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The Highly Conserved Protein Methyltransferase, Skb1, Is a Mediator of Hyperosmotic Stress Response in the Fission Yeast Schizosaccharomyces pombe*

Shilai BaoDagger §, Yibing QyangDagger §, Peirong YangDagger §, HyeWon KimDagger , Hongyan DuDagger , Geoffrey BartholomeuszDagger , Jenny HenkelDagger , Ruth PimentalDagger , Fulvia Verde, and Stevan MarcusDagger ||

From the Dagger  Department of Molecular Genetics and the Graduate Program in Genes and Development, University of Texas, M. D. Anderson Cancer Center, Houston, Texas 77030 and the  Department of Biochemistry and Molecular Biology, University of Miami, School of Medicine, Miami, Florida 33136-1015

Received for publication, February 20, 2001

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The p21-activated kinase, Shk1, is required for cell viability, establishment and maintenance of cell polarity, and proper mating response in the fission yeast, Schizosaccharomyces pombe. Previous genetic studies suggested that a presumptive protein methyltransferase, Skb1, functions as a positive modulator of Shk1. However, unlike Shk1, Skb1 is not required for viability or mating of S. pombe cells and contributes only modestly to the regulation of cell morphology under normal growth conditions. Here we demonstrate that Skb1 plays a more significant role in regulating cell growth and polarity under conditions of hyperosmotic stress. We provide evidence that the inability of skb1Delta cells to properly maintain cell polarity in hyperosmotic conditions results from inefficient subcellular targeting of F-actin. We show that Skb1 localizes to cell ends, sites of septation, and nuclei of S. pombe cells. Hyperosmotic shock results in substantial delocalization of Skb1 from cell ends and nuclei, as well as stimulation of Skb1 protein methyltransferase activity. Taken together, our results demonstrate a new role for Skb1 as a mediator of hyperosmotic stress response in fission yeast. We show that the protein methyltransferase activity of the human Skb1 homolog, Skb1Hs, is also stimulated by hyperosmotic stress in fission yeast, providing evidence for evolutionary conservation of a role for Skb1-related proteins as mediators of hyperosmotic stress response, as well as mechanisms involved in regulating this novel class of protein methyltransferases.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Skb1/Hsl7-related proteins have been highly conserved through evolution, with homologs identified in eukaryotes from yeast to human (1-3). The fission yeast Skb1 protein was identified from a two-hybrid screen for proteins that interact with the p21-activated kinase (PAK)1 homolog, Shk1 (also known as Pak1 and Orb2; Refs. 1, 4, 5). Shk1 is essential for cell viability, establishment and maintenance of cell polarity, and normal mating response in fission yeast (1, 2, 4-6). Genetic and molecular data suggest that Shk1 is a critical effector for the Rho-type p21 GTPase Cdc42, which like Shk1 is required for viability, morphological polarity, and normal mating of Schizosaccharomyces pombe cells (4-6). Cdc42 and Shk1 interact functionally with Ras1, the single known fission yeast homolog of the mammalian Ras p21 GTPase (1, 4, 5, 7-9). Ras1 is required for normal morphology and mating of S. pombe cells, but unlike Cdc42 and Shk1, it is not essential for cell viability (7, 8). Skb1 interacts with the amino-terminal regulatory domain of Shk1, and co-overexpression of the two proteins suppresses the morphological defect of ras1Delta cells (1). These and additional genetic data implicate Skb1 as a positive modulator of Shk1. However, unlike Shk1, Skb1 is not required for cell viability, morphological polarity, or mating of S. pombe cells (1). Indeed, the only defect previously attributed to skb1Delta mutants under normal growth conditions is that they divide at a length slightly shorter than that of wild-type S. pombe cells (1, 2). In contrast, overexpression of Skb1 results in a substantial delay in G2/M progression, suggesting that Skb1 has a dose-dependent mitotic inhibitory function. The G2/M delay caused by Skb1 overexpression is dependent on both Shk1 and the Cdc2 inhibitory kinase Wee1 (2).

