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1 Graduate Program in Biophysics and Biochemistry, School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
2 Division of Biology, Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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The fission yeast Schizosaccharomyces pombe is a good organism to study polarized growth and division of the cell. Fission yeast cells are rod-shaped and surrounded by a rigid cell wall, and grow by elongation at the ends where the cell wall is synthesized. In interphase cells, F-actin is organized as cortical patches that are localized at the growing ends of the cell, and also as cables that run along the long axis of the cell (Marks & Hyams 1985; Kanbe et al. 1989; Arai et al. 1998). In mitotic cells, the F-actin patches and cables almost disappear, and the contractile ring is formed at the middle region. Following the contractile ring assembly, F-actin patches reappear adjacent to the contractile ring, and these are thought to deliver materials for formation of the division septum. Around the end of anaphase when the mitotic spindle breaks down, the contractile ring constricts, and the primary septum is deposited in a centripetal manner following the invagination of the plasma membrane (reviewed by Le Goff et al. 1999). The division septum contains sugar polymers such as 1,3-ß-D-glucan, 1,3-
-D-glucan and -galactomannan (Ishiguro 1998). Subsequently, the secondary septa are assembled on either side of the primary septum. Finally, dissolution of the primary septum liberates two daughter cells. Thus, controlled organization of the actin cytoskeleton is required for the cell to undergo polarized growth, cytokinesis and septation (Gould & Simanis 1997; Le Goff et al. 1999). Six Rho GTPases, Cdc42, Rho1, Rho2, Rho3, Rho4 and Rho5 have been identified in S. pombe. The fission yeast Cdc42 and Rho1 are functional homologs of human Cdc42 and RhoA, respectively, and of budding yeast Cdc42p and Rho1p, respectively, and are essential for cell viability (Miller & Johnson 1994; Nakano et al. 1997). Cdc42 has been reported to be involved in determination of cell polarity (Miller & Johnson 1994), while Rho1 is involved in cell wall biosynthesis, maintenance of cell wall integrity and polarization of the actin cytoskeleton (Arellano et al. 1996, 1997, 1999; Nakano et al. 1997). On the other hand, Rho2, Rho3, Rho4 and Rho5 are not essential for cell growth, but they also play important roles in cell morphogenesis and septation (Hirata et al. 1998; Calonge et al. 2000; Nakano et al. 2002, 2003, 2005; Santos et al. 2003).
The S. pombe genome sequence analysis has revealed existence of seven Rho GEFs (Iwaki et al. 2003). Scd1/Ral1 and Gef1, which are GEFs for Cdc42, are not essential for cell viability and share an essential function for Cdc42 (Coll et al. 2003; Hirota et al. 2003). Scd1/Ral1 is necessary for mating and maintaining the cylindrical cell shape (Fukui & Yamamoto 1988; Chang et al. 1994). Gef1 is involved in control of cell polarity and cytokinesis (Coll et al. 2003; Hirota et al. 2003). Rgf3 has recently been shown to act as a GEF for Rho1 and to activate Rho1 function that coordinates cell wall biosynthesis to maintenance of cell wall integrity during septation (Tajadura et al. 2004). On the other hand, functions of other Rho GEFs, Rgf1, Rgf2, Gef2 and Gef3, have not yet been reported in detail. Here we analyzed functions of Rgf1, Rgf2, and Rgf3.
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Results |
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Amino acid sequence analyses of Rgf1, Rgf2 and Rgf3 revealed that they contained a putative DH (GEF) domain which located at the middle of the molecule followed by a PH (pleckstrin homology) domain, and a CNH (citron and NIK1 kinase homology) domain at the C-terminus. Rgf1 and Rgf2 contained a DEP (Dishevelled, Egl-10, and Pleckstrin) domain in the N-terminal half, but Rgf3 did not. All of these Rgfs had similar molecular structures to budding yeast Rho1p-GEFs Rom1p and Rom2p (Supplementary Fig. 1).
