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The Fission Yeast ES2 Homologue, Bis1, Interacts with the Ish1 Stress-responsive Nuclear Envelope Protein*

In fission yeast, nutrient starvation induces physiological, biochemical, and morphological changes that enable survival. Collectively these changes are referred to as stationary phase. We have used a green fluorescent protein random insertional mutagenesis system to isolate two novel stress-response proteins required in stationary phase. Ish1 is a nuclear envelope protein that is present throughout the cell cycle and whose expression is increased in response to stresses such as glucose and nitrogen starvation, as well as osmotic stress. Expression of Ish1 is regulated by the Spc1 MAPK pathway through the Atf1 transcription factor. Although overexpression of Ish1 is lethal, cells lackingish1 exhibit reduced viability in stationary phase. Bis1 is a novel interacting partner of Ish1. Bis1 is theSchizosaccharomyces pombe member of the ES2 nuclear protein family found in Mus musculus, Drosophila melanogaster, Homo sapiens, and Arabidopsis thaliana. Overexpression of Bis1 results in a cell elongation phenotype, whereas bis1− cells exhibit a reduced viability in stationary phase similar to that seen inish1− cells.

In fission yeast, nutrient starvation induces physiological, biochemical, and morphological changes that enable survival. Collectively these changes are referred to as stationary phase. We have used a green fluorescent protein random insertional mutagenesis system to isolate two novel stress-response proteins required in stationary phase. Ish1 is a nuclear envelope protein that is present throughout the cell cycle and whose expression is increased in response to stresses such as glucose and nitrogen starvation, as well as osmotic stress. Expression of Ish1 is regulated by the Spc1 MAPK pathway through the Atf1 transcription factor. Although overexpression of Ish1 is lethal, cells lacking ish1 exhibit reduced viability in stationary phase. Bis1 is a novel interacting partner of Ish1. Bis1 is the Schizosaccharomyces pombe member of the ES2 nuclear protein family found in Mus musculus, Drosophila melanogaster, Homo sapiens, and Arabidopsis thaliana. Overexpression of Bis1 results in a cell elongation phenotype, whereas bis1 ؊ cells exhibit a reduced viability in stationary phase similar to that seen in ish1 ؊ cells.
The regulation of cellular growth and proliferation in response to environmental stress is important for development as well as for maintenance of cell viability. Nutritional limitation causes cells to arrest cell growth and enter stationary phase (1)(2)(3). This is a metabolically quiescent state where expression of genes required for survival is induced, whereas expression of cell cycle genes is repressed (4,5). Fission yeast cells enter stationary phase from G 2 upon glucose starvation and from G 1 upon nitrogen starvation (6).
In eukaryotic organisms the mitogen-activated protein kinase (MAPK) 1 pathways are ubiquitous for sensing and responding to environmental stresses (7). In fission yeast the stress-activated MAPK, Spc1, like the mammalian p38 kinase responds to a variety of stresses including osmotic stress, heat stress, oxidative stress, nutritional limitation, UV radiation, and DNA damage. Spc1 (also known as Sty1 or Phh1) is activated by the Wis1 MAPK kinase, which is in turn activated by two MAPK kinase kinases, Wak1 (also known as Wik1 and Wis4) or Win1 (7)(8)(9)(10)(11)(12). Attenuation of Spc1 activity is accomplished by the actions of two tyrosine phosphatases, Pyp1 and Pyp2 (11,12). Pyp2 is regulated at the transcriptional level by the Atf1 transcription factor and participates in a down-regulation of the Spc1 MAPK via a negative feedback loop (8). Inactivation of Pyp1 activates the Spc1 pathway.
The completion of the fission yeast genome sequencing project has revealed the existence of a large number of open reading frames (ORFs) of no known function or apparent homologues. To provide information regarding the localization of some of these gene products in the cell, we have developed a GFP random insertional mutagenesis system (17). This system utilizes GFP-ura4 ϩ PCR-generated cassettes randomly integrated into the genome. GFP in-frame fusion integrants are expressed under the control of native promoters allowing us to examine expression levels and intracellular localization of their protein products under a variety of growth conditions. The affected genes from cells displaying specific intracellular GFP localizations are isolated by inverse PCR and are sequenced (17,18).
To search for novel genes expressed in stationary phase, we screened for GFP in-frame fusions that had increased expression under glucose limiting conditions. We report the isolation of a novel stress protein named Ish1 ϩ (induced in stationary phase) localized to the nuclear envelope and the plasma membrane. Ish1 expression is elevated in response to a number of environmental stimuli including glucose starvation and osmotic stress and is regulated by the Spc1 MAPK pathway through Atf1. We also report the isolation of a novel nuclear protein, Bis1 (binds to ish1), isolated by two-hybrid screening, and we show that it specifically interacts with Ish1 in vivo. Bis1 is the fission yeast homologue of the strongly conserved ES2 family of proteins known from Homo sapiens, Drosophila melanogaster, Arabidopsis thaliana, and Caenorhabditis elegans. They are of unknown function although we show that both ish1 and bis1 contribute to viability in stationary phase.
