Yeast Nap1-Binding Protein Nbp2p Is Required for Mitotic Growth at High Temperatures and for Cell Wall Integrity

Corresponding author: Akihiko Kikuchi, Research Institute for Disease Mechanism and Control, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-8550, Japan., aki{at}med.nagoya-u.ac.jp (E-mail)

Communicating editor: M. ROSE

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Nbp2p is a Nap1-binding protein in Saccharomyces cerevisiae identified by its interaction with Nap1 by a two-hybrid system. NBP2 encodes a novel protein consisting of 236 amino acids with a Src homology 3 (SH3) domain. We showed that NBP2 functions to promote mitotic cell growth at high temperatures and cell wall integrity. Loss of Nbp2 results in cell death at high temperatures and in sensitivity to calcofluor white. Cell death at high temperature is thought not to be due to a weakened cell wall. Additionally, we have isolated several type-2C serine threonine protein phosphatases (PTCs) as multicopy suppressors and MAP kinase-kinase (MAPKK), related to the yeast PKC MAPK pathway, as deletion suppressors of the nbp2 mutant. Screening for deletion suppressors is a new genetic approach to identify and characterize additional proteins in the Nbp2-dependent pathway. Genetic analyses suggested that Ptc1, which interacts with Nbp2 by the two-hybrid system, acts downstream of Nbp2 and that cells lacking the function of Nbp2 prefer to lose Mkk1, but the PKC MAPK pathway itself is indispensable when Nbp2 is deleted at high temperature.

NUCLEOSOME assembly protein 1 (Nap1) was identified in mammalian cell extracts by its intrinsic ability to facilitate nucleosome assembly in vitro in physiological ionic conditions (ISHIMI et al. 1983 ). Its homologs, including TAF1/set proteins, are expressed in abundance in most eukaryotes (NAGATA et al. 1995 , NAGATA et al. 1998 ; KAWASE et al. 1996 ; MATSUMOTO et al. 1999 ). Functional analyses, in vitro, have suggested that they are necessary to keep proper nucleosome structures in transcription and replication (WALTER et al. 1995 ; CHANG et al. 1997 ; ITO et al. 1997 ). In yeasts, additional functions have been ascribed to Nap1, as it has been shown to interact with Clb2 and Gin4, which are required for the proper control of mitotic events (KELLOGG et al. 1995 ; KELLOGG and MURRAY 1995 ; ALTMAN and KELLOGG 1997 ).

We first identified two genes (NBP1, NBP2) that encode proteins that interact with Nap1 by the two-hybrid system. NBP1, an essential gene with a coiled-coil structure in the center of the predicted amino acid sequence, encodes a protein localized in the nucleus as one or two tiny dots (SHIMIZU et al. 2000 ). On the other hand, NBP2, which contains a Src homology 3 (SH3) domain, encodes a novel protein consisting of 236 amino acids. SH3 domains constitute a family of protein-protein interaction modules that participate in diverse signaling pathways (e.g., cell cycle control, signal transduction, or cytoskeleton organization). From the Yeast Genome Database, 25 gene products in Saccharomyces cerevisiae contain at least one copy of the SH3 domain (MAYER 2001 ). Nbp2 shares structural homology with a Schizosaccharomyces pombe protein Skb5 (36% overall identity), a direct activator of Shk1 kinase (YANG et al. 1999 ).

Mitogen-activated protein kinase (MAPK) cascades control changes in gene expression, cytoskeletal organization, and cell division (HERSKOWITZ 1995 ; LEVIN and ERREDE 1995 ). In the PKC (protein kinase C) pathway, Pkc1 regulates a protein kinase cascade in which MAP-KKK Bck1 activates the redundant MAPKKs, Mkk1 and Mkk2, which in turn activate MAPK Mpk1. Mutants that perturb signaling through this pathway display phenotypes indicative of a defect in cell wall integrity. The polymorphic locus SSD1 is also important for promoting proper cell wall structure and integrity (KAEBERLEIN and GUARENTE 2002 ). Laboratory strains are divided into two types of SSD1 alleles, SSD1-V and ssd1-d. SSD1-V alleles can suppress the lethality due to a deletion of SIT4, while ssd1-d alleles are synthetically lethal in combination with sit4 deletion mutants (SUTTON et al. 1991 ). The SSD1 gene functions on a separate branch of the PKC MAPK pathway (KAEBERLEIN and GUARENTE 2002 ).

In this report, we show that cells lacking NBP2 exhibit cell death at high temperatures. In addition, nbp2 deletion mutants are sensitive to calcofluor white (CFW), indicating a defect in cell wall integrity. At high temperatures, nbp2 mutants failed to grow on medium supplemented with sorbitol, suggesting that NBP2 is required for additional functions other than cell wall integrity. Further, we used a new genetic approach in yeast to identify and characterize additional proteins in the Nbp2-dependent pathway. We isolated five genes (PTC1, PTC2, PTC4, MSB1, SKT5) as multicopy suppressors and six genes (GRE1, SEF1, MKK1, MKK2, YFR016C, PYK2) as deletion suppressors. A recent article also identified nbp2 synthetic lethal/sick genes (TONG et al. 2001 ). These data lead us to propose an NBP2 network (including NAP1), and we discuss a possible role of NBP2 in the cellular signaling system.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains, growth conditions, and transformations:
Yeast strains used in this study are listed in Table 1. Wild-type strains W303 and YPH499 are known to carry the defective SSD1 (C-terminal truncated) allele, called ssd1-d type (UESONO et al. 1997 ). Yeast cultures were grown in YPD (1% yeast extract, 2% peptone, 2% glucose, 400 mg/liter of adenine) and SD (0.67% yeast nitrogen base without amino acids, 2% glucose) supplemented with auxotrophic requirements. Yeast transformations were carried out by Frozen-EZ yeast transformation II (Zymo Research). Escherichia coli DH5 was used to propagate all plasmids. E. coli cells were cultured in Luria broth medium (1% tryptone, 0.5% yeast extract, 1% NaCl) and transformed by standard methods.