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To determine the function of the rgf genes (rgf1+, rgf2+ and rgf3+), we replaced each copy of the whole gene in a diploid strain by homologous recombination with the kanMX6 cassette to confer resistance to G418 on the cell. The cells lacking each of rgf1+ or rgf2+ grew normally on YEA plates at 25 °C, indicating that both rgf1+ and rgf2+ are not essential for cell viability. At 25 °C, however, the 
rgf1 cells grew slowly in a liquid medium, and showed some distorted or shrunken cells (8%; Fig. 1A). We could not detect F-actin structures in these shrunken cells (data not shown). The rgf2 cells did not show any growth or morphology defect in the liquid medium (Fig. 1B). These results suggested that rgf1+ and rgf2+ play less important roles than that of rgf3+ (see below) in the cell. Interestingly, rgf1rgf2 cells, however, showed synthetic lethality (data not shown).
Over-expression of rgf1+ or rgf2+ genes caused morphological changes of the cell
We cloned the rgf genes into the pREP1 plasmid and over-expressed these genes in wild-type cells in EMM in the absence of thiamine. It has been reported that most of the cells over-expressing one of the rgf genes show multiple septa and branched cell shape, and over-expression of Rgf3 interferes with septation and enhances cell-wall synthesis (Iwaki et al. 2003; Tajadura et al. 2004). We analyzed the deleterious effects of overproduction of Rgf1 and Rgf2 on cell wall synthesis and F-actin distribution, respectively (Fig. 1C). In the wild-type cells, both the newly synthesized cell wall region and the septum were stained with Calcofluor. At 18 h after removal of thiamine, most of the cells over-expressing rgf1- or rgf2-gene had multiple septa. Especially, rgf1-over-expressing cells showed dumpy cell shape (Fig. 1Ca). Strong Calcofluor-staining was observed at the septa and some ends of the cells, suggesting that formation of the cell wall and the septum were probably enhanced by over-expression of rgf1 or rgf2 gene. In the rgf1- or rgf2-over-expressing cells, the F-actin patches were randomly dispersed throughout the cortex independently of the cell cycle stage (Fig. 1Cb). In these cells, cytoplasmic MTs were randomly oriented (data not shown). These results confirmed that the Rgf1 and Rgf2 control septum formation, cell wall synthesis and localization of F-actin patches.
Rgf1 and Rgf2 physically and genetically interact with Rho1
The Rgf proteins contained a DH domain which stimulates the exchange of bound GDP to GTP to activate the Rho-family proteins. It has been reported that Rgf3 interacts with Rho1 (Tajadura et al. 2004). To test physical interactions of Rgf1 and Rgf2 with Rho proteins, we carried out a yeast two-hybrid assay. Both Rgf1 and Rgf2 interacted strongly with the dominant-negative Rho1 mutant, Rho1T20N, moderately with the wild-type Rho1, but not with the constitutively active Rho1 mutant, Rho1Q64 L (Table 1). Moderate interaction was seen between Rgf1 or Rgf2 and the dominant-negative Rho5. The results suggested that Rgf1 and Rgf2 serve as GEFs for Rho1 and Rho5 in vivo.
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rga1 cells show a phenotype similar to that of the cells in which Rho1 is excessively activated: the cells are swollen, multiseptated, sometimes branched, and have a thick cell wall (Nakano et al. 1997, 2001). We disrupted either rgf1+ or rgf2+ with kanMX6 cassette in a parental rga1 diploid strain (rga1+/rga1::ura4+). The diploid carrying one of the rga1+ and rgf1+ or rga1+ and rgf2+ copies was induced to sporulate, and subjected to tetrad analysis. Each tetrad analysis showed that Ura+ KanR progeny was recovered, suggesting that both rga1rgf1 cells and rga1rgf2 cells were viable. In these double mutants, the growth and morphology defects of rga1 cells were suppressed, although multiseptated or misshapen cells were occasionally observed (Fig. 2A,B). These results lead us to conclude that both Rgf1 and Rgf2 functionally interact with Rho1, and positively regulate the Rho1 signaling pathway in vivo.
