to be the Ran-interacting domain (Renault et al. 2001). Especially, the 201st amino acid, Phenylalanine (F201) is localized on the extra ß-sheet and it is conserved throughout evolution (Renault et al. 2001). The mutations, F256Y and I307M, of pim1-24^ts and pim1-21^ts, respectively, are also localized at sites similar to N135S and F201S on different RCC1-blades. Furthermore, the mutation site of S. cerevisiae srm1-1 is localized near N135S on the same RCC1-blade (Fig. 2). These mutations have a potential to abolish Pim1-Spi1 interaction directly.

The pim1-105^ts has another novel single point mutation, L278S, localized on the inter-blade between the 5th and 6th RCC1-blades. The mutations, P397S and P397L of pim1-6^ts and pim1-11^ts, respectively, were also localized at the same site as L278S of an inter-blade between the 6th and 7th RCC1-blades, thus suggesting these regions also to be important for the activity of RanGEF. These mutations may cause structural changes in the Pim1 proteins at non-permissive temperature as discussed later (see Discussion).

To estimate the relationship between Pim1 mutations and their Ran-binding abilities, the interaction of wild-type and mutated pim1 proteins, designated hereafter as Pim1^wt and Pim1^ts, to Spi1 was examined. Recombinant GST-Spi1 and, as a control, GST alone, both of which were purified as a single band (Fig. 3A), were mixed with the extracts from pim1⁺ and pim1^ts strains. After incubation at either 26 °C (permissive temperature) or 37 °C (non-permissive temperature) for 30 min, GST-Spi1 and GST were pulled down with glutathione beads. The amount of Pim1 bound to Spi1 was then examined by Western blotting. The amount of Pim1^wt bound to Spi1 after incubation at 37 °C was same as that at 26 °C. In contrast, the amount of Pim1^ts bound to Spi1 at 37 °C, however, showed a greater decrease from that at 26 °C. The representative Western blotting are shown in Fig. 3B. Since the total amount of Pim1 in the extracts derived from pim1⁺ and pim1^ts strains did not change after incubation at either 26 °C or 37 °C (Fig. 3C), these results indicated the Spi1-binding ability of Pim1 was affected by the mutation. As shown in Fig. 3D, the Spi1-binding ability of Pim1-7^ts and Pim1-121^ts was considerably reduced compared with other Pim1^ts. This finding is consistent with the fact that these Pim1^ts proteins contain amino acid changes in the Ran-binding domain of Pim1 (Fig. 2).

View larger version (40K): [in this window] [in a new window]   Figure 3  In vitro interaction of Spi1 with Pim1wt and Pim1ts proteins. (A) Five micrograms of purified GST-Spi1 and as a control, GST alone were separated in 5-10% SDS-PAGE and stained with Coomassie blue. (B) GST-Spi1 and as a control, GST alone bound to glutathione sepharose were mixed with the extracts derived from pim1+ (WT) and pim1ts strains, incubated at 26 °C (L) or at 37 °C (H) for 30 min. After incubation, the beads were collected by brief centrifugation. Pim1 co-precipitated with Spi1 was analyzed by immunoblotting with anti-Pim1 antibody. The arrow indicated a position of Pim1 protein. (C) The whole extracts were analyzed by immunoblotting with anti-Pim1 antibody. 0.5% of whole extracts (input) was applied. (D) Arbitrary estimation of the amount of Pim1 bound to Spi1. Three independent pull-down and immunoblotting experiments were carried out. The amount showed as percentage of that from pim1+ strain at 26 °C. Values indicate the mean ± SEM (n = 3).

Ran and its associated factors are required for many cellular processes such as nucleocytoplasmic transport, spindle formation and nuclear membrane fusion. Since we isolated a new series of pim1^ts mutants, it is interesting to ask how the loss of the RanGEF/Pim1 activity affects the Ran-mediated cellular functions in S. pombe in comparison with the previous reports (Matsumoto & Beach 1991; Matynia et al. 1996, 1998).

