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¹ Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
² Laboratory of Cell Recognition and Responses, Graduate School of Life Sciences, Tohoku University, Sendai 980-8578, Japan
³ Department of Integrated Genetics, National Institute of Genetics, SOKENDAI, Mishima, Shizuoka 411-8540, Japan
⁴ Laboratory of Biological Signaling, Graduate School of Life Science, University of Hyogo, 3-2-1 Kouto, Kamigori, Ako-gun, Hyogo 678-1297, Japan

	Abstract

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Snf2SR, a suppressor of rna1^ts, which is a temperature-sensitive mutation in Schizosaccharomyces pombe RanGAP (GTPase activating protein), possesses both the SNF2 and the helicase domains conserved in the chromatin remodeling SNF2 ATPase/helicase protein family. We have now clarified a function of Snf2SR. Snf2SR indeed showed DNA-stimulated ATPase activity, proving that it is a member of the SNF2 ATPase/helicase family. Consistent with this role, Snf2SR was localized in the nucleus and cell fractionation analysis revealed that Snf2SR was tightly associated with the nuclear matrix. The disruption of snf2SR⁺ was detrimental for a cell proliferation of S. pombe. Snf2SR that did not enhance RanGAP activity by itself, but abolished histone-H3-mediated RanGAP inhibition, as previously reported for the histone H3 methyltransferase, Clr4, another rna1^ts suppressor. In contrast to Clr4, Snf2SR directly bound to the GDP-bound form of the S. pombe Ran homologue Spi1 and enhanced the nucleotide exchange activity of Pim1, the S. pombe RanGEF (guanine nucleotide exchange factor). Over-expression of Spi1-G18V, a Ran GTPase mutant fixed in the GTP-bound form, was lethal to S. pombe snf2SR. Together, our results indicate that Snf2SR is involved in the Ran GTPase cycle in vivo.

	Introduction

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The Ran GTPase cycle functions in cellular spatially regulated events, such as nucleocytoplasmic transport, mitotic spindle formation, and nuclear membrane formation (for reviewed in Weis 2003; Mattaj 2004). The nucleotide exchange factor of Ran, RCC1, is localized on chromatin. In contrast, the Ran GTPase activating protein, RanGAP1/Rna1, is localized in the cytoplasm. Consequently, the concentration of Ran-GTP is high in the nucleus and low in the cytoplasm. This Ran-GTP concentration gradient from the nucleus to the cytoplasm is important for nucleocytoplasmic transport carried out by Ran. However, RanGAP/Rna1 has both a nuclear localization signal (NLS) and a nuclear export signal (NES) (Feng et al. 1999), suggesting that it functions even in the nucleus. Indeed, Segregation Distorter (SD), a naturally occurring meiotic drive system of Drosophila melanogaster that shows preferential transmission of the SD chromosome from SD/SD⁺ heterozygous males (Lyttle 1991), is caused by a mutant allele of RanGAP referred to as Sd-RanGAP, which is enzymatically active but lacks a functional NES (Kusano et al. 2001). In order to analyze the nuclear function of RanGAP, we have isolated a series of temperature-sensitive mutants of Schizosaccharomyces pombe RanGAP, that show a defect in chromosome segregation but not in mitotic spindle formation or spindle pole body localization, at the restrictive temperature (Kusano et al. 2004). Clr4, a histone H3 methyltransferase (Nakayama et al. 2001), and Snf2SR, a putative member of the Snf2 family (Lusser & Kadonaga 2003), rescued the temperature-sensitive lethality of rna1–15^ts (Kusano et al. 2004). Clr4 is required to organize heterochromatin that is essential for centromeric, telomeric, and mating type locus functions (Nakayama et al. 2001). On the other hand, in mammalian cells SNF2 re-arranges or mobilizes nucleosomes depending on transcriptional regulation, DNA repair, homologous recombination, and chromatin assembly (Lusser & Kadonaga 2003). Previously, we showed a tight functional relationship between RanGAP/Rna1 and Clr4 (Nishijima et al. 2006), proving that nuclear RanGAP is involved in heterochromatin formation.

In this study, we found another rna1^ts suppressor, Snf2SR that is a new member of the SNF2 family that possessed the DNA-stimulated ATPase activity characteristic of the SNF2 family and abolished histone-H3-mediated RanGAP inhibition, similarly to Clr4 (Nishijima et al. 2006). Furthermore, Snf2SR directly bound S. pombe Ran GTPase, Spi1-GDP, and enhanced the nucleotide exchange activity of Pim1 S. pombe RanGEF. Since Snf2SR is localized in the nucleus, these results suggest a functional relationship between the Ran GTPase cycle and the SNF2 ATPase/helicase in the nucleus.