Cloning of the ish1 Gene-Standard genetic methods and molecular biology techniques were used (1,37). The ish1 gene was isolated using our GFP insertional mutagenesis screen (17). ish1 was previously reported as an ORF upstream of the hba1 gene (21) and by the fission yeast genome project (Sanger data base).
Overexpression of Ish1 and Ish1-GFP Proteins Using the nmt1 Promoter-The ish1 gene was PCR-amplified using High Fidelity Taq polymerase (Roche Molecular Biochemicals) with genomic DNA as template and primers sporf9 and sporf12 (Table II). The 2052-bp fragment was cloned into pREP1 under the control of the thiamine-repressible nmt1 promoter (22,23), resulting in pLT1-1. pLT1-1 was transformed into Schizosaccharomyces pombe strains, and transformants were selected under repressing conditions (15 M thiamine).
pREP1-GFP was constructed by removing the NdeI restriction site residing in the GFP(S65T) open reading frame using a silent mutation created by overlap extension PCR (24). The resulting fragment with BamHI and XmaI sites added by PCR was subcloned into pREP1.
The ish1 ORF was PCR-amplified using primers sporf9 and sporf10 (Table II) that incorporated AseI and SalI restriction sites, and the product was cloned into pREP1-GFP, resulting in pREP-ish1-GFP and is referred to as pLT1-2.
pLT1-2 was digested with BglII and BamHI to excise a fragment encoding Ish1 amino acids 581-684. The remaining vector backbone was gel-purified and then self-ligated to create pREP1-ish1⌬C104-GFP and is referred to as pLT1-3.
An N-terminal deletion of Ish1 comprising amino acids 279 -684 was made using primers sporf11 and sporf10 (Table II) that incorporate NdeI and SalI restriction sites. The product ⌬N278ish1 was then cloned into pREP1-GFP to create pREP1-⌬N278ish1-GFP and is referred to as pLT1-4.
Construction of the ish1 and ish1-GFP Expression Vectors Using the ish1 Native Promoter-The entire ish1 gene from Ϫ351 to ϩ2052 bp was PCR-amplified using primers sporf5 with sporf12 or sporf10 (Table II) incorporating PstI and SalI restriction sites. pREP1 was digested with PstI and SalI to excise the 1200-bp fragment encoding the nmt1 promoter sequence. The ish1 PCR products were cloned into the gelpurified vector backbones pREP1 or pREP1-GFP to create pLT2-1 or pLT2-2, respectively.
Construction of ish1 Disruption-PCR-based gene targeting (25)(26)(27) was used to replace a 1934-bp fragment of the ish1 coding region with the ura4 ϩ gene leaving 118 bp of the C-terminal ORF of ish1 (Fig. 1A). The PCR primers used, sporf1 and sporf2 (Table II), included 80 bp of flanking sequence homologous to the ish1 sequence in the genome. Gene replacement was confirmed by PCR using primers sporf3 and sporf4 (Table II) and Southern blot analysis. Sporulation of the diploid and extensive out-crossing (6 times) was performed to ensure that no background mutations were present.
Construction of bis1 Disruption-Bis1 was identified as a two-hybrid target interacting with Ish1. The bis1 gene from Ϫ254 to ϩ1330 base pairs relative to the 1154-bp ORF was PCR-amplified using primers lanko1 and lanko2 (Table II) with High Fidelity Taq polymerase (Roche Molecular Biochemicals). The PCR product was subcloned into pGEM-T (Promega) to create pGEM-T-bis1. pGEM-T-bis1 was digested with XbaI, and the ends were filled in using the Klenow fragment of DNA polymerase, and then the plasmid was digested with ClaI. The ura4 ϩ gene was excised from pZA25 using SmaI and ClaI restriction enzymes and subcloned into the XbaI (blunt-ended)/ClaI site of pGEM-T-bis1 to generate pGEM-T-bis1::ura4 ϩ . The bis1::ura4 ϩ cassette in this recombinant vector was then PCR-amplified using High Fidelity Taq polymerase (Roche Molecular Biochemicals) and used to replace bis1 in a haploid strain (ura4-D18 h Ϫ ) by one-step gene disruption (20) (Fig. 1B). Stable ura4 ϩ haploids were selected, and exact gene replacement was confirmed by PCR. The strain was out-crossed extensively to ensure that no background mutations were present.
Overexpression of Bis1 and Bis1-YFP Proteins Using the nmt1 Promoter-The bis1 ORF was PCR-amplified from genomic DNA using primers lan1 and lan2 or lan1 and lan3 (Table II), incorporating NdeI and SalI restriction sites, with High Fidelity Taq polymerase (Roche Molecular Biochemicals). The PCR products were cloned into pREP2 or pREP2-YFP containing the ura4-selectable marker (23), resulting in pREP2-bis1 and pREP2-bis1-YFP referred to as pLT3-1 and pLT3-2, respectively. pLT3-1 and pLT3-2 were transformed into cells, and positive transformants were selected in the presence of 15 M thiamine.