First, we examined nucleocytoplasmic transport of isolated pim1^ts mutants. As a reporter protein, the GFP-NLS-NES protein containing NLS (nuclear localization signal) and NES (nuclear export signal) was expressed in pim1^ts mutants. GFP-NLS-NES was distributed mainly in the nucleus, but also in the cytoplasm of pim1⁺ and pim1^ts cells at 26 °C, the permissive temperature (Fig. 4A, 26 °C). After incubation at 37 °C for 4 h, however, it was accumulated in a dotted manner on the nuclear periphery of pim1^ts but not pim1⁺ (Fig. 4A and 37 °C), thus suggesting the reporter proteins were stuck in nuclear pores of pim1^ts at the non-permissive temperature. In some cases, they were concentrated as a single spot in the nuclear membrane (Fig. 4B–D). Based on these findings, the nucleocytoplasmic transport of proteins was thus concluded to be defective in pim1^ts mutants.

View larger version (48K): [in this window] [in a new window]   Figure 4  Nucleocytoplasmic protein transport and mRNA export. (A) The indicated pim1ts mutants expressing pREP 3X-NLS-NES-GFP were cultivated until log phase at 26 °C, and then at 37 °C for 4 h, fixed with 3.6% paraformaldehyde. The indicated images were taken using conforcal laser microscope. (B) Several observed patterns of NLS-NES-GFP accumulation in the nuclear envelope. (a) pim1-103ts, (b) pim1-21ts, (c) pim1-7ts and (d) pim1-9ts are representative patterns of single or two spots accumulation of NLS-NES-GFP at 37 °C for 4 h. No clear allele specificity of spot numbers or its location was identified. The images were taken with a conforcal laser microscope. Bar, 2.5 µm. (C) pim1ts mutants were cultivated at 26 °C until log phase, and then incubated at 36 °C for 4 h. Cells were fixed and treated as described in the Experimental procedures. As a control, S. pombe ptr8 was similarly treated. The images were taken with a conforcal laser microscope. Bar, 2.5 µm.

Cell cycle analysis of pim1^ts

Since pim1 has been identified as a cell cycle mutant which is either defective for the coupling S-phase and mitosis (Matsumoto & Beach 1991) or defective for mitosis to interphase transition (Sazer & Nurse 1994), a cell cycle analysis of these present mutants would be the next main issue.

When randomly growing cultures of pim1^ts were incubated at 37 °C for 4 h, most of pim1^ts mutants showed chromosomal DNA mis-segregation (Fig. 5). The observed DNA staining patterns and frequencies are summarized in Table 2. To investigate how these phenotypes appeared, cultures of pim1^ts were synchronized with hydroxyurea (HU) at 26 °C, and then a half of cultures was incubated at 37 °C or at 26 °C without HU. The progression of cell cycle was monitored by a flowcytometry analysis and by calculating the frequency of cells showing divided nucleus, septum, and mitotic spindle (Fig. 6A).

View larger version (130K): [in this window] [in a new window]   Figure 5  Abnormal nuclear divisions of pim1ts. Indicated pim1ts mutants were cultivated at 26 °C until log phase and then incubated at 37 °C for 4 h. The cells were fixed and stained with Hoechst 33342. Bar, 5 µm.

