Isolation and Characterization of Nrf1p, a Novel Negative Regulator of the Cdc42p GTPase in Schizosaccharomyces pombe

Corresponding author: Douglas I. Johnson, Department of Microbiology and Molecular Genetics, 202B Stafford Hall, University of Vermont, Burlington, VT 05405., dijohnso{at}zoo.uvm.edu (E-mail)

Communicating editor: P. G. YOUNG

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The Cdc42p GTPase and its regulators, such as the Saccharomyces cerevisiae Cdc24p guanine-nucleotide exchange factor, control signal-transduction pathways in eukaryotic cells leading to actin rearrangements. A cross-species genetic screen was initiated based on the ability of negative regulators of Cdc42p to reverse the Schizosaccharomyces pombe Cdc42p suppression of a S. cerevisiae cdc24^ts mutant. A total of 32 S. pombe nrf (negative regulator of Cdc forty two) cDNAs were isolated that reversed the suppression. One cDNA, nrf1⁺, encoded an ~15 kD protein with three potential transmembrane domains and 78% amino-acid identity to a S. cerevisiae gene, designated NRF1. A S. pombe nrf1 mutant was viable but overexpression of nrf1⁺ in S. pombe resulted in dose-dependent lethality, with cells exhibiting an ellipsoidal morphology indicative of loss of polarized cell growth along with partially delocalized cortical actin and large vacuoles. nrf1⁺ also displayed synthetic overdose phenotypes with cdc42 and pak1 alleles. Green fluorescent protein (GFP)-Cdc42p and GFP-Nrf1p colocalized to intracellular membranes, including vacuolar membranes, and to sites of septum formation during cytokinesis. GFP-Nrf1p vacuolar localization depended on the S. pombe Cdc24p homolog Scd1p. Taken together, these data are consistent with Nrf1p functioning as a negative regulator of Cdc42p within the cell polarity pathway.

MANY cellular events occur at a specific site within the cell and the ability to orient these events in a non-random, spatially directed manner to generate cellular asymmetry is termed cell polarity. Cell polarity is crucial for the control of many cellular and developmental processes, including the generation and maintenance of cell shape, morphological events during differentiation, intracellular movement of organelles, and directed secretion and incorporation of cell surface constituents (DRUBIN and NELSON 1996 ). An initial step in the cell polarity process is the establishment of an axis of polarity, which is followed by the cell-cycle-dependent distribution of cellular components to points along that axis.

The Cdc42p GTPase plays a critical role in the establishment of cell polarity in most, if not all, eukaryotic organisms (JOHNSON 1999 ). Cdc42p activity is regulated through its guanine-nucleotide-bound state and by its subcellular localization. A number of proteins that function as regulators and effectors of Cdc42p activity have been identified, including guanine-nucleotide exchange factors (GEFs) that activate Cdc42p, such as Saccharomyces cerevisiae Cdc24p and its Schizosaccharomyces pombe homolog Scd1p, and GTPase-activating proteins (GAPs) that deactivate Cdc42p, such as S. cerevisiae Bem3p and Rga1p (JOHNSON 1999 ). In S. pombe, cdc42 mutants are inviable and arrest as small, round cells (MILLER and JOHNSON 1994 ), indicating that Cdc42p has an essential function in directing polarized growth to the ends of cells. However, scd1 mutants are viable and display normal-sized round cells (CHANG et al. 1994 ), indicating a role for Scd1p in polarized cell growth but raising the possibility that other Cdc42-GEFs may exist in S. pombe. Two downstream effectors of S. pombe Cdc42p, the Pak1p/Shk1p and Pak2p/Shk2p protein kinases, show a high degree of similarity to mammalian and S. cerevisiae PAK (p21-activated protein kinase)-like kinases (MARCUS et al. 1995 ; OTTILIE et al. 1995 ; SELLS et al. 1998 ; YANG et al. 1998 ). Pak1p is an essential protein, with the pak1 mutant arresting with small, round cells similar to cdc42 cells, suggesting that Pak1p plays an essential positive role in the pathway. Pak2p is not essential and pak2 cells do not exhibit abnormal morphologies.