The YFP gene from pEYFP (Invitrogen) was excised from the vector by digesting with EcoRI and blunt-ended using the Klenow fragment of DNA polymerase. The pREP2 vector was digested with SalI, and the This study blunt-ended YFP fragment was cloned into the SalI/SmaI sites of pREP2 to create pREP2-YFP. Production of GST-Bis1 Fusion Protein-The full-length GST-Bis1 protein fusion was degraded in Escherichia coli. Therefore, a 354-bp fragment encoding amino acids 267-384 of the C-terminal region of bis1 was fused to the C terminus of GST in pGEX-2T (Amersham Biosciences). Expression was induced in E. coli with 1 mM isopropyl-1-thio-␤-D-galactopyranoside at 37°C for 1 h because this fusion protein was also rapidly degraded by proteases. The GST-Bis1 fusion protein was purified on a glutathione-agarose column and eluted with 10 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0, as described in the manufacturer's manual (Amersham Biosciences).
Immunochemical Analysis-To generate polyclonal antibodies against Bis1, the GST-Bis1 fragment was separated on a 12% SDSpolyacrylamide gel, excised, eluted, and mixed with Titer Max Gold Adjuvant (Cedarlane) as described in the manufacturer's instructions. The rabbit was boosted at day 28 and day 40. Serum was collected on day 50 and used as a source of antibody for Western blot analysis and immunofluorescence experiments.
Fluorescence Microscopy-A Leica fluorescence microscope equipped with a high performance CCD camera (Sensicam) and Slidebook software (Intelligent Imaging System) was used for all imaging. Cells were collected onto Whatman 934-AH glass microfiber filters (Fisher) and fixed with 100% ice-cold methanol at Ϫ20°C for 20 min. The immunofluorescence protocol used is described in Sawin and Nurse (28). Rabbit GST-Bis1 polyclonal antiserum generated in the lab (1:5000) was used with Alexa TM 488 goat anti-rabbit IgG (H ϩ L) conjugate (1:250) (Molecular Probes). Stained cells were counterstained with 1 g/ml DAPI.
Protein Lysates-Protein extracts (29) (20 g or 50 g, Bio-Rad protein assay) were separated by 7.5 or 10% SDS-PAGE, electroblotted to a polyvinylidene difluoride membrane (Santa Cruz Biotechnology), and detected by polyclonal anti-GST-Bis1 antibody or monoclonal anti-GFP antibody (1:1000) (Roche Molecular Biochemicals). Immunoreactive bands were detected with a horseradish peroxidase-conjugated secondary goat anti-rabbit IgG antibody (Santa Cruz Biotechnology) or goat anti-mouse IgG antibody (1:2000) and the luminol-based ECL detection kit (Santa Cruz Biotechnology). Protein loading was monitored by Coomassie Blue staining of gels.
Immunoprecipitation-Harvested cells in HB buffer (25 mM MOPS, pH 7.2, 60 mM ␤-glycerophosphate, 15 mM p-nitrophenyl phosphate, 15 mM MgCl 2 , 15 mM EGTA, 1 mM dithiothreitol, 0.1 mM sodium vanadate, 1% Triton X-100) (20), supplemented with complete protease inhibitor mixture (Roche Molecular Biochemicals), were broken by vortexing with glass beads and centrifuged to prepare a cleared whole-cell extract. Cell extracts (300 g, Bio-Rad protein assay) were incubated with 10 l of polyclonal anti-GST-Bis1 antibody in a 500-l volume in HB buffer at 4°C for 3 h, and 60 l of protein G-Sepharose beads (Amersham Biosciences) were added for 1 h at 4°C. Beads were washed extensively with HB buffer, resuspended in 2ϫ SDS loading buffer, and analyzed by SDS-PAGE. For immunoblot analysis, 25 g of total protein was loaded for detection of proteins in the total yeast lysate; 10% of total immunoprecipitated material was loaded for detection of the immunoprecipitated protein, and 90% was loaded for detection of the coimmunoprecipitate. The proteins were subjected to immunoblot analysis as described above.
Viability in Stationary Phase-Cells were grown to stationary phase in YEA or EMM medium, and incubation was continued for 6 days. A portion of each culture was removed at day 0, 1, 2, 4, and 6 and plated on YEA or EMM after appropriate dilution to determine cell viability. Samples were taken in duplicate over a 6-day period, and the experiment was independently repeated two times.
FIG. 1. Gene disruptions of ish1 ؉ and bis1 ؉ . Restriction maps of the ish1 ϩ and bis1 ϩ genomic DNA showing the fragment deleted by ura4 ϩ in the construction of the ish1::ura4 ϩ and bis1::ura4 ϩ mutant strains. A, a 1.94-kb fragment of ish1 ϩ was replaced with the 1.8-kb ura4 ϩ gene. B, the ura4 ϩ gene was introduced into the ClaI-XbaI sites of bis1 ϩ to generate bis1::ura4 ϩ .
Taq polymerase incorporating BamHI and NotI restriction sites. pEG202-⌬N90bis1lexA was constructed as outlined above. Approximately 2,000,000 cDNA clones were screened using this bait.