View larger version (166K): [in this window] [in a new window]   Figure 6  Cell cycle analysis of pim1ts. Cultures of pim1+ (WT) and indicated pim1ts mutants: pim1-7ts, pim1-105ts, and pim1-138ts were synchronized with HU (hydroxyurea) at 26 °C for 4 h as described in the Experimental procedures, washed out HU, and then (time point 0 h) a half of cultures was incubated either at 26 °C or 37 °C with the indicated schedules. At the indicated time points, cells were processed for cell cycle analysis as follows. (A) DNA contents of cell cultures incubated at (a) 26 °C or (b) 37 °C were examined by flowcytometry as described in the Experimental procedures. The vertical line indicates the time when cells were collected. (B) Cells collected as shown in (A) were stained with Hoechst 33342 (DAPI) and calcofluor. (A) A synchronous cell culture released from HU arrest at 26 °C was counted for cells with divided nuclei and septum. Closed symbols: pim1+ () and pim1-7ts () indicate the frequency of nuclear divided cells. The cell showed uneven-divided nuclei was counted as divided. Open symbols: pim1+ () and pim1-7ts () indicate the frequency of septated cells. A cell with multisepta was counted one. The insert is the representative nuclear staining taken at 1 h (upper panel) and 3 h (lower panel) after release from HU arrest. DAPI fluorescence images were superimposed on phase contrast images. (b,c) Temperature was shifted to 37 °C after the release from HU arrest. Symbols in (b) and (c): pim1+ (), pim1-7ts (), pim1-105ts () and pim1-138ts () indicate the frequencies of nuclear divided cells (b) and septated cells (c), respectively, at 37 °C. More than 200 cells were examined for each. (C) Mitotic spindle formation. Cells incubated at 26 °C (a) and 37 °C (b) were stained with antibody to tubulin and DAPI. The normal spindle, bold and straight staining with high intensity, was distinguished from abnormal one or interphase microtubules. More than 200 cells were examined. (D) The representative staining with Hoechst 33342 and calcofluor. Three hours after release from HU-arrest at 37 °C, pim1+ (a), pim1-7ts (b), pim1-105ts (c) and pim1-138ts (d) were stained. The white arrows and arrowheads indicated evenly divided nuclei and normal septum, respectively. Cells with abnormal mitosis were indicated by yellow arrows for unevenly divided nuclei or undivided nucleus, and by arrowheads for septa, respectively. WT, ma, mb and mc showed the patterns of cell cycle progression schematized in a model (Figure 7). Bar, 2.5 µm. (E) The representative results of microtubule staining at 37 °C. Three hours after being released from HU arrest, pim1+ (a,b), pim1-105ts (c,d), pim1-7ts (e,f,i,j), and pim1-138ts (g,h,k,l) were stained. The same fields of DAPI staining image superimposed on the phase contrast images (a,c,e,g,i,k) and microtubule images were shown (b,d,f,h,j,l). The white arrows and arrowheads in (a) and (c) indicated evenly divided nuclei and normal septum, respectively. The blue arrow in (g) indicated an enucleated cell. In the tubulin stained cells, normal spindles (white arrows), abnormal spindle-like stainings (yellow) and interphase shape microtubules (green) were identified. WT, ma, mb and mc showed the patterns of cell cycle progression schematized in a model (Figure 7). Bar, 2.5 µm.

At non-permissive temperature, 37 °C, pim1^ts mutants completed DNA replication with similar kinetics to that of wild-type cells (Fig. 6A,b), but showed abnormal mitotic events. The frequencies of the nuclear divided cells and septum formed cells were calculated (Fig. 6B,b,c). Representative staining images of nuclear DNA and septum at 3 h after HU release are shown (Fig. 6D). In the culture of pim1⁺ cells, nuclear division and septum formation peaked at 2.5–3 h after HU-release and declined thereafter (Fig. 6B,b,c). Most of the pim1⁺ cells completed mitosis by 4 h. However, pim1-7^ts strain showed delayed nuclear division and then, an accumulation of chromosomal DNA mis-segregation, due to non-disjunction or uneven-segregation/division of chromosomal DNA (Fig. 6D,b; a cell having unevenly divided nuclei and septum is indicated by mb with yellow arrows and arrowhead, and a cell with the undivided nucleus and septum by mc with yellow arrow and arrowhead). The cells were arrested without cytokinesis, and thus septated cells accumulated. Cells with divided nuclei and multisepta also appeared (Fig. 6D,b; MS). In pim1-138^ts, the nuclear division was strongly abolished but septation took place, thus resulting in an accumulation of the cells with single, undivided nuclei and a thick and wide septum (Fig. 6D,d; mc). As previously reported (Demeter et al. 1995; Matynia et al. 1998), abnormally wide septa were observed in all of presently isolated pim1^ts cells, most prominently in the pim1-138^ts strain. In addition, in the pim1-105^ts strain, unevenly divided nuclei and undivided nucleus appeared (Fig. 6D,c; mb and mc, respectively), but some populations showed a normal nuclear division and septation (Fig. 6D,c; white arrow and arrowhead-ma). A flowcytometry analysis also showed some populations of the pim1-105^ts strain to complete 1st mitosis (Fig. 6A,b; 3 h). These data indicate that the penetration of the defective phenotype caused by the loss of function in Pim1 may be weak in pim1-105^ts.