A cross-species genetic screen was developed to identify negative regulators of Cdc42p in S. pombe. This screen was based on the observations that S. pombe cdc42⁺ could suppress a cdc24^ts mutation in S. cerevisiae and that the S. cerevisiae Bem3p or Rga1p GAPs could reverse this suppression. Thirty-two S. pombe cDNAs were isolated by their ability to reverse this suppression. One of these cDNAs, nrf1⁺, encoded a novel ~15 kD putative transmembrane protein that was lethal when overexpressed in S. pombe, resulting in ellipsoidal cells indicative of a loss-of-polarity phenotype. nrf1⁺ also displayed synthetic overdose phenotypes with cdc42 and pak1 mutants and green fluorescent protein (GFP)-Nrf1p and GFP-Cdc42p colocalized. These data suggest that Cdc42p and Nrf1p functionally interact within the cell polarity pathway.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains, plasmids, media, and growth conditions:
S. pombe cells were grown in yeast extract and supplements (YES) complex media or in Edinburgh minimal media (EMM) lacking uracil (Ura), leucine (Leu), or both (MORENO et al. 1991 ). EMM and EMM agar were purchased from Bio101 (Vista, CA). The S. pombe strains used were ED668 (h⁺ ade6.M216 leu1.32 ura4-D18; provided by P. Fantes) and SPSCD1U (h⁹⁰ ade6.M210 leu1.32 ura4-D18 scd1::ura4⁺; provided by E. Chang). The nrf1⁺ coding region along with 34 bp downstream of the stop codon were replaced with the S. pombe ura4⁺ gene, using a polymerase chain reaction (PCR) approach to generate the nrf1::ura4⁺ allele. The nrf1::ura4⁺ strain JM2 (h⁺ ade6.M216 leu1.32 ura4-D18 nrf1::ura4⁺) was generated by transforming a nrf1:: ura4⁺ PCR product into ED668, selecting for Ura⁺ transformants. Integration of the nrf1::ura4⁺ allele at the nrf1 locus was verified by PCR analysis of Ura⁺ transformants. S. cerevisiae cells were grown in YEPD complex media or in SD synthetic complete drop-out media lacking specific amino acid(s) and containing 2% glucose as a carbon source. S. cerevisiae strains used were Y147 (MATa cdc24-4 his3 leu2 ura3; BENDER and PRINGLE 1989 ), W303-1A (MATa ade2 his3-11,5 leu2-3,112 trp1-1 ura3-1; THOMAS and ROTHSTEIN 1989 ). Yeast transformations were performed as previously described (SHERMAN et al. 1986 ; MORENO et al. 1991 ; PRENTICE 1991 ). Escherichia coli strain MH1066 (lacX74 hsr^- ppsL pyrF::Tn5 leuB600 trpC9830 galF galK) and SURE cells were used as plasmid hosts (SAMBROOK et al. 1989 ). Complementation of the pyrF::Tn5 mutation in MH1066 by the S. cerevisiae URA3 gene was used to isolate URA3-based pDB20 plasmids on M9-media lacking uracil. Plasmids used in this study are listed in Table 1. Enzymes, PCR kits, and other reagents were purchased from standard commercial sources and used as specified by the suppliers. Oligonucleotide primers were obtained from Genosys Biotechnologies Inc. (The Woodlands, Texas). Standard methods were used for recombinant DNA manipulations (SAMBROOK et al. 1989 ) and details of the specific recombinant DNA manipulations used to generate the plasmids in this study are available upon request. The DNA sequence of all PCR products was determined by ABI automated sequencing through the Vermont Cancer Center DNA Sequencing Facility.

Reversal of Cdc42p-dependent suppression screen:
To isolate possible negative regulators of cdc42⁺, 2.5 µg of a pDB20-based S. pombe cDNA library (FIKES et al. 1990 ) was transformed into S. cerevisiae Y147[pRS315-cdc42⁺] cells, and transformants were selected on SD-Ura-Leu media at 23°. A total of 2500 transformants were picked, resuspended into 96-well microtiter dishes, and tested for temperature sensitive (ts) growth by replica plating onto SD-Ura-Leu + 1 M sorbitol media at 23° and 37°, with confirmation by zone streaking of ts transformants (e.g., Fig 1). Of the original 2500 transformants, 38 displayed a ts phenotype, and plasmids from these transformants were rescued into E. coli MH1066 cells, selecting for an Ura⁺ phenotype on M9-Ura media. From these 38 transformants, 33 plasmids containing a library insert were identified; these were retransformed into Y147[pRS315-cdc42⁺], confirming their ability to confer a ts phenotype at 38°. DNA sequence analysis of the 33 plasmids revealed the presence of 32 complete or partial cDNAs, whose sequences were analyzed using the BLAST search engine (ALTSCHUL et al. 1990 , ALTSCHUL et al. 1997 ). cDNAs with nucleotide identity to known S. pombe sequences are indicated in Table 2 along with new S. pombe DNA sequence information, which was entered into the GenBank database with the listed accession numbers. New S. cerevisiae sequence information (i.e., designation of S. cerevisiae NRF1) was entered into the Saccharomyces Genome Database (http://genome-www.stanford.edu/Saccharomyces/).