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It has been reported that Rho1 activates the cell wall synthesizing enzyme, ß-glucan synthase, and Rgf3 increases amount of ß-glucan through the Rho1 activity (Arellano et al. 1996; Tajadura et al. 2004). We therefore tested the sensitivity of the other rgf-null or -over-expressing cells to ß-glucanase which lyses the cell wall (Fig. 2Ca,b). Both rgf1 and rgf2 cells were more sensitive to ß-glucanase than wild-type cells, even though rgf2 cells did not show any morphology defect (Fig. 1A). On the other hand, both rgf1- and rgf2-over-expressing cells were more resistant than the cells having empty plasmids. These results, together with those of the two-hybrid assay and the morphological alterations of rgf1-over-expressing cells and rgf2-over-expressing cells as mentioned above, suggested that the Rgf proteins are involved in controlling cell wall synthesis and septum formation through activating Rho1, and thereby ß-glucan synthase activity.
Disruption of rgf3+ causes phenotype distinct from that of rgf1+ or rgf2+
Only one or two spores per tetrad from rgf3/rgf3+ diploid strain (IMRG300) gave rise to colonies which were not resistant to G418. Microscopic observation of rgf3 spores revealed that they could germinate, grow up to some extent, and sometimes branch, but the germs then underwent lysis (Fig. 3A). Since cell wall biosynthesis mutants which tend to lyse can sometimes grow in the presence of osmotic stabilizer sorbitol, we analyzed about 80 tetrads on YE-1.2M sorbitol plates at 25 °C. Only three spores resistant to G418 gave rise to colonies. Interestingly, the rgf3 cells picked up from these colonies could grow without sorbitol although the rate was very slow, suggesting that the growth or cell division after germination is the step very sensitive to osmotic pressure in the rgf3 disruptants. In YE-1.2M sorbitol liquid medium, the growth rate of the rgf3 cells increased. However, it was still slower than that of wild-type cells (Fig. 3B). Ninety two percent of the rgf3 cells were shrunken in YE liquid medium without sorbitol, as compared to 3% with sorbitol. Therefore, the slow growth of the rgf3 cells in YE-1.2M sorbitol medium was not only due to cell lysis, but also to a growth defect.
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rgf3 cells could septate and divide, but these cells exhibited improper deposits of septum materials and incomplete separation of daughter cells (Fig. 6C; marked with small arrowheads and arrows, respectively): the separation initiated from one side of the septum and stopped on the way. We found that contractile ring was not formed in 16% of mitotic cells (Fig. 6C, large arrowhead), while F-actin patches localized normally in interphase cells. F-actin structures were not detected in shrunken cells. These observations suggest that Rgf3 is involved in regulation of assembly of the contractile ring and in deposition of septum materials.
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rgf3 cells are suppressed by Rho1
Next, we investigated whether the severe growth and morphology defects of the rgf3 cells described above were due to reduction of the Rho1 activity. We transformed the rgf3 strain with pSP1rho1 to over-express Rho1 in this strain (Fig. 5). As expected, the over-expression of Rho1 entirely suppressed the defects of septation and contractile ring formation in the rgf3 cells although this transformant grew more slowly than the rgf3 cells containing pSP1rgf3. Similar results were obtained with the rgf3 cells expressing Rho1 or a constitutively active mutant Rho1Q64 L under control of nmt81 promoter in the absence of thiamine (data not shown).
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rgf phenotypes
To examine which part of the Rgf3 molecule is important for its function in vivo, we made a series of plasmids that carried a modified Rgf3 allele. The structures of these plasmids, namely parental pSP1rgf3, pSP1rgf3CNH, pSP1rgf3PH-CNH, pSP1rgf3DH-CNH, pSP1rgf3DH, pREP81rgf3N and pREP81rgf3DH are shown schematically in Fig. 6A. The rgf3 strain or the rgf3+-shut off strain was transformed with each plasmid. The transformants were tested for growth in EMMA at 25 °C (Fig. 6B). Wild-type cells containing pSP1 and those containing pSP1rgf3 grew at the same rate at 25 °C, and wild-type cells and the rgf3+-shut off cells containing pSP1rgf3 also grew at the same rate at 25 °C (data not shown). Expresssion of Rgf3, Rgf3CNH, Rgf3PH-CNH, Rgf3N or Rgf3DH, all of which contained the DH domain, could rescue the growth defect of the rgf3 strain. In contrast, the rgf3 cells expressing Rgf3DH-XNH or Rgf3DH could rarely grow as well as the rgf3 cells. Therefore, the DH domain is most important for the Rgf3 function.