Construction of deletion mutants:
All gene disruptions were constructed using the described plasmids (Table 2). To delete NBP2, the following plasmid was constructed. The 790-bp MunI-HindIII fragment containing the 3' noncoding region of the NBP2 and a 506-bp EcoRI-HincII fragment containing the 5' end were blunt-end ligated into the SalI and KpnI sites of pMB-7 (provided by Neil A. R. Gow; see FONZI and IRWIN 1993 ) to produce pKO110, which contains the nbp2::hisG-URA3-hisG allele. This plasmid was digested with SacI and SphI and transformed into the wild-type strains. Deletion of NBP2 in Ura⁺ transformants was confirmed by PCR. The nbp2::hisG allele was derived from the nbp2::hisG-URA3-hisG allele after selection on 1.0 mg/ml 5-fluoroorotic acid.

For construction of the ptc1 and nap1 alleles, the 0.7-kbp NdeI-AseI fragment of PTC1 and the 0.6-kbp RsaI-XhoI fragment of NAP1 were replaced with LEU2. For construction of the mkk1 and nap1 alleles, the 1.4-kbp BsrGI-NdeI fragment of MKK1 and the 0.7-kbp NspV-XhoI fragment of NAP1 were replaced with URA3. For construction of the ptc2, ptc4, mkk1, and mkk2 alleles, the 1.2-kbp XbaI-NcoI fragment of PTC2, the 0.3-kbp NruI fragment of PTC4, the 1.0-kbp AatII fragment of MKK1, and the 1.5-kbp NcoI fragment of MKK2 were replaced with HIS3. For construction of the bck1 and mpk1 alleles, the 3.8-kbp BamHI-SphI fragment of BCK1 and the 1.3-kbp HindIII fragment of MPK1 were replaced with TRP1. Deletion mutants of components of the PKC MAPK pathway (bck1, mkk1, mkk2, mpk1) in W303 background were constructed on SD supplemented with 20% sorbitol. All of the deletion alleles were confirmed by PCR.

Plasmids:
Plasmids used in this study are listed in Table 2. To examine the subcellular localization of Nbp2, green fluorescent protein (GFP) was fused to the carboxy terminus of Nbp2 and expressed from its own promoter in a multicopy plasmid. To produce Nbp2-GFP (YEUpNBP2-GFP), the 0.76-kbp NheI-HindIII fragment containing GFP was ligated into the NheI-HindIII gap of YEUpNBP2.

Screening for multicopy suppressors:
The nbp2 cells (YPH499 nbp2::URA3: MATa ura3 leu2 trp1 his3 ade2 lys2 ssd1-d nbp2::URA3) were transformed with a yeast genomic library (provided by Y. Ohya) constructed in the multicopy vector YEp13. After 3 days of growth on SD-Leu plates at 30°, Leu⁺ transformants were transferred onto SD-Leu plates and colonies growing at 37° were obtained. Plasmids were isolated from the candidate transformants and transformed back into the nbp2 cells. A total of eight positive candidates were finally confirmed. The insert sequences in the candidate plasmids were sequenced from both ends to identify the region of the genome present. For those plasmids with more than one gene in the DNA insert, restriction fragments were subcloned to identify the responsible gene. DNA corresponding to the genes of interest was cloned into YEp13 or YEp51B and then transformed into various nbp2 cells.

Viability assay:
Yeast cells were precultured on YPD plates at 26° for 2 days, yielding stationary cells. These cells were collected and washed with deionized water. Approximately 1 x 10³ cells were spread on prewarmed (37°) YPD plates and cultured at 37°. After that, cells were shifted at 26° and live cells were counted after 3–5 days.

Screening for deletion suppressors:
A mutagenized yeast genomic library was provided by Michael Snyder (see BURNS et al. 1994 ). The mutagenized yeast DNA sequences were released from vector DNA by digestion with NotI and introduced into the nbp2 cells (YPH499 nbp2::URA3) by transformation and selection for the LEU2 marker in the transposon. After 7 days growth on SD-Leu plates at 36°, growing cells were selected. A total of five positive candidates among 8000 Leu⁺ transformants were finally confirmed. To determine the identity of sequences whose lacZ-Leu2-Amp fusion proteins localize to discrete sites within the cell, TaKaRa LA PCR in vitro cloning kit was used. The sequences fused to lacZ or AMP were determined using primers S2(lacZ) or S2(Amp(r)). Oligonucleotide primer sequences were as follows: S1(lacZ), 5'-AAAGCGCCATTCGCCATTCAGGCTG-3'; S2(lacZ), 5'-TTGGGTAACGCCAGGGTTTTCCCAG-3'; S1(Amp(r)), 5'-AAGTTGCAGGACCACTTCTGCGCTC-3'; S2(Amp(r)), 5'-TTATCTACACGACGGGGAGTCAGGC-3'.