The budding yeast homolog of the skb1 gene, HSL7, was discovered from a screen for mutations that are synthetically lethal in combination with a deletion of the amino terminus of histone H3 (3). The same screen resulted in the identification of mutations in genes encoding budding yeast homologs of the fission yeast cyclin-dependent kinase Cdc2 (Cdc28) and the Wee1 inhibitory kinase Nim1 (Hsl1). Loss-of-function of Hsl7 results in a delay in G2/M progression, suggesting that Hsl7, in contrast to Skb1, functions as a mitotic inducer (3). The G2/M delay of the hsl7 mutant can be suppressed by a null mutation in the swe1 gene, suggesting that Hsl7 is an inhibitor of Swe1 (3). This role for Hsl7 is supported by the findings of McMillan et al. (10), who provided evidence that Hsl7 acts in concert with Hsl1 to target Swe1 for degradation in S. cerevisiae, and by those of Shulewitz et al. (11), who showed that phosphorylation and ubiquitinylation of Swe1, modifications that target Swe1 for degradation, are substantially reduced in cells lacking Hsl7.

The cellular functions of metazoan Skb1/Hsl7-related proteins have yet to be defined, however, a human Skb1/Hsl7 homolog, Skb1Hs (also known as IBP72 (13) and JBP1 (14)), can substitute for Skb1 in fission yeast, suggesting that Skb1 protein function has been substantially conserved through evolution (2). Skb1Hs has been shown to associate with several different proteins in mammalian cells, including the tyrosine kinase JAK2 (14), the subtype 1 somatostatin receptor (15), and a protein of unclarified function, pICln (13). The biological significance of the interactions between Skb1Hs and these various proteins has not yet been established.

Skb1/Hsl7-related proteins lack significant structural homology to any other characterized proteins. However, Pollack et al. (14) noted that among the proteins with which Skb1Hs exhibits relatively weak homology (E >10-5) were several related protein arginine methyltransferases. These investigators also demonstrated that immunoprecipitates of Skb1Hs contain protein methyltransferase activity, suggesting that Skb1Hs either possesses an intrinsic protein methyltransferase function or associates with a protein methyltransferase (14).

In this report, we show that Skb1, which is largely dispensable for the regulation of cell morphology under normal conditions, is a hyperosmotic shock stimulated enzyme required for normal cell viability and morphological polarity under conditions of hyperosmotic stress. We also provide molecular evidence for evolutionary conservation of a role for Skb1/Hsl7-related proteins as mediators of hyperosmotic stress response, as well as Skb1/Hsl7 regulatory mechanisms in eukaryotic organisms. Our results provide important new insights into the cellular roles of this novel class of protein methyltransferases.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
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Yeast Strains, Manipulation, and Plasmids-- S. pombe strains used in this study were SP870 (h90 ade6-M210 leu1-32 ura4-D18) (D. Beach), SKB1U (h90 ade6-M210 leu1-32 ura4-D18 skb1::ura4) (1), CHP428 (h+ ade6-M210 his7-366 leu1-32 ura4-D18) (from C. Hoffman), and SMMG100 (h+ ade6-M210 his7-366 leu1-32 ura4-D18 skb1::ura4) (1). S. pombe cultures were grown in either rich medium (YEA) or in synthetic minimal medium (EMM) with appropriate auxotrophic supplements (16). The plasmids pAAUGST (2), pAAUGST-Skb1 (2), pART1CM (4), pART1CMSkb1 (2), and pART1CMSkb1Hs (2) have been described. pREPUHA-Skb1 was constructed by cloning a 2.4-kilobase BamHI-SacI fragment of the Skb1 protein coding sequence isolated from pART1CMSkb1 into the ura4-based plasmid, pREP4XHA (a gift from E. Chang). This plasmid allows for expression of a triple HA epitope-tagged Skb1 protein from the nmt1 promoter (17).

F-actin Staining and Indirect Immunofluorescence Microscopy-- F-actin was visualized using rhodamine-phalloidin as described (16). Indirect immunofluorescence microscopy of HA-Skb1 was performed using mouse monoclonal anti-HA antibody 12CA5 (19) and goat anti-mouse fluorescein isothiocyanate-conjugated secondary antibody (Pierce), essentially as described (18).