We next tested the ability of rgf genes to rescue the slow growth rate and the low cell viability of the rgf1 and rgf3 strains, respectively, because it was possible that the Rgf proteins have functional redundancy due to structural similarities (Supplementary Fig. 1). Actually, both Rgf2 and Rgf3 suppressed the slow growth rate of rgf1 cells (Fig. 6Ca). Proportion of shrunken cells in the rgf1 strain was decreased by expression of Rgf2 from 8% to 1%, and by expression of Rgf3–4%. In addition, rgf1 cells containing pSP1rgf1 grew at the same rate as wild-type cells containing pSP1 or pSP1rgf1 (data not shown). Both Rgf1 and Rgf2 partially suppressed the growth defect of rgf3 cells. Shrunken cells in the rgf3 strain were decreased by expression of Rgf1 from 92% to 26% and by expression of Rgf2 to 30%.
Localization of the Rgf proteins
To study the in vivo localizations of Rgf1 and Rgf3, the chromosomal copy of each gene was modified to add Yellow fluorescent protein (YFP) to the C-terminus of each Rgf protein. For localization of Rgf2, we utilized rgf2 cells containing pSP1rgf2-yfp. Both Rgf1-YFP and Rgf2-YFP were localized to the cell tips in interphase cells, and the division septum in mitotic cells (Fig. 7A,B). Interestingly, the localization of Rgf3-YFP was different from those of Rgf1-YFP and Rgf2-YFP. Rgf3-YFP did not show any specific localization in interphase cells as described by Tajadura et al. (2004), but it was localized to the division site forming a ring structure in mitotic cells, whose diameter became smaller as the division progressed (Fig. 7A,B). We next investigated the localization of Rgf3 in detail.
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We next examined whether the formation and/or maintenance of the Rgf3 ring was dependent on the contractile ring. We treated rgf3-yfp cells with the actin-depolymerizing drug latrunculin-A (Lat-A). The addition of the drug at 10 µM for 10 min resulted in the disappearance of actin patches and contractile ring, but the Rgf3-YFP signals were still detected at the division site (Fig. 7F).
Although we mentioned above that Rgf3 was involved in contractile ring formation and/or maintenance, it was not clear whether Rgf3-YFP accumulates at the division site during early mitosis. To examine the localization of Rgf3 in early mitosis, we utilized the nda3-KM311 mutant, which has a mutation in the ß-tubulin gene, to arrest cell cycle progression at metaphase (Hiraoka et al. 1984; Kanbe et al. 1989). When the nda3-KM311 rgf3-yfp cells were shifted to the restrictive temperature of 19 °C for 5 h, the Rgf3-YFP signals were detected at the middle of the cell (Fig. 7G). On the other hand, Rgf1-YFP and Rgf2-YFP signals were not detected at the middle of the metaphase-arrested rgf1-yfp and rgf2-yfp cells, respectively (data not shown). We concluded that Rgf3 was localized at the division site forming a ring at least during metaphase, and the ring shrunk following contraction of the contractile ring during cytokinesis.
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Discussion |
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rgf1 and rgf2 cells did not show marked phenotype, rgf1rgf2 cells showed synthetic leathality. The growth and morphology defects of rgf1 were suppressed by over-expression of rgf2, and Rgf1 and Rgf2 showed a similar localization, namely, at the cell tips in interphase and at the septum in dividing cells. Both rgf1 cells and rgf2 cells were susceptible to ß-glucanase, and both rgf1-over-expressing cells and rgf2-over-expressing cells were resistant to the enzyme. Moreover, Rgf1-YFP and Rgf2-YFP signals were not detected in the middle of the cell in metaphase-arrested rgf1-yfp and rgf2-yfp cells, respectively. All of these results suggest that Rgf1 and Rgf2 are functionally redundant, and both of them activate Rho1 to form cell wall and septum.