Cell lysis assay:
The cell lysis assay detecting extracellular alkaline phosphatase was described by CABIB and DURAN 1975 and applied with the modifications described by PARAVICINI et al. 1992 .

GeneChip analysis:
Yeast strains used in GeneChip analysis were isogenic pairs of RAY (wild type) and YKO213 (nbp2) or W303 (wild type) and YKO215 (nbp2). Cells were precultured in YPD medium to an OD₆₀₀ of 0.1 at 26°. The liquid culture was separated and then cells were grown for 4 hr at 26° or 37°. Total RNA preparation, poly(A)⁺ RNA purification, and GeneChip analysis were carried out as described (OHKUNI et al. 2003 ). Oligonucleotide arrays (GeneChip Yeast Genome S98 Arrays) were manufactured by Affymetrix.

Fluorescence microscopy:
Cells expressing GFP were grown in SD medium to an OD₆₀₀ of 0.5–0.7. To stain nuclear DNA, cells were incubated with 10 µg/ml 4',6-diamidino-2-phenylindole (Molecular Probes, Eugene, OR) for 15 min. Samples were examined with a fluorescence microscope (Olympus BX-60) using a x100 UPlan Apo objective equipped with phase-contrast optics. GFP fluorescence was detected with filter XF104-2 (Omega Optical, Brattleboro, VT).

Two-hybrid assay:
The strains and plasmids for two-hybrid analysis are listed in Table 1 and Table 2, respectively. Plasmids pGAD-NBP1 and pGAD-NBP2 were isolated by two-hybrid screening. Plasmids pBTM-SIR4 and pGAD-SIR4 were used as a positive control. Yeast strains were grown to stationary phase in SD medium lacking leucine and tryptophan, diluted to 5 x 10⁶ cells per milliliter, and then incubated at 30° for 3–4 hr. ß-Galactosidase activity was determined as described by VOJTEK et al. 1993 .

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

NBP2 is essential at high temperatures:
To characterize the function of NBP2, we introduced a disruption of NBP2 (nbp2) in several yeast strains of different backgrounds. Deletion alleles of NBP2 (nbp2::hisG-URA3-hisG) were constructed as described in MATERIALS AND METHODS. In five representative strains, the nbp2 mutants failed to grow at high temperatures (Table 3 and Fig 1A). These cells, once incubated at high temperatures for 2 days, could not recover their growth again at a room temperature (Table 3), suggesting that nbp2 cells are killed at restrictive temperatures. We therefore tested cell viability at 37° of nbp2 mutants, and a significant level of cell death was observed at 37° in the nbp2 cells (Fig 1B). These results indicate that NBP2 is essential for mitotic growth at high temperatures.

View larger version (29K): [in this window] [in a new window] [Download PPT slide]   Figure 1. nbp2 cells were observed for cell death at restrictive temperatures. Isogenic yeast strains 4795-408 (wild type) and YKO201 (nbp2) were cultured. (A) Yeast cells were streaked onto YPD medium and incubated at 37° for 2 days. (B) Yeast cells were precultured on YPD medium at 26° for 2 days and then dilutions of cells plated onto YPD medium were shifted to 37° for the indicated times. After growth at 26° for 3–5 days, living cells were determined. WT, wild type.

View this table: [in this window] [in a new window]   Table 3. Temperature sensitivity of the nbp2 strains

Isolation of multicopy suppressors of the nbp2:
To identify additional components of Nbp2-dependent cell viability at high temperature, we isolated genes that, when overexpressed from multicopy plasmids, are capable of suppressing the growth defect of nbp2 mutants (YPH499 in regard to nbp2) at high temperatures. Eight candidate genes cloned in the multicopy vector YEp13 were obtained. Sequence analysis of subclones containing the suppressor activity revealed that these DNA fragments are identical in sequence to PTC2, PTC4, MSB1, and SKT5. Next, we reexamined these suppressors in a wild-type SSD1 background (YKO201; 4795-408 in regard to nbp2). In this background, SKT5 could not suppress the nbp2 mutants (Fig 2). The wild-type strains W303 and YPH499 are known to carry defective SSD1 alleles (UESONO et al. 1997 ). In fact, a single-copy of SSD1 partially suppressed the temperature sensitivity of the nbp2 mutation in the W303 or YPH499 backgrounds (Table 4). This result suggests that SKT5 may be a multicopy suppressor of the ssd1 mutation.

View larger version (56K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Multicopy suppressors of the temperature-sensitive growth of the nbp2 mutant. The nbp2 mutant, YKO201, transformed with different plasmids, was streaked onto YPD medium and incubated at 36° for 3 days. Plasmids are YEp51B (Vector), pKO117 (PTC1), pKO100 (PTC2), pKO102 (PTC4), pKO104 (MSB1), pKO105 (SKT5), and RSL-NBP2 (NBP2).

PTC2 and PTC4 encode proteins belonging to the type-2C serine threonine protein phosphatase (PP2C) class of highly conserved protein family found in all eukaryotes (CHENG et al. 1999 ). Since PTC1 encoding a type-2C serine threonine protein phosphatase (JIANG et al. 1995 ) was shown to interact with Nbp2 in a comprehensive two-hybrid assay (ITO et al. 2000 ; UETZ et al. 2000 ), we tested whether a multicopy plasmid carrying PTC1 suppresses nbp2. PTC1 was found to suppress a temperature-sensitive nbp2 allele (Fig 2). These results suggest that these PP2C phosphatases may be important in the control of mitotic growth at high temperatures in nbp2 cells.