	Results

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Snf2SR is a novel member of the SNF2 family

Schizosaccharomyces pombe Snf2SR is a putative member of the SNF2 family, since it possesses both SNF2 and helicase domains conserved in the SNF2 family (Lusser & Kadonaga 2003) (Fig. 1A). A homology search revealed that S. pombe possesses 20 proteins belonging to the SNF2 family. Snf22 (Yamada et al. 2004b) is most similar to S. cerevisiae Snf2p. Snf2SR shows the highest similarity to S. cerevisiae Fun30 (gi171856) (Barton & Kaback 1994): the SNF2 and helicase domains are 84% and 90% similar to Snf2SR, including conserved amino acids (Fig. 1A). To prove that Snf2SR is a novel ATPase belonging to the SNF2 family, recombinant Snf2SR protein was purified as a single band that possessed a molecular mass of 107.9 kDa, the calculated molecular mass of His6-tagged Snf2SR (SPAC25A8.01c) (Fig. 1B, inset). Purified Snf2SR showed the ATPase activity that was further enhanced by the addition of DNA fragments, as reported for S. cerevisiae Snf2p (Laurent et al. 1993) (Fig. 1B). A majority of SNF2 family members are not essential for growth (Lusser & Kadonaga 2003), although they play an important role in chromatin remodeling. To address whether Snf2SR is essential for S. pombe, the snf2SR⁺ gene was disrupted as described in Experimental Procedures (Fig. 2A). The snf2SR strain (TO1) (Table 1) was viable at 26 °C and 30 °C, but neither at 15 °C nor 37 °C (Fig. 2B). Taken together, Snf2SR was found to be a new member of the SNF2 family, loss of which is detrimental for cell proliferation of S. pombe at both high and low temperatures.

View larger version (26K): [in this window] [in a new window]   Figure 1  Snf2SR is a novel member of the SNF2 family. (A) Homology of S. pombe Snf2SR with S. cerevisiae Fun30p and Snf2p. Snf2SR and Fun30p comprise 923 and 1131 aa residues, respectively, and have SNF2 and helicase domains conserved in the SNF2 family. Snf2p comprises 1703 aa residues and has a bromo domain in addition to SNF2 and helicase domains. Values (%) indicate percentage homology to the corresponding domain of Snf2SR that includes chemically conserved amino acid residues. (B) ATPase activity of Snf2SR. 10 nM of Snf2SR was pre-incubated in the presence or absence of plasmid DNA as indicated, and then incubated with 20 µM [-32P]ATP for the indicated times. After the reaction was completed, inorganic phosphate was separated from un-reacted ATP by subjecting the sample to PEI-cellulose thin layer chromatography (TLC). The ratio (%) of radioactivity to that of inputted [-32P]ATP is shown by vertical columns. The open column indicates the amount of inorganic phosphate present in the used [-32P]ATP stock. The inset shows recombinant Snf2SR proteins appearing as a single band. Numbers in the inset represent molecular mass.

View larger version (40K): [in this window] [in a new window]   Figure 2  Colony forming ability of snf2SR. (A) Manipulation of snf2SR+. As described in Experimental Procedures, S. pombe snf2SR+ was disrupted and tagged with HA at the C-terminus. (B) snf2SR+ TN1 (WT) and snf2SR TO1 (snf2SR) strains were cultivated in YE5S medium at 30 °C to OD600nm = 0.5 and then serially diluted one- to fivefold, as shown from left to right. A measure of 5 µL of diluted culture was spotted on YE5S plates containing 50 µg/mL G-418, and incubation was carried out at 15, 26, 30, and 27 °C for 5–7 days.

To determine the cellular localization of Snf2SR, we constructed plasmid pRHA41-Snf2SR containing snf2SR⁺ fused with HA (hemeagglutinin) in frame at the N-terminus (Table 2) that could be over-expressed under the nmt1 gene promoter (Maundrell 1993). In addition, we constructed S. pombe TO2 (Table 1) that expressed the endogenous snf2SR⁺ gene fused with HA in frame at the C-terminus, as shown in Fig. 2A. Immunofluoresence analysis using the mAb (monoclonal antibody) to HA revealed that both the endogenous and the over-expressed Snf2SR proteins were localized in the nucleus (Fig. 3A). Thus, Snf2SR is a nuclear protein.