View larger version (106K): In this window In a new window Download PPT slide   Figure 1. S. pombe cdc42+ suppression of the S. cerevisiae cdc24-4ts mutation can be reversed by Cdc42p-GAPs and other regulators. (Top) Plasmid combinations of pRS316 + Yep13 (- sector); pRS316-cdc42+ + YEp13 (cdc42+ sector); pRS316-cdc42+ + YEp13-RGA1 (cdc42+ + RGA1 sector); and pRS316-cdc42+ + YEp13-BEM3 (cdc42+ + BEM3 sector) were transformed into Y147 (cdc24-4ts) cells and selected on SC-Leu-Ura media at 30°. Individual transformants were streaked to SC-Leu-Ura + 1 M sorbitol media at 23° and 37°. (Bottom) Y147 cells containing S. pombe cdc42+, either pRS316-cdc42+(- sector) or pRS315-cdc42+, were transformed with YEp13-RGA1 (RGA1 sector); YEp13-BEM3 (BEM3 sector), five representative nrf genes (1, 13, 14, 20, and 29), and individual transformants were streaked to SC-Leu-Ura + 1 M sorbitol media at 23° and 37°.

S. pombe overexpression phenotypes:
Thiamine (Thi) was added to S. pombe growth media at 5 µg/ml to repress transcription from the nmt1 promoter. Combinations of nrf1⁺ and cdc42 or pak1 alleles, under different strength thiamine-repressible nmt1 promoters (FORSBURG 1993 ) were transformed into S. pombe ED668 cells and transformants were selected on EMMS (EMM + supplements)-Leu-Ura+Thi media at 30° (repressing conditions). The transformants were then serial-streaked twice to EMMS-Leu-Ura-Thi media (derepressing conditions) to determine viability. To examine cellular morphologies, the above transformants as well as scd1 cells containing pREP1-nrf1⁺ were grown under repressing conditions at 30°C overnight, diluted to OD₆₀₀ = 0.005 into derepressing media, and grown to mid-log phase (8–11 generations). Cells were harvested, briefly sonicated in the presence of 0.25 M NaCl (if necessary to relieve cell clumping), and examined.

Photomicroscopy:
Methods for preparing and staining S. pombe cells with rhodamine-conjugated phalloidin have been described previously (ALFA et al. 1993 ). Vacuolar staining was conducted using carboxy-DCFDA (Molecular Probes, Eugene, OR). The cells were grown to mid-log phase under derepressing conditions (described above) and incubated for 30 min in YE media, pH 4.5 containing 10 µM carboxy-DCFDA. GFP images were captured from mid-log phase cells after 18–28 hr in derepressing conditions. The photomicroscopy techniques employed have been described previously (RICHMAN et al. 1999 ).

Immunoblot analysis:
Total cellular protein was isolated from cells containing nmt1 promoter-driven HA-Nrf1 fusion proteins. The cells were grown in EMMS-Ura-Thi liquid media to mid-log phase, collected, washed with dH₂O, resuspended in 100 µl of 1x phosphate-buffered saline, and spheroplasted at 37° in the presence of 75–150 µg/ml zymolyase until a sample showing >80% lysis was observed upon the addition of SDS to 0.1%. The spheroplasts were then collected and resuspended in lysis buffer (0.3 M sorbitol, 140 mM NaCl, 50 mM Tris pH 8.0) with protease inhibitors (1:100 dilutions of 5 mg/ml aprotinin, 5 mg/ml leupeptin in water, 6 mg/ml phenylmethylsulfonyl fluoride, and 5 mg/ml pepstatin in methanol). SDS (0.1%) was added and the samples were vortexed briefly. Protein samples were diluted 1:2 in SDS-lysis buffer (LAEMMLI 1970 ) containing 40% ß-mercaptoethanol, heated at 100° for 5 min, separated on a 12.5% SDS-polyacrylamide gel, and protein was transferred to nitrocellulose paper (BA-S83, 0.02 µm; Schleicher and Schuell, Keene, NH). A total of 30 µg of protein was added to each lane as determined by the Bradford protein assay. Mouse anti-HA antibody 12CA5 (kindly provided by I. Rainville, University of Vermont) was used at 1:5000 dilution, and horseradish peroxidase-conjugated anti-mouse IgG secondary antibody (Amersham, Arlington Heights, IL) was used at 1:750 dilution. Immunoblots were developed using the Dupont Renaissance system.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