To examine genetic interaction between NBP2 and PTCs, single deletion mutants of PTCs (ptc1, ptc2, and ptc4) and double deletion mutants (nbp2 ptc1, nbp2 ptc2, and nbp2 ptc4) were constructed. Three mutants, nbp2, ptc1, and nbp2 ptc1, were normal in their growth on YPD at 26° but failed to grow at an elevated temperature (Fig 3A). When transferred to a high temperature, nbp2 gradually lost its viability, while the nbp2 ptc1 double mutant did so more rapidly—at the same rate as the ptc1 mutant (Fig 3B). We also tested the ability of a multicopy plasmid carrying NBP2 to complement the growth defect of the ptc1 cells at 37°. NBP2 could not suppress a temperature-sensitive ptc1 allele (Fig 3C). These results suggest that PTC1 is also essential at high temperatures and that PTC1 acts downstream of NBP2.

View larger version (37K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Genetic interactions between NBP2 and PTCs. Isogenic yeast strains used for A–C were as follows: KA31 (wild type), YKO214 (nbp2), YKO226 (ptc1), and YKO222 (nbp2 ptc1). (A) Yeast cells grown for 2 days on YPD medium were harvested, washed, normalized by OD600, and spotted onto YPD plates in a series of six 10-fold dilutions. Plates were allowed to grow for 3 days at 26° or 37°. (B) Yeast cells were precultured on YPD medium at 26° for 2 days and then dilutions of cells plated onto YPD medium were shifted to 37° for the indicated times. After growth at 26° for 3–5 days, living cells were determined. (C) The ptc1 mutant, YKO226, transformed with different plasmids, was streaked onto SD medium and incubated at 36.5° for 3 days. Plasmids are YEp24 (Vector), pKO116 (PTC1), and YEUpNBP2. Isogenic yeast strains used for D and E were as follows: RAY (wild type), YKO213 (nbp2), YKO278 (ptc2), YKO279 (nbp2 ptc2), YKO280 (ptc4), and YKO281 (nbp2 ptc4). (D) Yeast cells grown for 2 days on YPD medium were harvested, washed, normalized by OD600, and spotted onto YPD plates in a series of five 10-fold dilutions. Plates were allowed to grow for 3 days at 26° or 37°. (E) Yeast cells were precultured on YPD medium at 26° for 2 days and then dilutions of cells plated onto YPD medium were shifted to 37° for the indicated times. After growth at 26° for 3–5 days, living cells were determined. WT, wild type.

On the other hand, two single mutants, ptc2 or ptc4, were grown normally on YPD at 26° or 37°, but the double mutants nbp2 ptc2 or nbp2 ptc4 failed to grow at an elevated temperature (Fig 3D). These double mutants lost their viability more rapidly than the nbp2 mutant did (Fig 3E), suggesting that genetic interactions between NBP2 and PTC2 or PTC4 are parallel pathways to promote mitotic growth at high temperatures.

Isolation of deletion suppressors of the nbp2:
To search for genes involved in the function of NBP2, we tried to identify genes that could suppress the growth defect of the nbp2 mutants at high temperatures when deleted (see MATERIALS AND METHODS). A total of 8000 Leu⁺ transformants, carrying random deletions in addition to nbp2, were obtained and subsequently screened for their ability to grow at the restrictive temperature of 36°. Five transformants were capable of forming colonies (Fig 4). Sequence analysis (see MATERIALS AND METHODS) revealed that these five disruptants inactivated one of the following genes: SEF1 (protein with similarity to transcription factors; POCH 1997 ), MKK1 (MAPKK; IRIE et al. 1993 ), PYK2 (pyruvate kinase; BOLES et al. 1997 ), and the promoter regions of GRE1 (insertion at 14 bp upstream of the initiation codon; unknown function) and YFR016C (insertion at 470 bp upstream of initiation codon; unknown function). We also tested using another yeast background (KA31; wild-type SSD1). nbp2 pyk2 and nbp2 yfr016c double disruptants in the KA31 background could not suppress the growth defect of nbp2 cells (data not shown). These results indicate that PYK2 and YFR016C may behave as deletion suppressors of the ssd1 mutation.

View larger version (76K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Deletion suppressors of the temperature-sensitive growth of the nbp2 mutant. Deletion suppressors obtained were streaked onto SD medium and incubated at 35.5° for 5 days.

MKK has a negative effect on NBP2 function at high temperature:
To investigate genetic interactions between NBP2 and components of the PKC MAPK pathway, five deletion mutants, nbp2 bck1, nbp2 mkk1, nbp2 mkk2, nbp2 mkk1 mkk2, and nbp2 mpk1 were constructed in two backgrounds. We first examined the temperature sensitivity of nbp2 cells in a wild-type SSD1 background (Fig 5A). The nbp2 mkk1 and nbp2 mkk2 double disruptants could partially suppress the growth defect of nbp2 cells on YPD solid medium at a high temperature. The MKK1 and MKK2 genes are functionally redundant and are 31% identical at the N-terminal half and 80% identical at the C-terminal half (IRIE et al. 1993 ). In contrast, growth of the disruptants, nbp2 bck1, nbp2 mkk1 mkk2, and nbp2 mpk1, on YPD solid medium at high temperatures was more severely affected than that of the nbp2 cells. Wild-type cells could grow at elevated temperatures by addition of 20% sorbitol. The disruptants, nbp2, nbp2 bck1, nbp2 mkk1 mkk2, and nbp2 mpk1, could not grow on YPD supplemented with 20% sorbitol at 38°, while the bck1 and mpk1 single disruptants could. These results suggest that the loss of either MKK1 or MKK2 alleviates loss of the Nbp2 function at high temperatures, although the PKC MAPK pathway itself is indispensable for cell growth.