Since nuclear division is dependent on mitotic spindle formation, we next examined the cells after staining with anti-tubulin. At 26 °C, normal mitotic spindles appeared and then disappeared with similar kinetics between pim1⁺ and pim1^ts strains (Fig. 6C,a). However, at 37 °C, its frequency in pim1^ts strains was very low compared to wild-type cells (Fig. 6C,b). At 37 °C, normal spindles were rarely observed in pim1-7^ts and pim1-138^ts strains. In pim1-105^ts strain, though some populations completed mitosis normally, the frequency of the cells with normal spindles was also quite low (Fig. 6C,b and 6E,g,h,k,l). This should be considered later in our discussion. We showed the representative staining patterns of nuclear DNA and of microtubules at 3 h after HU release (Fig. 6E). In pim1⁺, bold and straight spindle was formed (Fig. 6E,b; WT with white arrows). In pim1^ts mutants at 3 h after HU release, a thin bundle of microtubules (Fig. 6E,f and l; mc with yellow arrow), bold but short bundle (d; ma with yellow arrow) or dispersed microtubule filaments (j; mb and d; ma with yellow arrow) appeared instead of normal spindle. These microtubules were not localized in the central axis of the cell. These defects in spindle formation caused non-segregation (Fig. 6E,e and k; mc with yellow arrow) or uneven division (i; mb with yellow arrows) of chromosomal DNA in all of pim1^ts strains examined, except for the fact that some cells showed evenly divided nuclei in pim1-105^ts (c; ma with white arrows). During cell wall digestion for immunostaining, septa were digested, thus producing smaller size cells with a nucleus and enucleated cells (ex. Figure 6E,g; mc with blue arrow). In both types of cells, microtubules were reorganized into interphase-structure (Fig. 6E,f,h,j,l; mc, mb with green arrow). Taken together, a mitotic spindle formation was concluded to be inhibited in pim1^ts cells at 37 °C. Based on these findings, the chromosomal DNA mis-segregation observed in random growing cultures upon temperature-shift, was thus suggested to be caused by a defect of mitotic spindle formation. In spite of such an aberrant mitosis, a septum was formed and microtubules were re-organized into interphase structures.

	Discussion

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Phenotype of pim1^ts

NLS-proteins are imported into nuclei as a complex containing importin and ß (Weis 2002; Lee et al. 2003) which is decomposed by the aid of nuclear Ran-GTP. For the nuclear export of proteins and the mRNA-protein complexes, Ran-GTP is also required. Therefore, a defect of RanGEF, Pim1, may cause a loss of both nuclear import and export of proteins. The presently obtained pim1^ts showed an accumulation of GFP-NLS-NES in the nuclear periphery, thus indicating a defect of either nuclear import or export, or both to occur. mRNA export seemed to be normal in the majority of pim1^ts. Probably, the residual nuclear Ran-GTP concentration in the pim1^ts cell after incubation at the high temperature should be enough to support mRNA export. It is remarkable that a reporter protein was concentrated at a few spots (Fig. 4B,c,d). This phenotype may be caused by nuclear envelope fragmentation and the altered distribution of nuclear pore complex as reported previously (Demeter et al. 1995).

All of pim1^ts mutants examined showed a defect in chromosomal DNA segregation when the random growing cultures shifted to 37 °C. To clarify this matter, we examined cells using synchronized cultures made by HU arrest and release. A flowcytometry analysis and calcofluor staining revealed that chromosomal DNA replication proceeded at the non-permissive temperature (Fig. 6A,b), and septum was formed in all pim1^ts mutants examined (Fig. 6B,c). However, chromosomal DNA segregation was severely impaired in pim1-7^ts and pim1-138^ts, and cells consistently possessing the DNA content of G1 phase did not appear (Fig. 6A,b). In pim1-105^ts, some population of cells showed normal chromosomal DNA segregation and cytokinesis by a flowcytometry analysis and staining of DNA, though the mitotic spindle formation was impaired. In almost all pim1^ts strains, we could not find a normal, bold and straight mitotic spindle (Hagan & Hyams 1988) with a strong fluorescence intensity. Instead, thin and curved microtubules were frequently observed in all of pim1^ts cells examined at non-permissive temperature. In S. pombe, tubulins have been reported to enter the nucleus prior to nuclear division (Zimmerman et al. 2004), and then mitotic spindle that is bold and straight were formed between divided nuclei as shown in Fig. 7 (Hagan & Hyams 1988). As a result, a defect of nucleocytoplasmic transport may affect mitotic spindle formation.

View larger version (26K): [in this window] [in a new window]   Figure 7  A model of spindle formation estimated based on the microtubule-staining pattern of pim1+ (WT) and pim1ts at 26 °C and 37 °C. The representative staining pattern of the indicated model was shown in Figure 6E by arrows.