View larger version (20K): [in this window] [in a new window]   Figure 3  Snf2SR is localized in the nucleus. (A) Exponentially growing cultures of S. pombe TN1 possessing pRHA41-snf2SR (a) and S. pombe TO2 expressing endogenous HA-tagged snf2SR+ (b) in medium lacking thiamine were fixed and stained with mAb to HA (red). DNA was stained with DAPI (blue). Scale bar: 10 µm. (B) Exponentially growing cultures of S. pombe, TO2, expressing endogenous HA-tagged snf2SR+ were harvested and fractionated as described in Experimental Procedures. The indicated fractions were separated by 5–20% gradient SDS-PAGE and blotted onto nitrocellulose membrane to be probed with mAb to HA (Snf2SR) and antibodies to indicated proteins.

In order to determine the chromosomal localization of Snf2SR, exponentially growing cultures of S. pombe TO2 were prepared and fractionated as described (Nishijima et al. 2006). The crude extracts of S. pombe TO2 prepared by lysing spheroplasts with Triton X-100, were divided into soluble and insoluble fractions (Fig. 3B, upper panel). The insoluble fraction containing chromatin was treated with 0.5% NP-40, and then with 0.4 m NaCl. After centrifugation, both precipitated fractions, P1 and P2, were digested with micrococcal nuclease (MNase). The resultant supernatant and precipitated fractions were analyzed by immunoblotting using the mAb to HA, and as controls, antibodies to Rna1, Pim1, Hht1 (S. pombe mammalian H3 homologue), and Spi1. The immunoblotting patterns of proteins used as controls were the same as previously reported (Fig. 3B, lower panel) (Nishijima et al. 2006). Under the same conditions in which Spi1 was easily solubilized into the cytoplasmic fraction, a significant amount of Snf2SR was in the chromatin fraction (Fig. 3B, P1). Although Pim1 was solubilized from the chromatin fraction by incubation in MNase or 400 mM NaCl, some chromosomal Snf2SR was tolerant to these extraction procedures (Fig. 3B, P2 and S2), indicating that it was bound to the chromatin matrix. Thus, there are two pools of cellular Snf2SR that differ in the strength of their association with chromatin.

Snf2SR abolishes histone-H3-mediated RanGAP inhibition

As previously reported (Kusano et al. 2004), over-expression of Snf2SR was shown to rescue rna1^ts in an allele-specific manner, similar to Clr4 (data not shown) (Kusano et al. 2004). These results indicate that Snf2SR might rescue rna1^ts in the same manner as Clr4, which abolished the histone-H3-mediated inhibition of RanGAP activity (Nishijima et al. 2006). As shown in Fig. 4A column 3, histone H3 inhibited the RanGAP activity of Rna1, as reported (Nishijima et al. 2006). Upon addition of Snf2SR, the histone-H3-mediated RanGAP inhibition was abolished (Fig. 4A, columns 4 and 5). The mechanism by which Snf2SR abolishes the histone-H3-mediated RanGAP inhibition seemed to be similar to that of Clr4, since Snf2SR bound core histones (Fig. 4B) but did not directly enhance the RanGAP activity of Rna1 (Fig. 5), as reported for Clr4 (Nishijima et al. 2006).

View larger version (30K): [in this window] [in a new window]   Figure 4  Snf2SR abolishes histone-H3-mediated inhibition of RanGAP activity. (A) A measure of 5 pmol of [-32P]GTP-Spi1 was mixed with 1 pmol of Rna1, 100 ng of histone H3 and an increasing amount of 0.13 and 1.3 µg of Snf2SR, as indicated. After incubation at 30 °C for 10 min, the remaining radioactivity on Spi1 was counted with a liquid scintillation counter. A measure of 5 pmol of [-32P]GTP-Spi1 was also incubated in buffer alone as control. The ratio (%) of radioactivity after incubation with Rna1 to that without Rna1 was shown by vertical columns. Means and SD are based on results of three separate experiments. (B) Snf2SR binds histones. Indicated histones were conjugated to NHS-activated Sepharose 4FF (Amersham Bioscience) in GAP buffer (25 mM Tris–HCl, pH 7.5, 50 mM NaCl, 20 mM MgCl2, 1 mM DTT, 0.05% gelatin) supplemented with 1 mM CHAPS at 4 °C. Purified recombinant His-tagged Snf2SR was incubated with histone beads for 2 h with gentle and continuous agitation. The resultant beads were washed with GAP buffer and boiled in SDS-PAGE loading buffer. Proteins eluted from the beads were examined on SDS-PAGE by immunoblotting with mAb to His-tag. Arrowhead indicates position of Snf2SR. Number on the left is the molecular mass.