RESULTS
Isolation of ish1 Using the GFP Insertional Mutagenesis System-An insertional mutagenesis cassette containing GFP on the 5Ј terminus was used to isolate in-frame insertional mutants that expressed GFP (17). A GFP fusion, lt13-26, was isolated based on the localization of the GFP to the nuclear envelope and the plasma membrane under glucose starvation conditions. In zygotic asci the GFP localized only to the nuclear envelope (17). GFP expression increased by ϳ5-fold following glucose and nitrogen starvation as well as during hyper-and hypo-osmotic stress (data not shown). lt13-26 is expressed throughout the cell cycle in growing cultures ( Fig. 2A). Sequencing revealed that lt13-26 was a single copy chromosomal fusion with an 873-bp 3Ј-truncation of the ish1 gene and expression remaining under the control of the ish1 native promoter. Cells expressing lt13-26 were indistinguishable from wild type cells with respect to growth and morphology. We will refer to the original integrant, lt13-26, as ish1⌬C291-GFP from this point.
Overexpression of Ish1 Is Lethal-In the presence of thiamine to repress the nmt1 promoter, cells containing ish1, ish1-GFP, ⌬N278ish1-GFP, or ish1⌬C104-GFP expression constructs displayed normal growth and morphology (Fig. 2B). In the absence of thiamine, overexpression of ish1, ish1-GFP, and ish1⌬C104-GFP was toxic, arresting cell proliferation (Fig. 2B). Microscopic examination showed no obvious morphological changes, and arrest appears to be in G 2 based on morphological criteria. Examination of the cells 15 h after induction showed similar localization of Ish1-GFP to that seen in the original integrant, ish1⌬C291-GFP ( Fig. 2A).
Overexpression of ⌬N278ish1-GFP was not toxic. ⌬N278Ish1-GFP appears to be retained in the endoplasmic reticulum (Fig. 2C). This suggests that the N-terminal region is necessary for nuclear envelope and plasma membrane targeting and/or localization of Ish1. Deletion of the N-terminal sequence renders the protein incapable of normal localization thus sparing the toxic effects observed with the full-length protein. We speculate that Ish1 at high concentration somehow interferes with membrane function or the function of some protein in the membrane and that the observed toxicity is the result. Deletion of the N terminus of Ish1 renders the protein incapable of being correctly targeted, and the toxicity is thus not observed.
Ish1 Expression Is Regulated by the Spc1 MAPK Pathway via Atf1-Expression of ish1⌬C291-GFP is under the control of its native promoter; therefore, it was used to examine the expression of the Ish1 protein in various mutant backgrounds. Western blotting performed using a monoclonal anti-GFP antibody shows that Ish1⌬C291-GFP has a molecular mass of ϳ90 kDa, whereas Ish1-GFP migrates at ϳ120 kDa (Fig. 3, A and B). The other bands in the Western blot are the result of nonspecific binding of the GFP antibody, which are detected in wild type cells not expressing GFP.
Ish1⌬C291-GFP expression was examined in a ⌬spc1 mutant strain to inactivate the MAPK pathway (14) and in a ⌬pyp1 mutant strain, an inhibitor of Spc1, to activate it (12).
Ish1⌬C291-GFP protein expression is almost undetectable in a ⌬spc1 mutant strain (Fig. 3A). However, increased activity of Spc1 as occurs in a ⌬pyp1 mutant strain resulted in an ϳ10fold increase in Ish1 expression (Fig. 3A).
Because expression and activity of Atf1 are regulated by Spc1 MAPK (13), we examined Ish1 expression in a ⌬atf1 mutant background. There is no detectable level of Ish1 protein in the ⌬atf1 mutant background (Fig. 3A) showing that Ish1 is regulated by the Spc1 MAPK pathway via Atf1. In a ⌬atf1 ⌬pyp1 mutant background, Ish1 is detectable but very low, suggesting that when Spc1 is hyperactivated a second transcription factor might make a small contribution.
The response to osmotic stress occurs at least in part through atf1 (13). Under 1.2 M KCl stress, Ish1 expression in a ⌬pyp1 mutant background is similar to that in wild type cells (Fig.  3C). However, in a ⌬atf1 and ⌬atf1 ⌬pyp1 mutant background Ish1 protein expression levels are very low but still present compared with wild type. In response to osmotic stress, expression is primarily through Atf1; however, low level Atf1-independent expression is also present.
Ish1 was isolated on the basis of its overexpression in response to glucose starvation. Glucose starvation results in both the activation of the Spc1 MAPK pathway and a reduction in activity of the cyclic AMP pathway (36 -40). We examined whether the cyclic AMP-Pka1 pathway regulated Ish1 expression. Ish1⌬C291-GFP expression in a ⌬pka1 (catalytic subunit of cAMP-dependent protein kinase) mutant background was similar to wild type cells following glucose starvation. This suggests that the Pka1 pathway does not regulate Ish1 expression (data not shown).
We sought to determine whether the toxicity observed with Ish1 overexpression could be rescued by deletion of elements in the Spc1 MAPK pathway. ⌬spc1, ⌬pyp1, or ⌬atf1 mutations could not rescue the toxic effect caused by constitutive overexpression of Ish1 (data not shown).