Protein Methylation Assays-- S. pombe cells transformed with the plasmids pART1CM, pART1CMSkb1, or pART1CMSkb1Hs were grown in 200 ml of EMM to about 107 cells/ml prior to harvesting of cells by centrifugation. Cultures were diluted with an equal volume of either EMM or EMM containing M KCl and grown for 15-60 min prior to harvesting. Cell lysates were prepared as described (6). Immunoprecipitations were performed by incubating extract volumes containing 2 mg of protein with 5 µl of anti-c-Myc monoclonal antibody 9E10 ascites (20) and 25 µl of protein A-agarose beads (Roche Molecular Biochemicals) for 2 h at 4 °C. Immune complexes were pelleted by centrifugation and washed three times with 1 ml of yeast lysis buffer and then twice with 1 ml of methylation assay buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA). Samples were divided into 2 equal volumes, one of which was pelleted and resuspended in 40 µl of methylation buffer and the other pelleted and resuspended in SDS-PAGE sample buffer. GST-Skb1 was expressed in the E. coli host strain BL21 from the plasmid pRP259 (a gift from E. Chang), a derivative of pGEX-1 (Amersham Pharmacia Biotech). GST-Skb1 was purified from bacterial cell lysates using glutathione-agarose beads following the manufacturer's recommendations (Amersham Pharmacia Biotech). Beads containing GST-Skb1 were resuspended in methylation buffer. Methylation assays were performed essentially as described (14). To initiate the methylation reaction, 5 µl of [3H]adenosyl methionine (specific activity, 78 Ci/mmol) (PerkinElmer Life Sciences) and 5 µl of myelin basic protein solution (12 mg/ml in methylation buffer) were added to each 40-µl volume of immune complex or GST-Skb1 beads in methylation buffer and incubated 30 min at 30 °C. Reactions were terminated by placing on ice, adding SDS-PAGE sample buffer, and boiling for 5 min. Reactions were resolved by SDS-PAGE and exposed to x-ray film for 3-6 days. The remaining portion of each immune complex was boiled for 5 min and then subjected to SDS-PAGE and subsequent Western blot analysis to measure the relative amount of Skb1 protein isolated from each GST precipitation or immunoprecipitation.

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Skb1 Is Required for Normal Cell Viability and Polarity in Hyperosmotic Medium-- Under normal growth conditions, S. pombe skb1Delta mutants are only modestly defective in cell growth and divide at a length just slightly shorter than that of wild-type S. pombe cells. We demonstrated previously that S. pombe mutants carrying a null mutation in the skb5 gene, which encodes an SH3 domain protein that directly activates Shk1, are unable to maintain cell polarity under conditions of hyperosmotic stress (21). Furthermore, PAKs have been implicated in the regulation of osmotic stress response in budding yeast and mammalian cells (22-24). We therefore determined whether Skb1 might play a more prominent role in regulating cell viability or morphology when S. pombe cells are subjected to hyperosmotic stress. Although not completely inhibited for growth, skb1Delta cells grew substantially slower than wild-type S. pombe cells on minimal medium containing 1.5 M KCl (Fig. 1A). We observed further that skb1Delta cells exhibited a marked defect in the ability to maintain cell polarity in hyperosmotic medium, becoming stubby to round in appearance at a high frequency when compared with wild-type S. pombe cells, which retained a primarily rod-like appearance under the same conditions (Fig. 1B). These observations demonstrate that Skb1, although largely dispensable for growth and maintenance of cell morphology under normal growth conditions, plays a more significant role in regulating cell growth and polarity under conditions of hyperosmotic stress.


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Fig. 1.   Skb1 is required for normal growth and control of morphological polarity in hyperosmotic medium. A, wild-type and skb1Delta S. pombe cells were streaked onto either EMM agar (top) or EMM agar containing 1.5 M KCl (EMM+KCl; bottom) and incubated at 30 °C (4 days for EMM plates and 7 days for EMM+KCl plates). B, wild-type and skb1Delta cells were grown in EMM or EMM+KCl liquid for 3 days prior to photomicroscopy.