View larger version (15K): [in this window] [in a new window]   Figure 5  Snf2SR has no effect on RanGAP activity of Rna1. A measure of 5 pmol of [-32P]GTP-Spi1 was pre-incubated with 0.3 µg of BSA (closed circles), 0.3 µg of Snf2SR (open circles) and 0.15 µg of Snf2SR (open squares), and then incubated with 1 pmol of Rna1 at 30 °C. At the indicated time, the reaction was stopped. The radioactivity remaining on Spi1 was counted with a liquid scintillation counter and normalized with the 0 min time point set at 100%. Means and SD are based on the results of three separate experiments. As control, the ratio of residual radioactivity of [-32P]GTP-Spi1 that was not incubated with Rna1 is shown as open triangles.

Based on the previous result that Rna1 enhanced the activity of Clr4, another suppressor of rna1^ts (Nishijima et al. 2006), we determined the effect of Rna1 on Snf2SR. In contrast to Clr4, Rna1 did not enhance the ATPase activity of Snf2SR (data not shown), suggesting that the relationship of Snf2SR with Rna1 differs from the case of Clr4 and Rna1. Next, we determined a functional relation of Snf2SR with the Pim1 RanGEF, since the accumulation of Ran-GTP in the absence of RanGAP activity might be toxic to S. pombe. The RanGEF activity of recombinant Pim1 was assessed by monitoring its nucleotide releasing and loading abilities, as described (Bischoff & Ponstingl 1991).

By the addition of Pim1, [³H] GDP was removed from Spi1-[³H] GDP, as reported (Bischoff & Ponstingl 1991). With the further addition of Snf2SR, the ability of Pim1 to remove [³H] GDP from Spi1-[³H] GDP was enhanced in a dose-dependent manner (Fig. 6A, open symbols). To further confirm the effect of Snf2SR on the RanGEF activity of Pim1, the ability of Pim1 to load nucleotide onto Spi1 was then examined as reported (Bischoff & Ponstingl 1991) and was found to be enhanced by the addition of Snf2SR (Fig. 6B). DNA fragments that stimulated Snf2SR-ATPase activity (Fig. 1B), showed no effect on Snf2SR mediated enhancement of RanGEF activity (data not shown). Thus, Snf2SR was able to enhance the RanGEF activity of Pim1, probably independent of its ATPase activity.

View larger version (20K): [in this window] [in a new window]   Figure 6  Snf2SR enhances Pim1 RanGEF activity. (A) A measure of 5 pmol of [3H]GDP-Spi1 was pre-incubated in buffer containing 0.3 µg of BSA (closed circles), 0.3 µg of Snf2SR (open circles) or 0.15 µg of Snf2SR (open squares) and then incubated with 0.1 pmol of Pim1 at 30 °C. At the indicated time, the reaction was stopped by adding cold stopping buffer. As control, 5 pmol of [3H]GDP-Spi1 was similarly incubated in buffer alone (without Pim1, closed triangle). The radioactivity remaining on Spi1 was counted with a liquid scintillation counter and normalized with the 0 min time point set at 100%. (B) An amount of 10 pmol of cold GDP-Spi1 was incubated with 18.5 kBq of [3H]GDP in buffer containing 0.02 pmol of Pim1 alone (closed circles) or 0.02 pmol of Pim1 and 8 pmol of Snf2SR together (open circles) at 30 °C. As control, mixtures of 10 pmol of cold GDP-Spi1 and 18.5 kBq of [3H]GDP were similarly incubated in buffer alone (closed squares), or in buffer containing 8 pmol of Snf2SR (open triangles) in the absence of Pim1. At the indicated time, the reaction was stopped and radioactivity loaded on Spi1 was counted with a liquid scintillation counter. Means and S.E. represent the results of three independent experiments.

Previously, Dis3 and histone H2A/2B were reported to enhance the RanGEF activity of Pim1 and RCC1 (Noguchi et al. 1996; Nemergut et al. 2001). While Dis3 binds Ran, histone H2A/2B binds RCC1. In order to address whether Snf2SR binds Spi1 the S. pombe Ran homologue, His-tagged Snf2SR was mixed with GST-fused Spi1. After incubation, His-tagged Snf2SR was significantly co-immunoprecipitated with GST-fused Spi1 as shown in Fig. 7A. The amount of Spi1-bound Snf2SR increased in the presence of the chromatin-associated fraction, but decreased in the presence of the cytoplasmic fraction. The chromatin-associated fraction was prepared as described in Fig. 7A legend, which corresponded to the P1-sup fraction of Fig. 3B. Thus, the chromatin associated fraction used in this experiment contained Pim1 but not Spi1 that is totally solubilized by cell disruption (Fig. 3B and Nishijima et al. 2006). In contrast, the cytoplasmic fraction contained a large amount of Spi1. Therefore, upon addition of the cytoplasmic fraction, the amount of Snf2SR bound to GST-Spi1 might decrease in a competitive manner. Since RanGAP is mainly localized in the cytoplasm, the cytoplasmic fraction likely contains the GDP-bound form of Spi1 rather than the GTP-bound form. To determine which form of Spi1 bound to Snf2SR, His-tagged Snf2SR was mixed with GST-fused Spi1 that bound to GDP, GMPPNP or no nucleotide, as shown in Fig. 7B. His-tagged Snf2SR was preferentially co-immunoprecipitated with GST-Spi1 bound to GDP, but not to GMPPNP. Thus, Snf2SR bound specially to Spi1-GDP that is a natural substrate of RanGEF. In contrast, Pim1 could not be co-immunoprecipitated with Snf2SR (data not shown), suggesting that the interaction of Snf2SR with Pim1 could be transient.