Time Course Experiment during Stress Response-Ish1 expression in response to KCl treatment increases within the initial 20 min of treatment and stays relatively constant for at least 4 h (Fig. 3D). A similar time course of expression was observed in response to sorbitol treatment (data not shown). Because all stress conditions examined appear to increase expression of ish1⌬C291-GFP, we examined its response to heat shock treatment at 36°C. Heat shock caused a relatively small increase in Ish1 expression (Fig. 3E).
ish1 Contributes to Viability in Stationary Phase-A null allele was constructed by replacing the ish1 gene with the ura4 ϩ gene in a diploid cell (Fig. 1A). Tetrad analysis revealed that the ⌬ish1 mutant is viable and did not exhibit a visible phenotype. We examined the ⌬ish1 mutant strain extensively under a variety of stresses including sorbitol, KCl, nitrogen starvation, glucose starvation, hydrogen peroxide, camptothecin, and caffeine treatment. In all cases there was no visible phenotype. The ability of the ⌬ish1 mutant strain to mate, conjugate, and sporulate was comparable with that of wild type.
⌬ish1 cells exhibited a phenotype upon streaking cells from rich to minimal medium plates. A population of cells appeared to grow and divide once and then stop growing. This phenotype is exaggerated when the initial cells are in stationary phase. ⌬ish1 cells after growth to saturation in YEA medium showed ϳ30 -40% reduction in viability compared with wild type when plated on YEA (Fig. 4) or EMM (data not shown). Exponentially growing ish1 mutant cells have essentially a 100% plating efficiency; however, viability is lost in stationary phase. These results suggest that Ish1 plays a role in stationary phase. Because it is known that Spc1 is essential for stationary phase viability and is involved in the regulation of Ish1, we examined the viability of the ⌬spc1 mutant strain. ⌬spc1 mutant cells grown to saturation in rich or minimal media showed extremely poor viability with Ͻ1% viable cells remaining after 2 days of incubation (Fig. 4).
We generated double mutants of ⌬ish1 with ⌬spc1, ⌬atf1, and ⌬pyp1 to see if ish1 deletion genetically interacts with these mutations. ⌬ish1 ⌬spc1, ⌬ish1 ⌬atf1, and ⌬ish1 ⌬pyp1 double mutants were indistinguishable from the single mutants on plates at a permissive temperature as well as on plates containing 1.2 M KCl or 1.2 M sorbitol (data not shown).
Isolation of bis1 Using a Two-hybrid Library Screen-To identify potential Ish1 interacting partners, we performed a yeast two-hybrid screen using ⌬N33ish1 as bait in which the potential transmembrane domain has been deleted (see "Experimental Procedure" ; Fig. 5A). A total of 3 million transformants were screened resulting in eight independent interactions representing only three different target genes (Table III). One of the targets was a fragment of Ish1, amino acids 384 -580. This region of Ish1 may be important for dimerization or oligomerization. Another target isolated was Rps6, a ribosomal protein repressed by ammonium starvation and regulated by the Spc1 MAPK and the Pka1 pathways (41). The third target, of which two different isolates were obtained, was Bis1. All of the targets induced expression of the ␤-galactosidase reporter gene (Table III).
To determine the region of Ish1 important for protein-protein interactions we generated ⌬N278ish1, ish1⌬C104, and ish1-(384 -580) constructs as baits (see "Experimental Procedures" ;  Fig. 5A). The Ish1-Ish1 interaction is largely unaffected by the N-terminal deletion. Although the interaction with the C-terminal deletion construct remains strong, it is reduced 2-fold compared with ⌬N33 (Table III). The results indicate that the central region of the protein from amino acid 384 -580 mediates the majority of the Ish1-Ish1 interactions. In contrast, Bis1 shows a somewhat increased binding affinity for the Ish1⌬C104 or Ish1-(384 -580) baits and slightly reduced binding affinity for ⌬N33ish1 or ⌬N278ish1 baits. These results indicate that Bis1 interact with different regions of the Ish1 protein. The main region of contact encompasses amino acids 384 -580.
Bis1 Is a Homologue of the ES2 Protein-The Bis1 (Gen-Bank™ accession number AL022243.3, locus SPCC364.02c) protein is 384 amino acids in length with a predicted molecular mass of 42.7 kDa. Bis1 is homologous to the ES2 nuclear protein family (ProDomain Data base) for which no function has been identified. The Bis1 protein shows extensive homology with ES2 family members from C. elegans (42), Drosophila (43), H. sapiens (44,45), and A. thaliana (Proteome data base) (Fig. 5E). There is no homologue in S. cerevisiae.
Characterization of Bis1 Protein-To characterize the expression level of endogenous Bis1, antibodies were generated to a GST-Bis1 fusion protein expressed in E. coli. By using our GST-Bis1 polyclonal antibody, we were unable to detect native Bis1 protein in cell extracts or following strong overexpression of native Bis1 protein (Fig. 5B). However, this antibody is able to detect a predominant band at ϳ80 kDa which corresponds to the full-length Bis1-YFP fusion protein (Fig. 5B). The ability to detect Bis1 following overexpression of Bis1-YFP suggests that the native protein is stabilized by YFP. Other smaller bands were detected by Western blot analysis, suggesting that the Bis1 protein is most likely susceptible to rapid proteolytic cleavage or degradation in vivo. Choosing different times of expression did not markedly affect this result.