Since DNA was replicated and a septum was formed in pim1^ts mutants at a rate similar to that of pim1⁺, the premature chromosome condensation observed in hamster tsBN2 cells (Nishitani et al. 1991) might not occur in pim1^ts mutants. However, in the presently isolated pim1^ts mutants, a septum was formed without a completion of nuclear division, and abnormal wide septum (Demeter et al. 1995; Matynia et al. 1998) was again observed. Taken together, these results indicate that a defect of RanGEF; RCC1/Pim1 abrogates the coupling of sequential mitotic events.

pim1^ts mutations

Most of pim1^ts mutants showed abnormal nuclear division as summarized in Table 2. The terminal segregation patterns differed among the mutants. According to an in vitro binding assay, however, some of pim1^ts mutants showed a significant defect in interaction with Spi1, thus suggesting them to have some structural defects. Based on the reported crystal structure of human RCC1 (Renault et al. 1998), the amino acid changes found in the presently isolated pim1^ts, along with those reported previously, were localized on the seven-blades. Except the N-terminal domain outside of the seven-blades, the positions of mutated amino acid residues were located at similar sites in the seven blades, even in the pim1^ts mutants possessing multiple amino acid changes. These findings suggested that each blade contributes equally to the RCC1; RanGEF activity. Among pim1^ts mutants, the frequency of independently isolated single point mutations, L113S and L278S, is quite high (Table 1). This fact is consistent with the argument that the amino acid change from Leucine (hydrophobic) to Serine (hydrophilic) may reduce the stability of hydrophobic core. Since RCC1 is well conserved functionally and structurally in a variety of organisms (Renault et al. 1998; Kliebenstein et al. 2002), the location of amino acid residues and the mutations of such residues which cause temperature sensitivity, should be conserved. Three of five presently found single point mutations, S. pombe, Asn¹³⁵, Phe²⁰¹ and Leu²⁷⁸, referred to hereafter as (SpAsn¹³⁵), (SpPhe²⁰¹) and (SpLeu²⁷⁸), are conserved and they correspond to human Asn⁹⁴, Phe¹⁴⁶ and Leu²³¹, respectively (Fig. 8B). These residues are important ones from a structural view of RCC1 protein as follows:

View larger version (44K): [in this window] [in a new window]   Figure 8  Localization of presently isolated single point ts mutations on the crystal structure of human RCC1. (A) Upper and lower panels indicated the top view and the bottom view of human RCC1 crystal structure. (B) Relationship of the amino acids between S. pombe Pim1 and human RCC1. (C) The magnified images of two mutation points. Upper panels: Leu231 (SpLeu278), and surrounding hydrophobic residues, Val229 (SpLeu276), Val240 (Ile281), Phe242 (SpSer283), Tyr262 (SpTyr301), and Pro283 (SpPro327). Lower panels: Asn94 (SpAsn135) and its hydrogen bonding partners, Leu99 (SpLeu140), Arg101 (SpArg142) and Thr103 (SpThr144).

Asn⁹⁴ (SpAsn¹³⁵) makes hydrogen bonds with Leu⁹⁹ (SpLeu¹⁴⁰), Arg¹⁰¹ (SpArg¹⁴²) and Thr¹⁰³ (SpThr¹⁴⁴) (Fig. 8C, dotted lines in the lower panel). The distances of these interacting atoms are 2.8Å (Asn⁹⁴-Thr¹⁰³), 3.0Å (Asn⁹⁴-Arg¹⁰¹) and 3.0Å (Asn⁹⁴-Leu⁹⁹), respectively. These amino acids are also highly conserved (Fig. 2) and localized just in front and behind of Asp⁹⁵ (SpAsp¹³⁶), which interacts with Ran (Renault et al. 2001). The mutations of these regions therefore are thus considered to destroy the interaction between RCC1 and Ran by destroying the hydrogen bond.

Compared to previously isolated pim1^ts mutants (Matsumoto & Beach 1991; Matynia et al. 1998), only one out of 16 presently obtained mutants overlapped with pim1-46^ts, suggesting our method to isolate pim1^ts is unique. Using these pim1^ts mutants, we could further clarify the molecular mechanism of how RanGEF couples Ran-mediated mitotic events to other cellular events. To further elucidate this issue, we are currently isolating a muticopy suppressor of pim1^ts mutants.

	Experimental procedures

Top Abstract Introduction Results Discussion Experimental procedures References

 
Yeast strains and methods

Standard S. pombe methods for genetics and culture are as described (Moreno et al. 1991). Yeast transformation was done by electroporation. S. pombe ptr8 was kindly donated by Dr T. Tani (Kumamoto University, Japan). The ts mutants were cultivated at 26 °C, the permissive temperature.