S. pombe cdc42⁺ suppressed the S. cerevisiae cdc24-4^ts mutation and the S. cerevisiae BEM3 and RGA1 GAPs reversed this suppression:
Previous studies have shown that S. cerevisiae CDC42 on a low-copy vector was able to suppress the cdc24-4^ts mutation in the presence of 1 M sorbitol (BENDER and PRINGLE 1989 ). Presumably, levels of activated GTP-bound Cdc42p are reduced in the cdc24-4 mutant at 37° and suppression is through an increase in levels of GTP-bound Cdc42p upon overexpression. Addition of a high-copy vector containing either the S. cerevisiae BEM3 or RGA1-encoded GAP reverses this suppression, presumably by inactivation of the Cdc42-GTP to a GDP-bound state (BI and PRINGLE 1996 ). S. pombe cdc42⁺ under S. cerevisiae CDC42 promoter control was inserted into a low-copy S. cerevisiae plasmid (pRS315-cdc42⁺) and transformed into cdc24-4^ts strain Y147. This plasmid was able to suppress the cdc24-4^ts mutant at 37° in the presence of 1 M sorbitol (Fig 1). In addition, high-copy plasmids containing either RGA1 or BEM3 reversed this suppression, returning transformants to a Ts^- phenotype. These results suggested that the S. cerevisiae GAPs could act on S. pombe Cdc42p and formed the basis for the isolation of S. pombe proteins that function in an analogous manner.

Identification of potential negative regulators of cdc42⁺:
An S. pombe cDNA library was transformed into Y147[pRS315-cdc42⁺] and Ura⁺ transformants were screened for growth at 37° in the presence of 1 M sorbitol. Of 2500 colonies screened, 38 transformants were isolated that displayed a Ts^- phenotype. Thirty-three plasmids were recovered from these transformants and were retransformed into Y147[pRS315-cdc42⁺]; upon retransformation, all resulted in a Ts^- phenotype (see Fig 1 for nrf1⁺ and other representative transformants) and displayed a cdc24-4^ts mutant phenotype of large, round, unbudded cells, indicating that a plasmid-encoded cDNA was responsible for the reversal of the Cdc42p-dependent suppression. These plasmids did not confer a Ts^- phenotype in wild-type S. cerevisiae cells (W303-1A) in the presence or absence of 1 M sorbitol and did not cause a phenotypic change in cdc24-4^ts cells lacking pRS315-cdc42⁺ at 32° (data not shown). These results suggested that the plasmid-mediated Ts^- phenotypes in Y147[pRS315-cdc42⁺] were not due to a general Ts^- overexpression phenotype or to an exacerbation of the cdc24-4^ts phenotype.