Ish1 Interacts with Bis1 in Vivo-To confirm that Ish1 and Bis1 interact in vivo, Ish1-GFP and Bis1-YFP were expressed under the control of the thiamine-repressible nmt1 promoter in wild type cells. Soluble proteins were prepared from cells that had been expressing these fusion proteins for no longer than   FIG. 4. ish1 and bis1 show reduced viability in stationary phase. Cells were grown to stationary phase in YEA medium, and incubation was continued for 6 days. A portion of each culture was removed at the indicated times, counted, and plated on YEA plates. The number of viable colonies forming cells was determined over a 6-day incubation period. Wild type (Q250), ⌬ish1 (Q1951), ⌬bis1 (Q1894), ⌬ish1 ⌬bis1 (Q1935), and ⌬spc1 (Q1699) mutant cells were used.
FIG. 5. Ish1 interacts with Bis1. A, deletion constructs of Ish1 in pEG202 as indicated. The positions of the first and last amino acids of each construct are shown relative to Ish1. B, Bis1 is undetectable in the absence of the YFP tag. ⌬bis1 mutant cells, wild type cells at 30°C and shifted to 36°C for 4 h, or cells producing high levels of Bis1 (nmt2:bis1), and (nmt2:bis1-YFP) were analyzed for Bis1 expression levels at 30°C. Following 18 h of growth, cell lysates were prepared. A portion of the extract (20 g) was separated by SDS-PAGE and used for Western blotting. The Bis1 protein was detected using our anti-GST-Bis1 antibody. C and D, Ish1 and Bis1 proteins interact in vivo. Extracts were prepared from wild type cells cotransformed with plasmids overexpressing Ish1-GFP and Bis1-YFP (Q1841) or Ish1⌬C104-GFP and Bis1-YFP (Q1847) under the control of nmt1 promoter. Cells were grown for 15 h in EMM at 30°C, and native extracts were prepared following centrifugation and bead lysis. Bis1 was immunoprecipitated from extracts using anti-Bis1-GST antibody. Bound proteins were eluted and subjected to immunoblot analysis with anti-GFP (C) or anti-Bis-GST (D) antibody. Lanes 1 and 6, ⌬bis1 mutant cells; lanes 2 and 4, nmt1:ish1-GFP and nmt2:bis1-YFP; and lanes 3 and 5,  nmt1:ish1⌬C104-GFP and nmt2:bis1-YFP. As a control 25 g of ⌬bis1 mutant cell lysates was probed directly for the presence of Bis1-YFP or Ish1-GFP. E, Bis1 is a member of a conserved ES2 coiled nuclear protein family. ClustalW alignment of conserved domains of S. pombe Bis1 (CAA18284), H. sapiens DGSI (NP_073210), C. elegans F42H10.7 (P34420), D. melanogaster DES2 (AAF46375), and A. thaliana F17A17.13 (AAF21189). Shaded boxes enclose regions with sequence identity to S. pombe Bis1. 15 h. Bis1 was immunoprecipitated using our GST-Bis1 polyclonal antibody and the presence of Ish1-GFP in the precipitates was assessed using an anti-GFP monoclonal antibody. An immunoprecipitated Bis1-YFP was able to coprecipitate Ish1-GFP or Ish1⌬C104-GFP (Fig. 5C). Our anti-GST-Bis1 polyclonal antibody immunoprecipitated Bis1-YFP (Fig. 5D). Approximately 30% of Bis1-YFP was immunoprecipitated from the yeast lysate, and ϳ1% of the total Ish1-GFP, a much more abundant protein, coimmunoprecipitated with the Bis1-YFP.
bis1 Contributes to Viability in Stationary Phase-A null allele was constructed by replacing the bis1 gene with ura4 ϩ in a haploid strain (Fig. 1B). The ⌬bis1 mutant is viable, and bis1 is nonessential for growth. We were unable to demonstrate a visible phenotype for the ⌬bis1 mutant under any of the conditions tested for ish1. However, ⌬bis1 mutant cells do exhibit a reduction in cell viability in stationary phase (Fig. 4). Similarly to ish1 mutant cells, bis1 mutant cells display a 100% plating efficiency during logarithmic growth but not from stationary phase. This result suggests that bis1 contributes to stationary phase viability as does ish1.
Localization of Bis1 Protein-We examined the phenotype and localization of Bis1-YFP expressed under the control of the nmt2 promoter in a wild type background. Overexpression of Bis1-YFP causes growth inhibition producing elongated cells exhibiting a cell cycle phenotype (Fig. 6A). Overexpression of the full-length native Bis1 protein exhibited a similar cell morphology (data not shown) suggesting that this phenotype is not a result of the tag. Bis1-YFP localizes to the nucleus as shown by DAPI staining (Fig. 6B). Interestingly, during mitosis Bis1-YFP appears to be associated with the mitotic spindle microtubules and possibly chromatin as the cells progress through mitosis (Fig. 6C).