View larger version (23K): [in this window] [in a new window]   Figure 7  Snf2SR binds S. pombe Ran, Spi1. (A) Purified recombinant His-tagged Snf2SR proteins (0.8 µg) were mixed with recombinant GST-Spi1 (2.2 µg) alone (Beads, lane 1), in the presence of either a chromatin-associated fraction (lane 3) or a cytoplasmic fraction (lane 4), as indicated. After incubation at 4 °C for 30 min, GST proteins were precipitated by adding Glutathione Sepharose 4B (Amersham Bioscience). The resultant beads were washed 3 times with PBS containing 1% Triton X-100, 1.0 mM dithiothreitol, 0.1 mg/mL aprotinin, 0.1 mg/mL leupeptin and 0.1 mg/mL pepstatin A, and boiled in SDS-PAGE loading buffer. Proteins eluted from the beads were resolved using 5–20% gradient SDS-PAGE, blotted onto a membrane, and probed with mAb to His-tag. Chromatin-associated and cytoplasmic fractions were prepared as follows: Exponentially growing cultures of S. pombe treated with 1 mM NaN3 were harvested and washed once with distilled water and 0.65 M KCl. Harvested cells were treated with 1 mg/mL Zymolyase (Seikagaku Corporation) at 30 °C in 0.65 M KCl. After incubation for 20 min, Tris–HCl, pH 7.5 (fin. 15 mM), sucrose (fin. 7.5%), and sorbitol (fin. 0.6 M) were added and the mixture was centrifuged. Resultant spheroplasts were washed 4 times with buffer (10 mM Tris–HCl, pH 7.5, 1.2 M sorbitol) and treated with buffer A (20 mM Hepes-NaOH, pH 7.9, 1.5 mM Mg acetate, 50 mM K acetate, 10% glycerol, 0.5 mM dithiothreitol, 0.1 mg/mL aprotinin, 0.1 mg/mL leupeptin and 0.1 mg/mL pepstatin A) supplemented with 1% Triton X-100 and 5 mM ATP. The resultant cell extract was divided into supernatant (cytoplasmic fraction) and precipitate by centrifugation at 20 000 x g for 15 min in a Hitachi RT5S4 rotor. The resultant pellet was finally incubated with buffer A containing 150 mM NaCl and 6 U/mL MNase at 25 °C for 60 min. After incubation, the pellet samples were centrifuged at 20 000 x g for 15 min in a Hitachi RT5S4 rotor. The supernatant was used as the chromatin-associated fraction (chromatin-associated fraction). The ratio of Snf2SR-bound Spi1 to the inputed Spi1 is as follows: lane 1 (10%); lane 2 (0%); lane 3 (31%); and lane 4 (2%). (B) Purified recombinant His-tagged Snf2SR was mixed with recombinant GST-Spi1 loaded with GDP, GMPPNP or none (nucleotide free), as indicated. After incubation at 4 °C for 1 h, samples were incubated with GST beads and then pulled down. The precipitated samples were resolved using 7.5% SDS-PAGE, blotted onto a membrane, and probed with mAb to His.

Over-expression of Spi1 is lethal to snf2SR

The fact that over-expression of Snf2SR rescued rna1^ts, suggested that the Ran-GTP concentration in this mutant might be increased due to a defect in RanGAP and that Snf2SR reduced the Ran-GTP concentration by enhancing the nucleotide exchange activity of Pim1 and abolishing the histone-H3-mediated RanGAP inhibition. To address this issue, Spi1 and its GTP-stabilized mutant form, Spi1-G18V (Lounsbury et al. 1996), were expressed in snf2SR⁺ and snf2SR (Table 1, TO4–TO7) from the weak version of the nmt1 gene promoter. Upon expression of Spi1, the colony forming ability of snf2SR was slightly impaired, compared to that of snf2SR⁺ (Fig. 8). However, when the mutated Ran-GTP form, Spi1-G18V, was expressed, snf2SR did not even papillate. Thus, the loss of Snf2SR was lethal upon over-expression of Spi1-GTP.