On the other hand, depletion of Rgf3 caused a tendency to cell lysis and defects in contractile ring formation and septation. Since these defects were suppressed by over-expression of either wild-type or activated Rho1, Rgf3 is likely to serve as GEF for Rho1, similar to Rgf1 and Rgf2. However, the localization of Rgf3 was completely different from those of Rgf1 and Rgf2, and overproduction of Rgf1 or Rgf2 could not completely suppress the defects of rgf3 cells. These results suggest that the role of Rgf3 is distinct from those of Rgf1 and Rgf2. Since Rgf3 was localized between the actomyosin contractile ring and the invaginating edge of the Rho1, it is likely that it activates Rho1 at the invaginating cell membrane in order to form and maintain the contractile ring.
The fission yeast Rho1 is essential for cell viability, and is required for maintenance of cell wall integrity and polarization of the actin cytoskeleton through regulating at least three targets: ß-1,3-glucan synthase and two PKC-related protein kinases Pck1 and Pck2 (Arellano et al. 1996, 1997, 1999; Nakano et al. 1997; Sayers et al. 2000). Our results, as well as those of Tajadura et al. (2004), indicate that the Rgf proteins also bound to the dominant-negative form of Rho5. Rho5 is a structural and functional homolog of Rho1 (Nakano et al. 2005). A double mutant obtained from the rgf3+-shut off strain and rho5 showed severer growth defect than the rgf3+-shut off strain, and both of rho5rgf1 and rho5rgf2 cells are more sensitive to ß-glucanase than each single mutant (unpublished observations). Therefore, the Rgf proteins also may serve as GEFs for Rho5. However, Rho5 may play a minor role than Rho1 probably because its expression level is lower than that of Rho1.
Rho GTPases are involved in cytokinesis in various aspects. In sand dollar eggs, Xenopus eggs and C. elegans embryos, Rho is required for formation of the contractile ring (Kishi et al. 1993; Mabuchi et al. 1993; Jantsch-Plunger et al. 2000). In Drosophila and mammalian cells, RhoA may regulate actomyosin contractility through effector kinases, ROCK/Rho-kinase and/or citron kinase (Kimura et al. 1996; Wissmann et al. 1997; Madaule et al. 1998; Kawano et al. 1999). Moreover, in Drosophila, a mutant in the pebble gene, which encodes a RhoGEF, causes defect in cytokinesis (Prokopenko et al. 1999). The analyses of the rgf3-shut off strain and localization of Rgf3 provided indirect evidence that Rho1 is involved not only in regulaton of septation, cell wall biosynthesis and localization of F-actin patches, but also in contractile ring formation. Rho1 has been localized to the middle cortex of the cell prior to the septum invagination (Nakano et al. 1997). The defect of contractile ring formation in the rgf3 cells was not suppressed by over-expression of known downstream effectors of Rho1, Pck1 and Pck2 (unpublished observation), although the defect was completely suppressed by Rho1. Therefore, Rho1 is involved in contractile ring formation through a pathway that does not contain PKCs. It has been reported that formin-related proteins which generally contain a GTPase-binding domain (GBD)/Rho-binding domain (RBD) at the N-terminus, are involved in cytokinesis in diverse organisms (for review see Wasserman 1998; Tanaka 2000). In budding yeast, Rho1p regulates formin-mediated contractile ring assembly during cytokinesis (Tolliday et al. 2002). In fission yeast, the formin-related protein Cdc12 is required for contractile ring formation (Chang et al. 1997; Arai & Mabuchi 2002). However, upstream regulators of Cdc12 remain unknown: Cdc12 does not bind any of the Rho proteins in fission yeast in two-hybrid analysis, and Cdc12 does not seem to contain an RBD. Our unpublished observation has shown that over-expression of Cdc12 increases the rate of contractile ring formation in rgf3 cells, although this might be a Rho1-independent function of Cdc12. Further analysis is required to elucidate relationship between the Rho proteins, especially Rho1, and Cdc12.