We examined whether overexpression of Bis1 is able to rescue the growth inhibition associated with Ish1 overexpression and found that Bis1 was not able to suppress the lethality.
Some Overlap in Localization of Bis1 and Ish1 Proteins-When Bis1-YFP and Ish1-GFP are co-overexpressed in wild type cells, they display partial overlap in their localization at the nuclear envelope (Fig. 6, D and E). To determine whether the localization of Bis1-YFP or Ish1-GFP is dependent upon one another, their localization was examined in null mutant cells of each partner. The localization of Bis1-YFP in the ⌬ish1 mutant background and Ish1-GFP in ⌬bis1 mutant background is similar to that seen in wild type backgrounds (data not shown).
Search for Synthetic Interaction between ish1 and bis1-We generated a double mutant of ⌬ish1 with ⌬bis1 to see if ish1 genetically interacts with bis1. The ⌬ish1 ⌬bis1 double mutant did not exhibit a visible phenotype. We examined the ⌬ish1 ⌬bis1 double mutant strain extensively under similar conditions to those used to test the ⌬ish1 mutant strain. In all cases there is no visible phenotype. Intriguingly, the ⌬ish1 ⌬bis1 double mutant strain behaves similarly to the ⌬ish1 or ⌬bis1 mutant strains and has no additive effect upon the level of survivability in stationary phase (Fig. 4).
Two-hybrid Screen Using Bis1 Bait-We performed a yeast Expression of LacZ was measured by assessing ␤-galactosidase activity as described in Evangelista et al. (32). Measurements are expressed in Miller units as the mean of three independent determinations (Ϯ S.D.). The first 33 amino acids (aa) of Ish1 were deleted since it contains a potential transmembrane domain. The Ish1⌬C104 bait plasmid is also deleted for the first 33 amino acids of Ish1 for same reasons above. DNA   two-hybrid screen using a Bis1 construct where 90 N-terminal amino acids (⌬N90bis1) have been deleted. ⌬N90bis1 was used because the full-length DNA encoding for bis1 transactivated on its own. A total of 2 million transformants were screened. The screen produced at least 71 reproducible interactions with at least 22 different targets as shown in Table IV. We have only included data where multiple hits have been verified. Bis1 clearly interacts with a broad array of mostly nuclear proteins.

DISCUSSION
Ish1 Is a Novel Stress-response Protein-We used GFP insertional mutagenesis to isolate genes up-regulated following glucose starvation, and we identified Ish1, a novel stressresponsive protein. The Ish1-GFP fusion protein localizes predominantly to the nuclear envelope as well as to the plasma membrane throughout the cell cycle making it a useful fluorescent marker for monitoring changes that occur in the nuclear membrane during mitosis and meiosis. Expression of Ish1 is strongly induced in response to a variety of stresses including nitrogen starvation and osmotic stress. Ish1 exhibits limited similarity to S. cerevisiae YML128c. YML128c has been characterized through a DNA microarray analysis of S. cerevisiae genes responsive to diverse environmental stresses. It is up-regulated ϳ8-fold in response to a number of stresses including heat shock, dithiothreitol, hydrogen peroxide, hyperand hypo-osmotic stress, stationary phase, and nitrogen depletion (47). The limited similarity between S. pombe Ish1 and S. cerevisiae YML128c and the fact that both are responsive to stress suggest the possibility that they may be functional homologues. Both YML128c and Ish1 have putative transmembrane domains at their N termini although the cellular localization of YML128c has not yet been determined. The YML128c deletion mutant has been shown to have a 3-fold increase in meiotic unequal sister chromatid recombination (35). Whether Ish1 has a similar role in S. pombe is not known at present. However, ⌬ish1 mutant cells do not appear to exhibit a major defect in mating, conjugation, or sporulation compared with wild type cells. Its constitutive expression during growth and the wide variety of stresses capable of inducing Ish1 suggests that its role is certainly not limited to meiosis.
Ish1 Is Regulated by the Stress-activated Spc1 MAPK Pathway-In S. pombe the Spc1 MAPK pathway is known to play a role in adaptation to a variety of cellular stresses and to nutrient limitation (8). Ish1 expression is modulated through the stress-activated Spc1 MAPK pathway via Atf1. There is also a low level of constitutive expression of Ish1. The response to glucose starvation involves the activation of Spc1 with a concomitant deactivation of the cAMP-Pka1 pathway. However, we found Ish1 expression in a ⌬pka1 mutant background in response to glucose starvation to be similar to wild type levels, indicating Ish1 levels are not modulated directly by the cAMP-Pka1 pathway.
Ish1 Is Important for Stationary Phase Viability-Although overexpression of Ish1 is toxic, ⌬ish1 mutant cells are viable and exhibit no observable phenotype in response to a variety of stress conditions tested. Whereas spc1 and atf1 have been shown to be essential for stationary phase viability (11,15), ⌬ish1 mutant cells display a reduction of 30 -40% in cell survival. This moderate effect of the ish1 gene on cell survival in stationary phase suggests the existence of stress proteins with overlapping functions.