skb1Delta Mutants Are Defective in Restoring Localization of F-actin Patches to Cell Ends After Hyperosmotic Shock-- During interphase, cortical F-actin patches are concentrated at the growing ends of S. pombe cells (25). When S. pombe cells are subjected to hyperosmotic shock, F-actin patches become transiently delocalized from the cell ends and randomly distributed but eventually redistribute to the cell ends after continued incubation in hyperosmotic medium (26). To determine whether the inability of skb1Delta mutants to properly maintain cell polarity in hyperosmotic medium might correlate with a defect in regulating the polarization of F-actin patches, we examined the appearance of F-actin in wild-type and skb1Delta S. pombe cells after subjecting them to hyperosmotic stress. Consistent with previously reported observations (26), we found that F-actin patches became delocalized from cell ends and randomly distributed in both wild-type and skb1Delta cells within 30 min of exposure to EMM containing 1 M KCl (Fig. 2, EMM+KCl). After 2.5 h of incubation in EMM + 1 M KCl, F-actin patches became substantially redistributed to the cell ends in wild-type S. pombe cultures but remained depolarized in skb1Delta cells (Fig. 2). After 3.5 h of incubation in 1 M KCl, cortical F-actin was redistributed to the ends of skb1Delta cells but at a lower frequency than in cultures of wild-type cells. These results suggest that the failure of skb1Delta mutants to properly maintain morphological polarity in hyperosmotic medium is likely to be due, at least in part, to a defect in effectively repolarizing the localization of F-actin patches to the cell ends, a process viewed as essential for establishing and maintaining morphological polarity in S. pombe cells (25).


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Fig. 2.   Skb1 is required for effectively redistributing F-actin to cell ends after hyperosmotic shock. A and B, wild-type and skb1Delta S. pombe cells were grown in EMM and then were transferred to EMM containing 1 M KCl and incubated at 30 °C. Cells were fixed and stained with rhodamine-phalloidin at the indicated times for visualization of F-actin by fluorescence microscopy. A, fluorescence photomicrographs of F-actin in wild-type (left) and skb1Delta (right) cells at 0, 60, and 150 min after exposure to hyperosmotic medium. B, percentages of wild-type (black bars) and skb1Delta (gray bars) cells with F-actin patches at the cell ends at indicated times after exposure to hyperosmotic medium. See text for description of results.

Skb1 Localizes to Cell Ends, Sites of Septation, and Nuclei in S. pombe Cells-- To obtain additional insights into the role of Skb1 as a morphological regulator, we performed experiments to examine its subcellular localization. To do this, we constructed the plasmid pREPUHASkb1 for expressing Skb1 as a triple hemagglutinin epitope-tagged protein (HA-Skb1) from the thiamine-repressible nmt1 promoter (17). S. pombe cells transformed with pREPUHASkb1 were grown in medium containing thiamine to repress expression of HA-Skb1, then transferred to medium lacking thiamine, and grown for 11 h to derepress HA-Skb1 expression prior to immunofluorescence microscopy. HA-Skb1 protein was detected at either one or both cell ends in interphase cells and at what appeared to be the nuclear periphery in both interphase and mitotic cells (Fig. 3, A and B). In a small percentage of dividing cells, we were able to detect HA-Skb1 at the septum-forming region (Fig. 3B). The localization of Skb1 to cell ends is consistent with its role as a regulator of morphological polarity, because a number of S. pombe proteins required for proper control of cell polarity has been shown to localize to the cell ends (27, 28).


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Fig. 3.   Skb1 localizes to cell ends, sites of septation, and nuclei of S. pombe cells. A, indirect immunofluorescence photomicrograph of HA-Skb1 in wild-type S. pombe cells. Inset, cells transformed with HA control plasmid. B, HA-Skb1-expressing cells were immunostained for detection of HA-Skb1 and counterstained with 4',6-diamidino-2-phenylindole (DAPI) to visualize nuclei. I, interphase; M, mitosis; S, septation.

Hyperosmotic Shock Induces Rapid Delocalization and Enzymatic Stimulation of Skb1-- To obtain additional molecular evidence of a role for Skb1 as a mediator of hyperosmotic stress response, we determined whether its subcellular localization or protein methyltransferase activity are affected by hyperosmotic shock. S. pombe cells expressing HA-Skb1 were grown in EMM and then shifted to EMM with 1.5 M KCl. Culture samples were then processed for immunostaining at various time points after transfer to hyperosmotic medium. We found that the frequency of cells exhibiting HA-Skb1 at cell ends and nuclei was markedly reduced within 15 min of hyperosmotic shock (Fig. 4). After this initial delocalization, HA-Skb1 protein returned substantially to the cell ends and nuclei within 1 h of exposure to 1.5 M KCl.