From the 33 plasmid inserts, 32 different cDNAs were identified (suggesting that the screen was not saturated) and termed nrf (negative regulator of Cdc forty two; Table 2). Five of the cDNA products showed no similarity to known protein sequences and 27 cDNAs encoded polypeptides with varying levels of sequence similarity to known proteins, including: (i) two proteins (Ubc4p and Let1p) that have been shown to be involved in protein degradation; (ii) 13 ribosomal protein subunits; (iii) a human translationally controlled tumor protein (TCTP) homolog; (iv) thioredoxin (Trx2p) and thioredoxin reductase (Trr1p) homologs; (v) a S. cerevisiae Asc1p protein kinase C-like receptor homolog; (vi) a 6-phosphogluconate dehydrogenase (Gnd1p) homolog; (vii) hexokinase 2 (Hxk2p); (viii) a putative heat-shock protein (Scf1p); (ix) a phosphatidylserine decarboxylase (Psd2p) homolog; (x) a mammalian UV-damage repair protein (XP-E) homolog; (xi) a putative seven-transmembrane domain protein; (xii) a Spt5p putative transcription factor homolog; and (xiii) a novel ~15-kD protein, Nrf1p (Fig 2). Overexpression of nrf1⁺ led to several morphological defects in S. pombe (see below) and therefore it was chosen for further study. Nrf1p was predicted to contain three transmembrane domains using the PHDsec algorithm from the PredictProtein online server (http://www.embl-heidelberg. de/predictprotein/predictprotein.html; Fig 2A). S. cerevisiae encodes a protein (YER072w; accession no. P40046) that is 78% identical (91% similar) in predicted amino-acid sequence to S. pombe Nrf1p (Fig 2B), but its function is not known.

View larger version (49K): In this window In a new window Download PPT slide   Figure 2. DNA and amino-acid sequence of nrf1+ and comparison of S. pombe and S. cerevisiae Nrf1p amino-acid sequences. (A) nrf1+ DNA and predicted amino-acid sequence; shaded, boxed amino acids contain the putative transmembrane domains. Numbers on left indicate the nucleotide position; numbers on right indicate the amino-acids position. (B) Nrf1p from S. pombe (Sp) and S. cerevisiae (Sc) were aligned with the DNASTAR/MEGALIGN program; identical amino acids are boxed; overlines indicate the putative transmembrane domains.

High-level expression of nrf1⁺ was lethal in S. pombe:
A S. pombe nrf1 deletion strain (see MATERIALS AND METHODS) was viable and displayed wild-type cellular and vacuolar morphologies at 16°, 23°, 30°, and 37° (data not shown). To examine the effects of nrf1⁺ overexpression in S. pombe, the nrf1⁺ cDNA was inserted into three pREP plasmids that contain different-strength thiamine-repressible nmt1 promoters. The nmt1 promoters in plasmids pREP1, pREP41X, and pREP81X result in overexpression rates of 300x, 25x, and 8x, respectively (FORSBURG 1993 ). Expression of nrf1⁺ under the high-strength promoter (pREP1-nrf1⁺) conferred a lethal phenotype in wild-type ED668 cells, but expression from the medium- (pREP41X-nrf1⁺) and low- (pREP81X-nrf1⁺) strength promoters did not (Fig 3A), suggesting that the lethality was dose dependent. It should be noted that the lethality associated with the highest-level expression was only observed after serial-streaking onto derepressing media, suggesting that the lethality depended on high-level accumulation of Nrf1p. To corroborate the different levels of nrf1⁺ expression from the nmt1 promoters, nrf1⁺ was inserted into the pSLF series of plasmids (Table 1) to generate high-, medium-, and low-strength nmt1 promoter-driven HA-tagged nrf1⁺ constructs. These plasmids were transformed into ED668 cells and immunoblot analysis indicated that the levels of HA-Nrf1p varied according to the strength of the nmt1 promoter (Fig 3B).

View larger version (62K): In this window In a new window Download PPT slide   Figure 3. Characterization of dose-dependent Nrf1p lethality and morphological defects. (A) Plasmids pREP1, pREP1-nrf1+, pREP41X-nrf1+, and pREP81X-nrf1+ were transformed into ED668 cells and selected at 30° on EMMS-Leu+Thi media (repressing conditions). Individual transformants were streaked at 30° on EMMS-Leu+/-Thi media. (B) Immunoblot analysis of HA-nrf1+ overexpressing strains was with the 12CA5 anti-HA antibody. The indicated plasmids were transformed into ED668 and total cell protein was isolated. A total of 30 µg of protein was loaded into each lane. (C) The indicated transformants were grown in EMMS-Leu+Thi liquid media overnight, then diluted to OD600 = 0.005 in EMMS-Leu-Thi, and grown at 30° to mid-log phase (~48 hr) prior to examination. Arrowheads indicate cells with large vacuoles. (D) The indicated transformants were grown as described in C and stained with rhodamine-conjugated phalloidin to visualize the cortical actin structures. Scale bars, 10 µm.