A plasmid, pREP81X-pim1 was constructed by cloning the pim1⁺ cDNA after PCR amplifying. pIRT2-pim1L that contains the genomic pim1⁺ DNA fragment was constructed by amplifying a 3.5 kb fragment with PCR from S. pombe genomic DNA using as primers, 5' primer (pim1-06), GGG GGA TCC ATTA CCG ATA GAT AAA ACCGC, and 3' primer (pim1-07), GGG GAG CTC CAA GGA GAT CCT GTA AC.

Construction of pim1^ts mutants

A diploid strain SP1054 (h+/h– pim1::ura4+/+leu1-32/leu1-32 ura4-D18/ura4-D18 ade6-210/ade6-216) (Matsumoto & Beach 1991), a gift from Dr T. Matsumoto (Kyoto University, Japan), was transformed with pREP81X-pim1. After sporulation, a strain that was depleted of pim1⁺ and kept alive with pREP81X-pim1, was isolated, and named MM-1 (h– pim1::ura4 + leu1-32 ura4-D18 ade6-210 or 6-216, pREP81X-pim1). To isolate pim1^ts mutants, an error-prone PCR method was applied using rTaq polymerase (Takara) in the presence of 3.0 mM Mg²⁺, 0.5 mM Mn²⁺, 0.2 mM dATP and dGTP, and 1.0 mM dCTP and dTTP as previously reported (Oki et al. 1998). As described in Fig. 1, MM-1 cells were transformed with 3.5 kb fragment amplified by error-prone PCR using pIRT2-pim1L as a template. The cells were cultured in EMM2 medium supplemented with adenine, uracil, leucine and thiamine at 26 °C for 5 h, papillated on EMM2 plate containing adenine, uracil, leucine, thiamine and 5-FOA (1.5 mg/mL), and then were incubated at 26 °C. The colonies were replica-plated on YE5S phloxine B plates, incubated at 36 °C, and selected for ts growth. Isolated ts mutants were backcrossed with the wild-type 975 h+cell, and the ts mutants with suitable selection markers were selected for further characterization.

Protein transport assay

pREP-NLS-NES-GFP was transferred into pim1^ts mutants in minimal medium containing 10 µg/mL thiamine plate at 26 °C. The resulting transformants were cultivated in liquid medium until log phase at 26 °C, and then at 37 °C. After incubation for 2, 4 and 8 h, cells were washed with PEM buffer (100 mM PIPES, 1 mM EGTA, 2 mM MgSO₄), fixed with 3.7% paraformaldehyde in PBS for 30 min, washed twice with PEM buffer, stained with Hoechst 33342 (3 µg/mL) in PEM buffer. After washing with PEM buffer, the cells were observed using Conforcal Microscope (Bio-Rad Radiance 2100).

mRNA export assay

We used a modified method of that reported by Kadowaki et al. (1993) and Azad et al. (1997). pim1^ts mutants were cultivated until log phase at 26 °C, and then incubated at 37 °C for 4 h, fixed with 4% paraformaldehyde/0.1 M PBS (pH. 6.9) for 1 h, washed three times with PEMS buffer containing 1.2 M sorbitol and then were treated with PEMS buffer containing zymolyase and novozyme (1 mg/mL each) for 30 min at 37 °C. After washing with PEMS buffer, the cells were loaded on poly lysine coated glasses, fixed with 70%, 90%, 100% Ethanol for each 5 min, treated with 0.3% TritonX-100/4 x SSC (pH 7.0) for 5 min and then were blocked with blocking solution (4x SSC, 5x Denhardt's, 1 mg/mL tRNA (Boehringer), 3% BSA (Sigma), 0.5% Fish Gelatin (Sigma)) at 37 °C for 2 h to be hybridized with digoxygenin labeled (3'-end) oligo-dT(50) (500 pg/mL). After washing with 2x SSC at 42 °C, with 1x SSC and then with 0.1% TritonX-100/4x SSC, cells were incubated in blocking buffer containing 4x SSC, 3%BSA, 0.5% Fish Gelatin, and anti-digoxygenin antibody (Roche) for 1 h. After washing with 4x SSC containing 1% BSA, cells were stained with the secondary antibody, which is Alexaflour 488 conjugated anti-mouse Ig (Molecular Probe). After another three times washing with 4x SSC containing 1% BSA and then with 4x SSC containing 0.1% Triton X-100, cells were mounted with VECTASHIELD (Vector). Cells were observed using Conforcal Microscope (Bio-Rad Radiance 2100).