View larger version (145K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Genetic interaction between NBP2 and components of PKC MAPK pathway. Yeast cells grown for 2 days on YPD medium were harvested, washed, normalized by OD600, and spotted onto the designated media in a series of five 10-fold dilutions. Plates were allowed to grow for 3 days. (A) Cells were cultured on YPD and YPD supplemented with 20% sorbitol at 36°–38°. Isogenic yeast strains in a SSD1-V background were as follows: RAY (wild type), YKO213 (nbp2), YKO233 (nbp2 bck1), YKO234 (nbp2 mkk1), YKO232 (nbp2 mkk2), YKO244 (nbp2 mkk1 mkk2), YKO235 (nbp2 mpk1), YKO247 (bck1), YKO237 (mkk1), YKO238 (mkk2), YKO254 (mkk1 mkk2), and YKO236 (mpk1). (B) Cells were cultured on YPD and YPD supplemented with 20% sorbitol at 26°. Isogenic yeast strains in ssd1-d background were as follows: W303 (wild type), YKO215 (nbp2), YKO268 (nbp2 bck1), YKO264 (nbp2 mkk1), YKO262 (nbp2 mkk2), YKO266 (nbp2 mkk1 mkk2), and YKO253 (nbp2 mpk1). WT, wild type.

We next examined the temperature sensitivity of the nbp2 cells in defective SSD1 alleles (Fig 5B). In this background, the nbp2 mkk1 and nbp2 mkk2 double disruptants could partially suppress the growth defect of the nbp2 cells on YPD solid medium at high temperatures (data not shown). On the other hand, the nbp2 bck1, nbp2 mkk1 mkk2, and nbp2 mpk1 disruptants could no longer grow on YPD solid medium at 26°. These disruptants could grow on YPD supplemented with 20% sorbitol, indicating that they are deficient in cell wall integrity. Thus, Nbp2 may play a role in promoting cell wall stability.

NBP2 also regulates cell wall integrity:
Mutations that perturb signaling through the PKC MAPK pathway can result in sensitivity to changes in external osmolarity, defective budding, and cell lysis (LEVIN and ERREDE 1995 ; KAEBERLEIN and GUARENTE 2002 ). To test a cell wall defect in nbp2 mutants, we first examined sensitivity to CFW, a cell wall perturbing agent. The nbp2 mutants were more sensitive to CFW than were wild-type cells (Table 3 and Fig 6A). Cell lysis was next examined on the basis of the extracellular release of alkaline phosphatase (Fig 6C). Two background nbp2 mutants were grown on YPD plates at 26° and incubated overnight at 37°, and then cell lysis assays were performed. The mpk1 mutant in a ssd1-d background turned dark blue after cell lysis assay, indicating that the cells lyse rapidly, while wildtype remain white. On the other hand, the mpk1 in a SSD1-V background and the nbp2 in a ssd1-d background turned pale blue, indicating that the cells lyse slowly. The PKC MAPK pathway and Ssd1 are defined as parallel to regulate cell wall integrity (KAEBERLEIN and GUARENTE 2002 ). Thus, Nbp2 may influence cell wall integrity, but this effect is smaller than that of the PKC MAPK pathway.

View larger version (78K): [in this window] [in a new window] [Download PPT slide]   Figure 6. Mutation of NBP2 results in cell wall integrity. (A and B) Yeast cells grown for 2 days on YPD medium were harvested, washed, normalized by OD600, and spotted onto the designated media in a series of five 10-fold dilutions. Plates were allowed to grow for 3 days. (A) Media in each plate were YPD, YPD supplemented with 0.1 mg/ml CFW (+CFW), YPD supplemented with 20% sorbitol (+Sorbitol), and YPD supplemented with 0.1 mg/ml CFW and 20% sorbitol (+CFW+Sorbitol). Isogenic yeast strains RAY (wild type) and YKO213 (nbp2) were used. (B) Cells were cultured on YPD, YPD supplemented with 0.05 or 0.1 mg/ml CFW, and YPD supplemented with 0.1 mg/ml CFW and 20% sorbitol at 26°. Isogenic yeast strains used were as follows: RAY (wild type), YKO213 (nbp2), YKO233 (nbp2 bck1), YKO234 (nbp2 mkk1), YKO232 (nbp2 mkk2), YKO244 (nbp2 mkk1 mkk2), YKO235 (nbp2 mpk1), YKO247 (bck1), YKO237 (mkk1), YKO238 (mkk2), YKO254 (mkk1 mkk2), and YKO236 (mpk1). (C) Cell lysis assay of the nbp2 cells. Approximately 105 cells were spotted onto YPD plates, cultivated for 2 days at 26°, and then incubated overnight at 37°. The plates were then overlaid with an alkaline phosphatase assay solution. Isogenic yeast strains in a SSD1-V background were as follows: RAY (wild type), YKO236 (mpk1), and YKO213 (nbp2). Isogenic yeast strains in a ssd1-d background were as follows: W303 (wild type), YKO252 (mpk1) and YKO215 (nbp2). WT, wild type.