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Fig. 4.   Hyperosmotic shock triggers a rapid delocalization of Skb1 from cell ends and nuclei. HA-Skb1 expressing S. pombe cells were grown in EMM and then were shifted to EMM containing 1.5 M KCl. Culture samples were fixed at the indicated times for detection of HA-Skb1 protein by indirect immunofluorescence microscopy. The percentage of cells exhibiting HA-Skb1 localization at cell ends (black bars) and nuclei (gray bars) is indicated in the graph.

Immune complexes of both human and budding yeast homologs of Skb1 have been shown to possess protein methyltransferase activity. We determined that recombinant Skb1 protein purified from either fission yeast or bacterial cells likewise possesses protein methyltransferase activity (Fig. 5A), thereby demonstrating for the first time that this activity represents an intrinsic enzymatic function of this class of proteins. To examine whether Skb1 methyltransferase activity is stimulated by hyperosmotic shock, cells expressing a c-Myc epitope-tagged Skb1 protein were grown in EMM and were then shifted to either EMM or EMM + 1.5 M KCl and incubated for 15 to 60 min prior to lysing the cells and assaying for Skb1 protein methyltransferase activity. As shown in Fig. 5B, Skb1 methyltransferase activity was stimulated within 15 min of hyperosmotic shock. We conclude from these experiments that hyperosmotic shock induces both a delocalization of Skb1 protein from cell ends and nuclei, as well as a concomitant increase in Skb1 protein methyltransferase activity.


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Fig. 5.   The protein methyltransferase activities of S. pombe and human Skb1 proteins are rapidly stimulated by hyperosmotic shock of S. pombe cells. A, fission yeast Skb1 possesses intrinsic protein methyltransferase activity. The panel at the far left shows MBP methylation (top) and immunoblot analysis (bottom) of GST and GST-Skb1 isolated from S. pombe cells. The adjacent panel shows results of the same analyses for GST and GST-Skb1 isolated from E. coli cells. The far right panels show MBP methylation (top) and immunoblot analysis of c-Myc epitope-tagged S. pombe (CM-Skb1) and human (CM-Skb1Hs) proteins expressed and purified from S. pombe cells. B, S. pombe cells expressing CM-Skb1 were grown in EMM and were then shifted to EMM containing 1.5 M KCl and incubated for the indicated times before assaying for CM-Skb1 methyltransferase activity. C, S. pombe cells expressing CM-Skb1Hs were subjected to hyperosmotic shock and assayed for protein methyltransferase activity as described in B.

The Human Skb1 Homolog, Skb1Hs, Is Stimulated by Hyperosmotic Shock in Fission Yeast-- To address whether the methyltransferase activity of Skb1Hs is stimulated by hyperosmotic shock in fission yeast cells, we subjected cells expressing Skb1Hs to hyperosmotic shock and then prepared cell lysates and assayed Skb1Hs protein methyltransferase activity. As shown in Fig. 5C, Skb1Hs methyltransferase activity, similar to that of fission yeast Skb1, was rapidly stimulated by hyperosmotic shock, suggesting that mediation of hyperosmotic stress response is likely to represent a conserved function of Skb1-related proteins.

In conclusion, we have demonstrated that a highly conserved protein methyltransferase, Skb1, is required for normal growth and maintenance of cell polarity under conditions of hyperosmotic stress in fission yeast. We have shown that skb1Delta mutants are defective in redistributing F-actin patches to cell ends after hyperosmotic stress-induced F-actin depolarization. It is likely that the failure of skb1Delta cells to properly maintain cell polarity in hyperosmotic medium is caused, at least in part, by this defect. Correlating with these findings, we found that in response to hyperosmotic stress, the Skb1 protein delocalizes from cell ends and nuclei and becomes markedly stimulated with respect to its protein methyltransferase activity. Cumulatively, these data establish a role for Skb1 as a mediator of hyperosmotic stress response in fission yeast. Previous genetic studies suggested that Skb1 functions as a positive modulator of the fission yeast PAK, Shk1. Whereas Shk1 plays an essential role in regulating the localization of F-actin patches and cell polarity under normal growth conditions, we consider it likely that it also shares with Skb1 a role in mediating hyperosmotic stress response because of the following. (i) Its kinase activity is rapidly stimulated by hyperosmotic stress. (ii) Similar to Skb1, it becomes delocalized from cell ends in response to hyperosmotic stress, and (iii) it is required for proper subcellular targeting of Skb1.2