View larger version (42K): [in this window] [in a new window]   Figure 8  Over-expression of Spi1-G18V is lethal to snf2SR. pREP42X-spi1-G18V and pREP42X-spi1 as control were introduced into snf2SR+ and snf2SR as indicated, and then incubated on EMM plate supplemented with leucine. The resultant transformants were spread on medium with or without thiamine, and then incubated at 30 °C for 5 days.

	Discussion

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Nucleosome consists of approximately 146 bp of DNA wrapped around a histone octamer containing histone H2A, H2B, H3 and H4. On the other hand, DNA binds to the nuclear matrix through regions called MAR or SAR (for a review, see Hart & Laemmli 1998). These regions are distributed throughout the genome and organize the chromatin into loops of variable sizes. Snf2SR/Fft3 was bound to the nuclear matrix, although it lacks a DNA-binding bromo domain of Snf2p (Fig. 1A) (Lusser & Kadonaga 2003). How Snf2SR/Fft3 binds the nuclear matrix remains to be investigated. It is notable that like Pim1, some Snf2SR/Fft3 was solubilized upon 400 mM NaCl, suggesting that there is some structural differences between the solubilized and nonsolubilized forms of Snf2SR/Fft3 that influences its interaction with the nuclear matrix. Snf2SR/Fft3 abolished histone-H3-mediated RanGAP inhibition, similar to the histone H3 methyltransferase, Clr4 (Nishijima et al. 2006). It directly bound the GDP-bound form of Spi1, S. pombe homologue of Ran GTPase, and stimulated the nucleotide exchange activity of the S. pombe RanGEF, Pim1. The ability of Snf2SR/Fft3 to bind Spi1 was enhanced by the addition of the chromatin-associated fraction of S. pombe that contains chromosomal proteins extractable with high salt or DNase treatment, but not Spi1, as shown in Fig. 3B P1. Although the chromosomal fraction contained Pim1, the S. pombe RCC1 homologue, Pim1 was not co-immunoprecipitated with Snf2SR/Fft3. Thus, there are unknown chromosomal proteins that enhance the interaction of Snf2SR with Spi1.

As shown in Fig. 9, nuclear RanGAP bound with histone H3 could be activated by either Clr4 or Snf2SR/Fft3, while its activation should be transient since RanGAP binds exportin1/Crm1 with the aid of Ran-GTP (Nishijima et al. 2006). Upon activation, RanGAP/Rna1 hydrolyzes Ran/Spi1-GTP. Subsequently, Snf2SR/Fft3 binds the resultant Ran/Spi1-GDP to enhance the nucleotide exchange of Ran/Spi1-GDP carried out by RanGEF/Pim1. Over-expression of Spi1-G18V, the mutated Ran-GTP form, was lethal in S. pombe lacking Snf2SR/Fft3, although the nucleus contains Mog1, a GTP releasing factor for Ran-GTP (Oki & Nishimoto 2000), in addition to a large amount of RanGEF/Pim1. Thus, the lethality of snf2SR to over-expression of Ran-GTP indicates that Snf2SR/Fft3 functionally contributes to the nucleotide exchange of Ran GTPase in vivo. Nuclear Ran-GDP is mainly supplied from the cytoplasm with the aid of NTF2 (Ribbeck et al. 1998) that specifically binds Ran-GDP and inhibits RanGEF activity (Yamada et al. 1998). Nuclear imported Ran-GDP could be released from NTF2 by a putative Ran-GDP dissociation inhibitor (GDI) displacement factor (GDF), in an ATP-dependent manner (Yamada et al. 2004a) that is thought to involve the binding of Ran-GDP for stimulating nucleotide exchange. In this regard, an important question is whether Snf2SR acts like the putative GDF for Ran GTPase.

View larger version (16K): [in this window] [in a new window]   Figure 9  Working hypothesis of Snf2SR in the Ran GTPase cycle. Nuclear RanGAP/Rna1 silenced by histone H3 is released by either Clr4 or Snf2SR to hydrolyze Ran/Spi1-GTP. The GDP to GTP exchange of resultant Ran/Spi1-GDP carried out by RanGEF/Pim1 is enhanced by Snf2SR.