Our unpublished observation showed that Rho1 localized on the cell cortex throughout the cell cycle, and it was accumulated at the division site during septation in rgf3 cells. The activity of Rho1 in the cell may change at regions where RhoGEF or RhoGAP existed. It was found that the rgf3 cells showed severer defect in contractile ring assembly than the rgf3+-shut off cells (final 6% of mitotic cells). This is likely to be due to the fact that the nmt promoter allows some level of expression even under thiamine repressive conditions (Forsburg 1993). Thus, rgf3 may be expressed slightly in rgf3+-shut off cells in the presence of thiamine. On the other hand, the proportion of lysed cells was the same between rgf3 cells and rgf3+-shut off cells. Moreover, it has been reported that the rgf3+ mRNA level changes during division cycle showing a peak before septation (Tajadura et al. 2004). In addition, Rgf1 and Rgf2 localized at the division site during septation. Thus, it is suggested that the activity of Rho1 increases during mitosis and reaches the maximum level during septation.
In the experiment of complementation of the rgf3 phenotype, Rgf3CNH was more effective than the other Rgf3 truncates having a DH domain (Fig. 4A,B). This truncate contains a region necessary for localization to the division site: we have shown that YFP tagged N-terminus region of Rgf3 (1–468 amino acids) is able to localize to the division site (unpublished observation). Rho1 is likely to be activated at the division site since Rgf3 is localized there. Therefore, it is very important to find Rgf3-binding partners in order to understand the Rgf3 signaling pathway, which in turn leads us to understand the Rho1 signaling pathway.
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Experimental procedures |
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S. pombe strains used in this study (Table 2) were grown in YE medium or EMM with appropriate supplements (Moreno et al. 1991). Expression of constructs under control of the thiamine repressible nmt promoter was performed as previously described (Maundrell 1993). MEA was used for induction of conjugation and sporulation. All plates contained 2% agar. Standard procedures for S. pombe genetics were carried out as described in Alfa et al. (1993) and Moreno et al. (1991). Standard methods of DNA manipulations were carried out as described in Sambrook et al. (1989).
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Deletion of each rgf ORF was achieved by homologous recombination (Bähler et al. 1998): kanMX6 cassette was amplified by PCR from kanMX6 plasmid using a forward oligonucleotide corresponding to 60 bp right upstream of each rgf+ ATG codon and a reverse oligonucleotide corresponding to 60 bp right downstream of each rgf+ stop codon, and transformed into a diploid strain obtained by mating JY333 and JY336 or JY741 and JY746 strains. Stable transformants were then selected, and correct integration was verified by Southern blotting and PCR. To analyze the progeny of the rgf/rgf+ strain, sporulation was induced on MEA plates for 3 days and tetrads were dissected using a tetrad dissector (MSM, Singer Instruments). Spores were allowed to germinate and form colonies at 25 °C on YE medium. Finally, colonies were replicated on YEA plates containing 0.1 g/L G418. In order to generate random spores, parental diploid cells were digested by an overnight treatment with 10 µL/mL glusulase (Sigma) dissolved in H2O.
Cloning of rgf genes
The open reading frame (ORF) of each rgf gene was amplified by PCR from a cDNA and/or genomic library. The PCR primers used are as follows. The underlined sequences are sites for restriction enzymes indicated in the parentheses.