We also examined the relationships between cell death at high temperatures and cell wall integrity. Interestingly, the sensitivity to CFW at 26° caused by the loss of Nbp2 is completely suppressed by addition of 20% sorbitol to the medium (Fig 6A). At a high temperature (38°), however, the nbp2 mutant failed to grow on YPD supplemented with CFW and 20% sorbitol, suggesting that NBP2 at high temperatures is required for additional functions other than cell wall integrity.

We further examined the CFW sensitivity of the nbp2 cells related to the PKC MAPK pathway (Fig 6B). All of the disruptants except for mkk1 and mkk2 single mutants were sensitive to CFW. The CFW sensitivity of these disruptants except for the nbp2 mkk2 double mutant could be suppressed by addition of 20% sorbitol to the medium, indicating that NBP2 and components of the PKC MAPK pathway play similar roles in promoting cell wall stability. On the other hand, the CFW sensitivity of the nbp2 mkk2 double disruptant could be partially suppressed by loss of Mkk1. These observations indicate that cells lacking the function of Nbp2 grow better in the absence of MKK1 and MKK2 for maintaining cell wall integrity.

GeneChip analysis:
It is possible that the suppression of temperature-sensitive growth in nbp2 cells was the consequence of gene expression of PTCs and the PKC MAPK pathway. To test this, we measured the mRNA levels of PTCs (PTC1, PTC2, PTC4) and PKC MAPK pathway genes (BCK1, MKK1, MKK2, MPK1) in wild-type and nbp2 cells in the cells with two genetical backgrounds (SSD1-V and ssd1-d) at two growth temperatures, 26° or 37°, using the GeneChip method. In either background, by shifting growth temperature from 26° to 37°, no significant differences (within twofold up- or downregulation) in mRNA levels of those genes were observed (data not shown).

Nbp2 localizes in the cytoplasm:
To investigate the subcellular localization of Nbp2, we constructed a fusion protein with GFP. GFP was fused to a C terminus of the Nbp2 and expressed from a multicopy plasmid in a nbp2 strain. This multicopy Nbp2-GFP plasmid complemented the temperature sensitivity of the nbp2 strain at 37° (data not shown). In most cells, Nbp2-GFP was found in cytoplasm; in some cells, several small spots were also observed in the cytoplasm (Fig 7). An experiment to determine the location of these small spots is in progress. Thus, the localization of Nbp2 is in the cytoplasm of a cell, suggesting that Nbp2 may play a role in a cellular signaling system in cytoplasm.

View larger version (45K): [in this window] [in a new window] [Download PPT slide]   Figure 7. Subcellular localization of Nbp2. The subcellular localization of a Nbp2-GFP fusion protein expressed from a multicopy plasmid (YEUpNBP2-GFP) was examined in a nbp2 strain (YKO214). Cells were grown in SD medium to an OD600 of 0.5–0.7 and were visualized using a fluorescence microscope. Bar, 5 µm.

Synthetic sick interaction between Nbp2 and Nap1:
In a previous article (SHIMIZU et al. 2000 ), we isolated Nbp2 that interacts with Nap1 by the two-hybrid system (Fig 8A). Here, we tried to determine the genetic interaction between NBP2 and NAP1 (Fig 8B). Cells lacking Nap1 could grow well at 37° and on YPD solid medium supplemented with 0.1 mg/ml CFW (data not shown). When tested for various phenotypes characteristic of the nbp2 nap1 double mutant, this disruptant was more sensitive at high temperatures and to CFW than were either nbp2 or nap1 mutants. But the nbp2 nap1 double mutant was slightly temperature sensitive compared with the nbp2 mutant, and new morphological defects at high temperatures were not observed (data not shown). On the other hand, the CFW sensitivity of the nbp2 nap1 double mutant could not be suppressed by addition of 20% sorbitol to the medium, indicating that this mutant may produce another defect in cellular integrity. Thus, these observations demonstrate that the relation between Nbp2 and Nap1 is a synthetic sick-type interaction.

View larger version (50K): [in this window] [in a new window] [Download PPT slide]   Figure 8. Synthetic sick interaction between NBP2 and NAP1. (A) Strain L40 carrying pBTM-NAP1 was transformed with pGAD, pGAD-NBP1, or pGAD-NBP2. Exponential-phase yeast cells were assayed for ß-galactosidase activity as described by VOJTEC et al. (1993). Plasmids pBTM-SIR4 and pGAD-SIR4 were used as a positive control. (B) Yeast cells grown for 2 days on YPD medium were harvested, washed, normalized by OD600, and spotted onto YPD, YPD supplemented with 0.05 mg/ml CFW, and YPD supplemented with 0.1 mg/ml CFW and 20% sorbitol in a series of five 10-fold dilutions. Plates were allowed to grow for 3 days at 26° or 35°. Isogenic yeast strains used were as follows: RAY (wild type), YKO213 (nbp2), YKO227 (nap1), and YKO240 (nbp2 nap1). WT, wild type.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Nap1 is necessary to keep proper nucleosome structures in vitro (WALTER et al. 1995 ; CHANG et al. 1997 ; ITO et al. 1997 ) and is required for the mitotic cyclin Clb2 to be able to execute a subset of its normal mitotic activities (KELLOGG and MURRAY 1995 ). Yet little is known about Nap1 in vivo, although in an earlier report we indicated that Nbp2, containing an SH3 domain, interacts with Nap1 by a two-hybrid system (SHIMIZU et al. 2000 ). In the present study, we demonstrate that the Nbp2 regulates mitotic cell growth at high temperatures and cell wall integrity. Furthermore, we have used a new genetic approach to identify the function of Nbp2. These genetic analyses suggest that Ptc pathways regulate cell death at high temperatures and that the PKC MAPK pathway regulates cell wall integrity together with Nbp2.