The Interaction between Ish1 and Bis1-Bis1 was isolated as an Ish1-interacting protein in a two-hybrid screen. To confirm this finding, we have shown that Ish1 and Bis1 physically interact in vivo. The relative band intensities for the coimmunoprecipitation of the two proteins suggest a stoichiometry of Bis1-Ish1 interaction in the complex of ϳ1:1 or 1:2. Ish1-GFP and Bis1-YFP proteins show some overlapping localization on the nuclear envelope. However, cellular localization of the bulk of Ish1 or Bis1 is not interdependent. We presume that the interaction occurs mostly when Bis1 contacts the nuclear envelope and that this represents only a portion of total Bis1 protein. It is of interest that immunoprecipitation from cell lysates recovered ϳ30% of total Bis1 protein. This fraction of the protein appears to be associated with Ish1. We do not know whether this percentage is representative of the status in vivo.
The two-hybrid data shows that Ish1 can homodimerize. We do not know whether the Bis1 interaction is to such a dimer (or multimer) or whether Ish1 and Bis1 compete in binding Ish1. Ish1 is stress-responsive and may function to bind and hold out of circulation proteins such as Bis1 or recruit Bis1 to the membrane in times of stress. Bis1 interacts with different regions of the Ish1 protein; however, the main region of contact encompasses amino acids 384 -580 of Ish1 as shown by the two-hybrid data. Sequence analysis identified a strong coiledcoil region (Coils version 2.1) from amino acids 384 to 580 in Ish1. This region of the protein also contains an LEA domain that is found in LEA proteins in higher plants. Interestingly, LEA proteins are induced under similar stress conditions to Ish1. We postulate that the LEA domains are involved in protein-protein interactions.
As discussed previously, ish1 plays a role in stationary phase viability. The interaction of ish1 with bis1 suggests that bis1 plays a similar role. The ⌬bis1 mutant cells show a reduction in cell viability similar to ⌬ish1 mutant cells alone. The ⌬ish1 ⌬bis1 double mutant, however, was indistinguishable from the single mutants in the reduction of cell viability in stationary phase. They may both play a role in stationary phase viability, but their effects are not additive, suggesting they may be members of the same pathway.
Possible Roles of Bis1-Bis1 is the S. pombe homologue of the ES2 family of nuclear proteins that have been found in various organisms from A. thaliana to H. sapiens. Notably there does not appear to be an ES2 homologue in S. cerevisiae. Similar to the results in mouse, Bis1-YFP localizes to the nucleus (43). At present no function has been identified for the ES2 family of proteins. Our findings of an effect on stationary phase viability are the first suggestion of a phenotype. The C. elegans homologue, F42H10.7, has been shown to interact with Mpk1 in a two-hybrid screen (46). Mpk1 has regions of similarity to the kinase domains of Spc1, Spm1, and Spk1 in fission yeast. Coupled with the existence of several putative MAPK phosphorylation sites in the Bis1 sequence, this suggested that Bis1 activity could be regulated by a MAPK. Although we could not show that Bis1 interacts with Spc1, we have not ruled out the possibility of Bis1 interacting with Spm1 or Spk1.
In an effort to gain insight into the function of Bis1, we performed a two-hybrid screen. As might be expected from the Bis1 localization, ϳ80% of the two-hybrid targets of Bis1 are nuclear or nuclear envelope proteins. They fall broadly into several classes as follows: 1) RNA processing/modification; 2) protein degradation; and 3) centromere/chromatin structure. Interestingly, we found that Bis1-YFP localizes to the mitotic spindle microtubules during mitosis starting in anaphase B. These results are in line with the functions of the third class of Bis1 targets.
The second class of Bis1 targets is involved in protein degradation. The high turnover of Bis1 implied by these results may explain why our antibody was unable to detect endogenous Bis1 protein. However, it was able to detect Bis1-YFP. GFP tags have been shown to increase protein stability in some cases (41). It is possible that the YFP tag increases resistance to proteolytic degradation allowing the proteins to be detected.
Although we have not carried out any direct experimental promoter analysis on ish1 and bis1, we have identified a number of elements upstream of the start site that suggests that they are stress-responsive genes. We have found at least four putative nitrogen-response elements (48) in both ish1 and bis1. Presumably, these nitrogen-response elements account for the stress response of ish1. This is also likely to be the case for bis1. In addition, we have found two putative Ste11 binding motifs (48) in ish1. Ste11 is essential in the mating and meiosisresponse pathway (49). Ste11-binding motifs are found in a number of genes regulated by Ste11, including mat1-P, mat1-M, mei2 (49), esc1 (50), ste6 (51), and fus1 (52). It is conceivable that ish1 may also be regulated by Ste11.
In conclusion, we have presented data that identifies a novel nuclear envelope protein, Ish1, whose expression is mediated by Atf1 in response to a variety of stresses. We also identified a novel nuclear protein, Bis1, that interacts with Ish1. Although neither ish1 nor bis1 is essential for viability during growth, they both play a role in maintaining stationary phase viability. The mechanism by which Ish1 or Bis1 maintains cell viability during glucose starvation remains to be determined.