Preparation of recombinant Spi1

GST fused Spi1 was prepared as previously described (Kusano et al. 2004). Purified GST-fused Spi1 was separated with 5-10% gradient gel (ATTO) and analyzed with immunoblot using anti-Pim1 antibodies (kindly provided by Dr. S. Sazer, Baylor College of Medicine, TX, USA).

Preparation of S. pombe crude extracts

Cells were digested with zymolyase 100T (400 units/mL) in 0.65 M KCl for 15 min at 30 °C, treated with lysis buffer (20 mM HEPES pH 7.9, 150 mM NaCl, 15 mM MgCl₂, 50 mM potassium acetate, 10% glycerol, 5 mM DTT, 100 µM NaVO₄, 1% Triton X-100, 10 µg/mL DNase I, and protease inhibitors, 1 µM pAPMSF, 20 µg/mL aprotinin, 20 µg/mL leupeptin, 20 µg/mL pepstatin, 20 µg/mL antipain) at 25 °C for 10 min.

Flowcytometry analysis

Cells of OD₆₀₀ = 0.5 were incubated with 20 mM HU for 4 h, and then incubated at 26 °C or at 37 °C without HU. At the indicated times, cells were collected, washed with distilled water, treated with 50 µg/mL RNaseA and then stained with 50 µg/mL propidium iodide (Sigma) to be analyzed with FACScan (Becton Dickinson). Analyzed data was shown using Cell Quest program ver. 3.1.

Staining of DNA and septum

The collected pim1⁺ (WT) and pim1^ts cells were washed with PEM buffer, fixed with 3.7% paraformaldehyde for 30 min at room temperature, and then stained with 10 µg/mL of Hoechst 33342 and 4 µg/mL of Calcofluor (fluorescent brightener 28, Sigma) in PEM buffer for 1 h. After washing with PEMS buffer, cells were mounted with VECTASHIELD to be examined with ZEISS Axiophoto.

Immunostaining of tubulin

pim1^ts cultivated at 26 °C, and then at 37 °C was washed with PEM buffer, fixed with 4.0% paraformaldehyde in PBS (pH 6.9) for 60 min, washed three times with PEM buffer, treated with PEMS buffer containing Novozyme (2.5 mg/mL) and Zymolyase (3 mg/mL) for 1 h, washed with ice-cold PEMS buffer, and then mounted on poly lysine coated slide glass. After treatment with PEM buffer containing sodium borohydride (1 mg/mL), the cells were treated with blocking buffer containing 100 mM PIPES pH 6.9, 3% BSA, 0.5% Fish Gelatin, 100 mM lysine-HCl, 1 mM MgSO₄, 0.1% sodium azide for 2 h, and then stained with anti-Tat1 (Sherwin & Gull 1989) at 4 °C overnight. After incubation with the 1st antibody, the cells were treated with the secondary antibody, Alexaflour 488 conjugated anti-mouse Ig or Alexaflour 594 conjugated anti-rabbit Ig as indicated, in blocking buffer for 1 h at room temperature. After washing with PEMS buffer, cells were stained with PEM buffer containing 3 µg/mL of Hoechst 33342 for 3 min. After washing with PEMS buffer, cells were mounted with VECTASHIELD to be examined with ZEISS Axiophoto applied with digital Camera (SPOT, model1.4, DIAGNOSTIC).

	Acknowledgements

 
We would like to thank Drs T. Matsumoto (Kyoto University) for providing the pim1 strain, T. Tani (Kumamoto University) for the S. pombe ptr8 strain, S. Sazer (Baylor College of Medicine) for the anti-Pim1 antibody, and A. Wittinghofer (Max-Planck-Institut) for helpful discussions. We also thank Dr Brian Quinn (Kyushu Medical Communication) for proofreading the English of this manuscript. This work was supported by Grants-in-Aid for Specially Promoted Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

	Footnotes

 
Communicated by: Mitsuhiro Yanagida

^* Correspondence: E-mail: tnishi{at}molbiol.med.kyushu-u.ac.jp

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Received: 27 September 2005
Accepted: 10 October 2005

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