nbp2 cells have severe growth defects at high temperatures:
In nbp2 strains, cells could not grow at high temperatures and rapid loss of viability at a restrictive temperature was observed. This temperature sensitivity could not be suppressed by the addition of 20% sorbitol to the medium (Fig 6A), indicating that the major cause of cell death at high temperatures may reside in a process other than cell wall integrity. Cell death of nbp2 at high temperatures is regulated by several PP2Cs of the protein phosphatase class of enzymes. Genetic analyses suggest that PTC1 acts downstream of NBP2 and that both PTC2 and PTC4 act parallel to NBP2. PTC1, PTC2, and PTC3 are known as negative regulators of the HOG (high-osmolarity glycerol) MAPK pathway (WARMKA et al. 2001 ). We isolated MAPKK, related to the yeast PKC MAPK pathway, as deletion suppressors of the nbp2 mutant. Therefore, it is possible that two MAPK pathways, PKC and HOG, cross-talk through NBP2.

A role for Nbp2 in promoting cell wall integrity:
In all the nbp2 strains used in this study, cells could not grow on YPD supplemented with CFW, a cell wall perturbing agent, suggesting that nbp2 mutants have a primary defect in cell wall integrity. The strains we used can be divided into two groups, SSD1-V (active allele; 4795-408, RAY, KA31) and ssd1-d (inactive allele; W303, YPH499). SSD1 can act in parallel to the PKC MAPK pathway to promote cell wall integrity (KAEBERLEIN and GUARENTE 2002 ). For several reasons the genetic interactions between NBP2 and SSD1 and the PKC MAPK pathway are parallel to promote cell wall integrity. First, the NBP2 and SSD1 pathway generates a parallel signal at high temperatures. As in the case of the nbp2 mutants in W303 and YPH499 backgrounds, a single-copy vector carrying SSD1 could partially suppress the temperature-sensitive phenotype of the nbp2 mutant (Table 4). The NBP2, ssd1-d strains (W303, YPH499), transformed with the single-copy vector carrying SSD1, could grow to some extent at an elevated temperature (Table 4). These results suggest that the relation between NBP2 and SSD1 exhibits a synthetic sick phenotype. Second, the double disruptants, nbp2 bck1, nbp2 mkk1 mkk2, and nbp2 mpk1 were more sensitive than the single disruptants (Fig 5A), indicating that the relation between NBP2 and the PKC MAPK pathway must be a parallel pathway. Third, the disruptants in a W303 background, nbp2 bck1, nbp2 mkk1 mkk2, and nbp2 mpk1, could not grow on YPD at 26°, while they could grow on YPD supplemented with 20% sorbitol (Fig 5B), indicating that the synthetic lethality of these disruptants may come from a defect in cell wall stability. Finally, double-mutant cells carrying mpk1, nbp2, and ssd1-d were lysed more rapidly than single mutant cells (Fig 6C). Taken together, we believe that the NBP2, SSD1, and PKC MAPK pathways define three parallel pathways that regulate cell wall integrity.

We also suggest different roles for MKK1 and MKK2, which correspond to MEK in S. cerevisiae, in the cell signaling system under the influence of NBP2. The CFW sensitivity of the nbp2 mkk2 double mutant is more severe than that of nbp2 mkk1 cells. The CFW sensitivity of the nbp2 mkk2 double mutant could not be suppressed by addition of 20% sorbitol to the medium, but could be suppressed by the loss of MKK1 (Fig 6B). Thus, we propose that MKK1 and MKK2 are not necessarily redundant and that each gene may play a separate role in promoting cell wall integrity.

We have isolated mkk1 and mkk2 and loss of function of either MKK1 or MKK2 as deletion suppressors of nbp2 cells. But the PKC MAPK pathway itself is indispensable for cell wall maintenance in the nbp2 mutant. According to our GeneChip analysis, no significant differences in the mRNA amounts of components of the PKC MAPK pathway were observed between wild-type and nbp2 cells (data not shown). These results suggest that MKK1 and MKK2 may have roles other than in cell wall integrity in the Nbp2-dependent pathway.

A new genetic approach of deletion suppressor:
In this study, we have used a novel genetic approach, which we have called deletion suppressor, to pursue characterization of Nbp2. We isolated six candidate genes as deletion suppressors of nbp2 cells. Until now, the isolation of multicopy suppressor and synthetic lethal alleles has been practiced as a standard method. By isolating a deletion suppressor, we can argue for, for example, a balance of two opposite pathways, such as kinase and phosphatase. As in Fig 9A, we think that if a gene responsible for negative effect ("A" in Fig 9A) is defective, a positive effector should be removed at the same time. If the balance of "A" and "B" in Fig 9A is affected when negative regulators only are deleted, the accumulation of a poisonous factor might occur in this pathway. In the case of Nbp2, it is possible that A in Fig 9A is Ptcs, B in Fig 9A is Mkk1/2, and the poison is the hyperphosphorylation of the target of Ptcs. There could be another case in which, by inactivating one gene, the toxic gene products might accumulate and cause the growth arrest, in which case one should try to remove the toxic gene on deletion screening. We believe that this technique is a very useful genetic approach for the future.