Importantly, we have shown that the protein methyltransferase activity of the human Skb1/Hsl7 homolog, Skb1Hs, is induced by hyperosmotic stress in S. pombe cells. This result provides evidence for evolutionary conservation of a role for Skb1/Hsl7-related proteins in mediating cellular response to hyperosmotic stress. Moreover, the fact that the Skb1Hs enzyme is induced by hyperosmotic stress in S. pombe cells suggests that mechanisms involved in regulating the function of these highly conserved proteins may have also been substantially conserved through evolution.

Protein sequence analyses suggest that Skb1/Hsl7-related proteins belong to the protein arginine methyltransferase family (14, 29). A variety of eukaryotic proteins of diverse function have been shown to undergo methylation on arginine or lysine residues (30). Although the functional significance of this modification remains ill defined in all but a handful of cases, recent studies have implicated arginine methylation as being of potential significance in signal transduction (31), transcriptional regulation (32), and RNA processing (33, 34). The results presented in this report suggest that protein methylation is likely to play a role in cellular response to hyperosmotic stress. The continued characterization of Skb1/Hsl7-related protein methyltransferases and in particular the identification of Skb1 substrates in the evolutionarily distant fission and budding yeasts will undoubtedly shed substantial new insights into roles for protein methylation in eukaryotic organisms.

    FOOTNOTES

* This research was supported by National Institutes of Health Grant R01GM53239 (to S. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These authors contributed equally to this work and are listed alphabetically.

|| To whom correspondence should be addressed. Tel.: 713-745-2032; Fax: 713-794-4394; E-mail: smarcus@mdacc.tmc.edu.

Published, JBC Papers in Press, March 9, 2001, DOI 10.1074/jbc.C100096200

2 Y. Qyang, R. Pimental, and S. Marcus, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: PAK, p21-activated kinase; HA, hemagglutinin; EMM, essential minimal medium; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; MBP, myelin basic protein.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