The interaction of the Ran GTPase cycle with chromatin dynamics has been reported elsewhere. S. cerevisiae Ran GTPase, Gsp1p, affects telomeric function through the Sir4 protein (Clement et al. 2006) and is involved in the Tel1 pathway (Hayashi et al. 2007b). S. cerevisiae RanGAP, Rna1p, is also required for telomere silencing (Hayashi et al. 2007a). It is unknown whether the interaction of Gsp1, the S. cerevisiae Ran homologue, with Sir2 could enhance the RanGEF activity of Prp20, the S. cerevisiae RanGEF homologue (Clement et al. 2006).

Taken together with our previous report (Nishijima et al. 2006), the finding that Snf2SR/Fft3 enhanced the RanGEF activity of Pim1 and abolished histone-H3-mediated RanGAP inhibition, indicates that nuclear Ran functions not only for nucleocytoplasmic transport, but also for chromatin dynamics.

	Experimental procedures

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Yeast media and strains

Schizosaccharomyces pombe strains were grown in rich medium (YE5S) or Edinburgh minimal medium (EMM) with appropriate supplements. For selection of kan⁺ transformants, YE5S plate supplemented with final 50 µg/mL G-418 (Sigma-Aldrich, St. Louis, MO) was used. The strains used in this experiment are listed in Table 1.

Cloning of snf2SR⁺

Total S. pombe genomic DNA was obtained using Dr GenTLE^TM for Yeast (TaKaRa Biomedicals, Shiga, Japan). Full-length snf2SR⁺ fragment was amplified by polymerase chain reaction (PCR) as the SalI-BamHI fragment from genomic DNA using primers 5'-GGG GTC GAC TAT GGA TGG AAA AAG AAA AAT AGA GC-3' and 5'-GCG GGA TCC CTA ATC ATC GTC ATC TTC AGC CTC C-3', and then inserted into the SalI/BamHI site of pRHA41 and pRHA42 (Maundrell 1993).

Assay for ATPase

[-³²P]ATP was mixed with buffer containing 20 µM ATP in 12 mM HEPES-NaOH, pH 7.9, 60 mM KCl, 7 mM MgCl₂, 60 ng/mL BSA, 6% glycerol, and 10 nM Snf2SR in the presence or absence of 20 nM pBluescript II TKS⁺ plasmid DNA (Stratagene, La Jolla, CA) in a final volume of 10 µL. After incubation for 30 or 60 min at 30 °C, 2 µL aliquots were quenched in 5 µL of stopping buffer A (50 mM Tris–HCl, pH 7.5, 3% SDS, and 100 mM EDTA). Each sample was spotted onto PEI-Cellulose TLC plate (Sigma-Aldrich) and incubated in 0.5 M LiCl and 1 M formic acid. The radioactivity of separated inorganic phosphates was scanned with a Fuji Bioimage analyzer and quantified using NIH image.

Cloning of spi1⁺

Full-length spi1⁺ was amplified by PCR using primers containing restriction enzyme sites, XhoI and BamHI, 5'-CCG CTC GAG ATG GCT CAA CCA CAA AAC GTT CC-3' and 5'-CGC GGA TCC TTA CAA ATC AGC GTC ATC C-3', from S. pombe genomic DNA. The resultant fragments were digested with restriction enzymes XhoI and BamHI, and then inserted into SalI/BamHI and XhoI/BamHI sites of pRMH41 and pREP42X (Maundrell 1993), respectively. The mutant in which Gly18 was replaced with Val (spi1-G18V) corresponded to the GTP-mutated form (Ran-G19V) of Ran (Lounsbury et al. 1996). The spi1-G18V fragment was amplified by PCR using primers containing restriction enzyme sites, XhoI and BamHI, 5'-CCG CTC GAG ATG GCT CAA CCA CAA AAC GTT CC-3' and 5'-CGC GGA TCC TTA CAA ATC AGC GTC ATC C-3', from pGEX4T-2-spi1-G18V (Table 2). The resultant fragment was inserted into the XhoI/BamHI site of pREP42X (Maundrell 1993).

Disruption of snf2SR⁺

The SacI-SpeI fragment of 0.5 kbp (Fig. 2A, fragment A) downstream from snf2SR⁺ ORF (open reading frame) was amplified by PCR using primers 5'-CCC GAG CTC TTT ATA CAG GGT TTT TTA TGC ATT AG-3' and 5'-CCA CTA GTC TTA GCT TCC ACA TAT AAA GAT TCA GC-3', and then inserted into the SacI/SpeI site of pFA6a-kanMX6 (Bähler et al. 1998), resulting in pFA6a-kanMX6-snf2SR3down. On the other hand, the SalI-BamHI fragment of 0.5 kbp (Fig. 2A, fragment B) upstream from snf2SR⁺ ORF was amplified by PCR using primers 5'-CCC GTC GAC GTT TGC TTC ATC TTA AAC ATC AGG-3' and 5'-CGC GGA TCC AAT TAA GTA ACA ATC AAT GTA TCC-3', and then inserted into the SalI/BamHI site of pFA6a-kanMX6-snf2SR3down. Resultant pFA6a-kanMX6-snf2SR5up3down was digested with SalI and SpeI enzymes to give the SalI-kan-SpeI fragment (Fig. 2A, fragment D) that was introduced into S. pombe strain, TN1 (Table 1) through electroporation. Kan⁺ transformed cells were selected on YE5S plate supplemented with final 50 µg/mL G-418.