View larger version (32K): [in this window] [in a new window] [Download PPT slide]   Figure 9. The role of Nbp2. (A) Model for a deletion suppressor. "A" is a negative regulator. "B" is a positive regulator. (1) In wild-type cells, there is a balance between "A" and "B." (2) If "A" is deleted, the balance of "A" and "B" is lost. (3) When both "A" and "B" are deleted, there is a balance between "A" and "B" as in wild-type cells. (B) Genetic and biochemical interaction network of Nbp2. Genes are represented as nodes, and interactions are represented as edges that connect the nodes. All of the multicopy and deletion suppressor genes of the NBP2 deficiency and part of the protein-protein and synthetic lethal/sick relationships are shown. Overproduction of MSB1 partially suppresses bem1 or bem2 mutants. References for all the genes except our data can be found listed as synthetic lethal/sick interactions in TONG et al. 2001 , in the Yeast Protein Database, and in the Saccharomyces Genome Database. Nbp2, directly or indirectly, interacts with protein phosphatase (red) and kinase (green). The interaction of Nbp2 and Ssd1 or Nap1 is synthetic sick. SKT5, PYK2, and YFR016C are isolated using only a ssd1-d background (asterisk).

Nbp2 signaling network:
In this report, we isolated multicopy suppressors (PTC1, PTC2, PTC4, MSB1, SKT5) and deletion suppressors (GRE1, SEF1, MKK1, MKK2, YFR016C, PYK2) of the NBP2 deficiency. Previous work has identified several genes that show a synthetic lethal/sick interaction with nbp2 (TONG et al. 2001 ) and a two-hybrid interaction with Nbp2 (ITO et al. 2000 ; UETZ et al. 2000 ). The signaling network of Nbp2 shown in Fig 9B contains four interactions (multicopy and deletion suppressor, synthetic lethal/sick, and two-hybrid interactions). NBP2 has recently been shown to show a synthetic lethal/sick interaction with bni1 (TONG et al. 2001 ). We have noted that many of the genes interacting with NBP2 also shared an interaction with BNI1. BNI1 encodes members of the highly conserved family that control the assembly of actin cables, which guide myosin motors to coordinate the polarized cell growth and spindle orientation (EVANGELISTA et al. 2002 ). As TONG et al. 2001 suggested, one of the Nbp2 functions might be cytoskeletal organization. We believe that the role of Nbp2 is as a general cellular signaling system (e.g., cytoskeletal organization, cell wall maintenance, and cell cycle control). Deletion of NBP2 does not apparently affect actin staining (K. OHKUNI and A. KIKUCH, unpublished data). We showed that NBP2 is required for promoting cell wall integrity and is essential for mitotic growth at high temperatures. NBP2, which interacted and related with several genes (MSB1, SMI1, FAB1, BEM1, BEM2), is involved in cell polarity and cell wall organization. Furthermore, Nbp2 shows a synthetic sick interaction with Nap1, which binds to Clb2 (B-type cyclin; KELLOGG et al. 1995 ) and to Clb4 (B-type cyclin; TONG et al. 2001 ). Clearly, further studies are needed to more clearly determine the role of Nbp2 in the cellular signaling system.

Genetic screens in yeast have revealed that NBP2 directly or indirectly interacts with protein phosphatases and kinases (Fig 9B). Previous studies demonstrate that Nap1 binds Gin4 kinase and works with Cla4 and Elm1 kinase to control mitotic events (SREENIVASAN and KELLOGG 1999 ). Mammalian TAF1 proteins ( and ß), a Nap1 family protein, have the inhibitory activity of type-2A protein phosphatase (SAITO et al. 1999 ). It is interesting to note that the functions of Nap1 and Nbp2 are very similar, involving phosphorylation and dephosphorylation of proteins, which suggests that Nbp2 may be a part of Nap1's function through various signaling networks. We do not know yet what kind of proteins were phosphorylated or dephosphorylated as targets of the Nbp2 signaling network; however, a more precise understanding of the role of Nbp2 in these processes will emerge from further studies of the interactions of this protein.

	FOOTNOTES

¹ Present address: Mitsubishi Kasei Institute of Life Sciences, Tokyo 194-0031, Japan.

	ACKNOWLEDGMENTS

We thank Michael Snyder and Yoshikazu Ohya for providing the yeast genomic library and Neil A. R. Gow, Rolf Sternglanz, Tatsuya Maeda, and Yoshiko Kikuchi for providing strains and plasmids; Katsuhiko Shirahige for support in the GeneChip analysis; Yoshiyuki Nakagawa, Toshio Kanbe, Tomohiro Akashi, Motoshi Suzuki, Ikuyo Mizuguchi, Ayako Sakaguchi, Tsuya Taneda, Tomoko Tsuruta, and Mika Kawagishi for helpful discussions and encouragement; and Marie Fujino for her contributions to multicopy suppressor screening. Special thanks go to Douglas Kellogg for helpful discussions and extensive help with the manuscript. This study was supported in part by grants from the Ministry of Education, Science, Sports and Culture of Japan.

Manuscript received February 3, 2003; Accepted for publication June 17, 2003.

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