1. Gilbreth, M., Yang, P., Wang, D., Frost, J., Polverino, A., Cobb, M. H., and Marcus, S. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13802-13807[Abstract/Free Full Text]
2. Gilbreth, M., Yang, P., Bartholomeusz, G., Pimental, R. A., Kansra, S., Gadiraju, R., and Marcus, S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 14781-14786[Abstract/Free Full Text]
3. Ma, X. J., Lu, Q., and Grunstein, M. (1996) Genes Dev. 10, 1327-1340[Abstract]
4. Marcus, S., Polverino, A., Chang, E., Robbins, D., Cobb, M. H., and Wigler, M. H. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 6180-6184[Abstract/Free Full Text]
5. Ottilie, S., Miller, P. J., Johnson, D. I., Creasy, C. L., Sells, M. A., Bagrodia, S., Forsburg, S. L., and Chernoff, J. (1995) EMBO J. 14, 5908-5919[Abstract]
6. Yang, P., Kansra, S., Pimental, R. A., Gilbreth, M., and Marcus, S. (1998) J. Biol. Chem. 273, 18481-18489[Abstract/Free Full Text]
7. Fukui, Y., Kozasa, T., Kaziro, Y., Takeda, T., and Yamamoto, M. (1986) Cell 44, 329-336[Medline] [Order article via Infotrieve]
8. Nadin-Davis, S. A., Nasim, A., and Beach, D. (1986) EMBO J. 5, 2963-2971
9. Chang, E. C., Barr, M., Wang, Y., Jung, V., Xu, H. P., and Wigler, M. H. (1994) Cell 79, 131-141[Medline] [Order article via Infotrieve]
10. McMillan, J. N., Longtine, M. S., Sia, R. A., Theesfeld, C. L., Bardes, E. S., Pringle, J. R., and Lew, D. J. (1999) Mol. Cell. Biol. 19, 6929-6939[Abstract/Free Full Text]
11. Shulewitz, M. J., Inouye, C. J., and Thorner, J. (1999) Mol. Cell. Biol. 19, 7123-7137[Abstract/Free Full Text]
12. Deleted in proof
13. Krapivinsky, G., Pu, W., Wickman, K., Krapivinsky, L., and Clapham, D. E. (1998) J. Biol. Chem. 273, 10811-10814[Abstract/Free Full Text]
14. Pollack, B. P., Kotenko, S. V., He, W., Izotova, L. S., Barnoski, B. L., and Pestka, S. (1999) J. Biol. Chem. 274, 31531-31542[Abstract/Free Full Text]
15. Schwarzler, A., Kreienkamp, H. J., and Richter, D. (2000) J. Biol. Chem. 275, 9557-9562[Abstract/Free Full Text]
16. Alfa, C., Fantes, P., Hyams, J., McLeod, M., and Warbrick, E. (1993) Experiments with Fission Yeast: A Laboratory Course Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
17. Maundrell, K. (1990) J. Biol. Chem. 265, 10857-10864[Abstract/Free Full Text]
18. Bauman, P., Cheng, Q. C., and Albright, C. F. (1998) Biochem. Biophys. Res. Commun. 244, 468-474[CrossRef][Medline] [Order article via Infotrieve]
19. Field, J., Nikawa, J., Broek, D., MacDonald, B., Rodgers, L., Wilson, I. A., Lerner, R. A., and Wigler, M. (1988) Mol. Cell. Biol. 8, 2159-2165[Medline] [Order article via Infotrieve]
20. Evan, G. I., Lewis, G. K., Ramsay, G., and Bishop, J. M. (1985) Mol. Cell. Biol. 5, 3610-3616[Medline] [Order article via Infotrieve]
21. Yang, P., Pimental, R., Lai, H., and Marcus, S. (1999) J. Biol. Chem. 274, 36052-36057[Abstract/Free Full Text]
22. Raitt, D. C., Posas, F., and Saito, H. (2000) EMBO J. 19, 4623-4631[Abstract/Free Full Text]
23. Roig, J., Huang, Z., Lytle, C., and Traugh, J. A. (2000) J. Biol. Chem. 275, 16933-16940[Abstract/Free Full Text]
24. Clerk, A., and Sugden, P. H. (1997) FEBS Lett. 403, 23-25[CrossRef][Medline] [Order article via Infotrieve]
25. Le Goff, X., Utzig, S., and Simanis, V. (1999) Curr. Genet. 35, 571-584[CrossRef][Medline] [Order article via Infotrieve]
26. Rupes, I., Jia, Z., and Young, P. G. (1999) Mol. Biol. Cell 10, 1495-1510[Abstract/Free Full Text]
27. Mata, J., and Nurse, P. (1998) Trends Cell Biol. 8, 163-167[CrossRef][Medline] [Order article via Infotrieve]
28. Sawin, K. E., and Nurse, P. (1998) J. Cell Biol. 142, 457-471[Abstract/Free Full Text]
29. Ma, X. J. (2000) Trends Biochem. Sci. 25, 11-12[CrossRef][Medline] [Order article via Infotrieve]
30. Aletta, J. M., Cimato, T. R., and Ettinger, M. J. (1998) Trends Biochem. Sci. 23, 89-91[CrossRef][Medline] [Order article via Infotrieve]
31. Lin, W. J., Gary, J. D., Yang, M. C., Clarke, S., and Herschman, H. R. (1996) J. Biol. Chem. 271, 15034-15044[Abstract/Free Full Text]
32. Chen, D., Ma, H., Hong, H., Koh, S. S., Huang, S. M., Schurter, B. T., Aswad, D. W., and Stallcup, M. R. (1999) Science 284, 2174-2177[Abstract/Free Full Text]
33. Valentini, S. R., Weiss, V. H., and Silver, P. A. (1999) RNA 5, 272-280[Abstract/Free Full Text]
34. Shen, E. C., Henry, M. F., Weiss, V. H., Valentini, S. R., Silver, P. A., and Lee, M. S. (1998) Genes Dev. 12, 679-691[Abstract/Free Full Text]


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