HA-tagging at C-terminus of chromosomal snf2SR⁺

Fragment A (Fig. 2A) was cloned into the SacI/SpeI site of pFA6a-3HA-kanMX6 (Bähler et al. 1998), resulting in pFA6a-3HA-kanMX6-snf2SR3down. The SalI-SmaI fragment of 0.5 kbp (Fig. 2A, fragment C) downstream of snf2SR⁺ ORF was amplified by PCR using primers 5'-CCC GTC GAC AAA GAT GAA CCT TGG ATG GAC GCC-3' and 5'-TCC CCC GGG ATC ATC GTC ATC TTC AGC CTC CAC-3', and then inserted into the SalI/SmaI site of pFA6a-3HA-kanMX6-snf2SR3down, resulting in pFA6a-3HA-kanMX6-snf2SR3up3down. This was then digested with SalI and SpeI enzymes. The resultant fragment of SalI-3HA-kan-SpeI (Fig. 2A, fragment E) was introduced into S. pombe strain, TN1 (Table 1) through electroporation. Kan⁺ transformed cells were selected on YE5S plate supplemented with final 50 µg/mL G-418.

Immunolocalization of Snf2SR

Cells expressing HA-tagged Snf2SR were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 1 h at 37 °C, incubated in PBS containing 0.3 mg/mL Zymolyase (Seikagaku Corporation, Tokyo, Japan) at 30 °C, and then treated with 1% Triton X-100 in PBS for 30 s at room temperature. The resultant fixed cells were incubated with an mAb to HA (Sigma-Aldrich) at a dilution of 1 : 100 in PBS containing 10% FCS, for 12 h at room temperature, followed by washing with PBS and incubation for 8 h with Alexa-594-labeled anti-mouse IgG antibody (Molecular Probes, Eugene, OR) at a dilution of 1 : 2000. Stained cells were mounted in fluoromount G containing DAPI (4',6-diamido-2-phenylindole) (EM Sciences, Gibbstown, NJ).

Subcellular fractionation of S. pombe

Exponentially growing cultures of S. pombe were harvested and washed once with NaN₃, 5 times with distilled water, and 5 times with 0.65 M KCl. Harvested cells were fractionated as described (Nishijima et al. 2006).

Guanine nucleotide exchange assay

Nucleotide release
Recombinant Spi1 was incubated with 55.5 kBq [³H]GDP in nucleotide loading buffer (20 mM Tris–HCl, pH 7.5, 10 mM EDTA, 100 mM NaCl, 0.2% Lubrol, 0.5 mM MgCl₂, 1 mM dithiothreitol) for 30 min on ice. The reaction was stopped by the addition of 50 mM MgCl₂ (final concentration) and unloaded nucleotides were removed by passing through a PD-10 gel filtration column (Amersham Biosciences, Uppsala, Sweden). A measure of 5 pmol of resultant [³H]GDP-Spi1 was pre-incubated with Snf2SR and then incubated with 0.1 pmol of Pim1 for the indicated times at 30 °C.

Nucleotide loading
Ten pmol of Spi1 was incubated with Pim1 in buffer (20 mM Tris–HCl, pH 7.5, 2 mM EDTA, 100 mM NaCl, 0.2 mg/mL Lubrol, 50 mM MgCl₂, 1 mM dithiothreitol) containing 18.5 kBq [³H]GDP for the indicated times at 30 °C. The nucleotide exchange reactions were stopped by the addition of cold stopping buffer B (20 mM Tris–HCl, pH 7.5, 25 mM MgCl₂, 100 mM NaCl), and the mixtures were filtered through nitrocellulose filter (0.45 µm, Schleicher & Schuell). The remaining radioactivity on the filter was counted with a liquid scintillation counter.

	Acknowledgements

 
This work was supported by Grants-in-Aid for Specially Promoted Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank Dr Shelly Sazer (Baylor College of Medicine, Houston, USA) for preparing the manuscript.

	Footnotes

 
Communicated by: Fuyuki Ishikawa

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

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Accepted: 3 March 2008

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