(1) rgf1N-5', 5'-GCGGAATTCCCATATGGATTACCGGCATCC-3' (EcoRI and NdeI) and rgf1N-3', 5'-CGGCTCGAGGGTACCCTGTGAATCCACAATTTTTG-3' (XhoI and KpnI)
(2) rgf1C-5', 5'-GGCGAATTCCATATGGTACCCCAGGAGATTGC-3' (BamHI, NdeI and KpnI) and rgf1C-3', 5'-GCCGCATGCTCGAGTTACTTGTCTACATGCTGCTC-3' (SphI and XhoI)
(3) rgf2N-5', 5'-GCGGGATCCTCGAGTATGCTTCGCAATGGAGCTC-3' (BamHI and XhoI) and rgf2N-3', 5'-CCCCTGCAGGAAAGCTGTTCGTTTAAC-3' (PstI)
(4) rgf2C-5', 5'-CGCGGATCCTCGAGTAAGGTCCAACCTG-3' (BamHI) and rgf2C-3', 5'-CCCGCATGCAGATCTATCTTACACAACAAGAG-3' (SphI and BglII)
(5) rgf3N-5', 5'-CCCGTCGACTATGAAGCTCTCCAATGAACTTTTTC-3' (SalI) and rgfs3N-3', 5'-CCCGGATCCCTTAGCTCTAGATTGACAC-3' (BamHI)
(6) rgf3C-5', 5'-CGCGTCGACCGAATTCACTTCTCTGCCAGGAC-3' (SalI and EcoRI) and rgf3C-3', 5'-GCGCCCGGGTTAACTTGTAATAAATATG-3' (SmaI and HpaI)
The PCR products of 5' halves of the genes, rgf1N, rgf2N and rgf3N, and those of 3' halves of the genes, rgf1C, rfg2C and rgf3C, were digested with appropriate restriction enzymes, and then the full length of the rgf genes were cloned into pUC18 or pBluescript SK-.
Epitope-tagged strains
Strains expressing epitope-tagged proteins were constructed using a PCR-based approach (Bähler et al. 1998) and transformed into the diploid JY333/JY336 or JY741/JY746 strain, and parental diploid cells were subjected to the random spore analysis. Each ORF was tagged at the 3' end of its endogenous locus with a variety of epitope-tagged kanMX6 cassettes. Appropriate tagging was confirmed by PCR and either microscopic or immunoblot analysis of the cells.
Fluorescence microscopy
Cells were fixed in 3% paraformaldehyde for 1 h, and processed for immunofluorescence microscopy as previously described (Alfa et al. 1993) using an anti-tubulin monoclonal antiserum (TAT1; a kind gift from Dr K. Gull). The secondary antibody used was rhodamine-conjugated anti-mouse IgG IgG (goat) (Molecular Probes). F-actin was stained with BODIPY-FL-phallacidin (Molecular Probes). Cell wall and septum were stained with Calcofluor White (Sigma). DNA was stained with DAPI (Sigma). Stained cells were viewed by two approaches. Conventional fluorescence and DIC images were obtained using a Zeiss Axioskop fluorescence microscope (Carl Zeiss) equipped with a Plan Apochromat X63 objective lens. Three-dimensional (3-D) reconstitution of fluorescence images from optical sections was performed as described by Arai & Mabuchi (2002) using a Delta Vision system (Applied Precision) attached to an Olympus IX-70-SIF fluorescence microscope equipped with a UplanApo X100 objective lens (Olympus).
Two-hybrid system
The interaction between proteins was examined by a two-hybrid system utilizing the expression of the HIS3 reporter gene as previously described (Vojtek et al. 1993). rho1T20N, rho2T22N, rho3T27N, rho4T28N, rho5T20N, or cdc42T17N, in which the Cys residue in the CAAL-motif was replaced with Ser in order to abolish isoprenylation at the C-terminus, were cloned into the bait plasmid pBTM116, and each rgf-DH domain into the prey plasmid pGAD424. Rgf1-DH, Rgf2-DH or Rgf3-DH contained the amino acid residues 607
1009, 396653, or 431687 of each Rgf protein, respectively.
Sensitivity to ß-glucanase
Exponentially growing cells were washed with and resuspended in TE (10 mM Tris-HCl, pH 7.5 and 1 mM EDTA) at a concentration of 1 x 107 cells/mL. After the addition of ß-glucanase (Zymolyase-100T, Seikagaku Kogyo Co.) at 30 °C, lysis of the cells was monitored by measuring optical density at 600 nm.
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Acknowledgements |
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Footnotes |
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aPresent address: Institute of Biological Sciences, University of Tsukuba, 1-1-1 Tennohdai, Tsukuba, Ibaraki 305-8577, Japan.
* Correspondence: E-mail: mabuchi{at}ims.u-tokyo.ac.jp |
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Received: 13 July 2005
Accepted: 21 September 2005
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) were cultured in YE medium at 25 °C. b, wild-type cells containing pREP81 (
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) pSP1rgf3
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