Overexpression of nrf1⁺-generated ellipsoidal cells with delocalized actin and large vacuoles:
ED668 [pREP1-nrf1⁺] cells grown under derepressing conditions for ~48 hr showed an ellipsoidal cellular morphology (Fig 3C; 63% of cells, n = 100) similar to that seen with cells expressing the dominant negative cdc42^T17N allele (OTTILIE et al. 1995 ), suggesting that overexpression of nrf1⁺ had a negative effect on S. pombe cell polarity. In ~30% of the cells, large vacuoles were observed, which were not observed in cells expressing cdc42^T17N (Fig 3C). ED668 cells containing pREP41X-nrf1⁺ (Table 3) or pREP81X-nrf1⁺ also conferred a similar morphological phenotype, although the penetrance was not as great and the presence of large vacuoles was not observed (Fig 3C). ED668[pREP1-nrf1⁺] cells were stained with rhodamine phalloidin to observe nrf1⁺ overexpression effects on the actin cytoskeleton. Of the 77% of cells that showed an abnormal morphology, 89% had delocalized or partially mislocalized cortical actin patches while 11% had normal cortical actin distribution (Fig 3D). Again, this staining was similar to the pattern observed in cells expressing cdc42^T17N (MARCUS et al. 1995 ; OTTILIE et al. 1995 ).

These cells were also stained with the vacuolar-specific stain carboxy-DCFDA, which stains the vacuole lumen (NOVICK and BOTSTEIN 1985 ), to determine the nature of the observed large vacuoles. Although carboxy-DCFDA staining of small vacuoles was seen in wild-type cells, the large vacuoles seen in nrf1⁺-overexpressing cells at ~48 hr postderepression showed no accumulation of carboxy-DCFDA, and there were no carboxy-DCFDA-stained small vacuoles observed in cells containing large vacuoles (data not shown). Carboxy-DCFDA is believed to be a substrate for esterases in the vacuole and is frequently used as a vital stain. Therefore, the lack of carboxy-DCFDA staining in nrf1⁺-overexpressing cells suggested that the cells containing these large vacuoles were dead. Carboxy-DCFDA was also used to determine the percentage of nrf1⁺-overexpressing cells that were dead at ~24 hr postderepression. High-level expression (pREP1-nrf1⁺) resulted in an approximately threefold increase in ellipsoidal morphology at ~24 hr (Table 3) with a concomitant increase in cell inviability, suggesting a possible causal relationship between abnormal morphology and viability. However, when individual cells at ~24 hr postderepression were micromanipulated onto repressing media, similar percentages of normal and ellipsoidal cells recovered to form viable colonies (data not shown), suggesting that the presence of an ellipsoidal morphology was not indicative of cell inviability and that the nrf1⁺-overexpressing phenotypes of abnormal morphology and inviability were separable.

nrf1⁺ displayed synthetic overdose phenotypes with cdc42 and pak1 alleles:
In (pREP1-nrf1⁺)-containing cells, a lethal phenotype was observed in combination with wild-type cdc42⁺, activated cdc42^G12V, and cdc42^T17N alleles. Cells co-overexpressing nrf1⁺ and wild-type cdc42⁺ showed the nrf1⁺ morphologies, suggesting that overexpression of wild-type cdc42⁺ cannot reverse the nrf1⁺ phenotype. Cells co-overexpressing nrf1⁺ and cdc42^T17N were ellipsoidal (which is characteristic of both cell types), and the nrf1⁺ phenotype of large vacuoles was observed (data not shown). High-level nrf1⁺ overexpression eliminated the cdc42^G12V large, round cell phenotype, and only the nrf1⁺ overexpression morphology was observed (Fig 4A; Table 3). Medium-level expression of nrf1⁺ (pREP41X-nrf1⁺) with cdc42^G12V led to a lethal phenotype not observed in cells overexpressing the individual genes with the cells displaying both nrf1⁺ and cdc42^G12V morphological phenotypes (Table 3). A similar "synthetic overdose" phenotype was observed upon overexpression of cdc42 and pak1 mutant alleles (OTTILIE et al. 1995 ). When nrf1⁺ was expressed from the low-strength promoter (pREP81X-nrf1⁺) with the different cdc42 alleles, the morphological phenotypes observed were those of the cdc42 alleles. These data are consistent with a functional interaction between Nrf1p and Cdc42p that is sensitive to Nrf1p levels.

View larger version (69K): In this window In a new window Download PPT slide   Figure 4. nrf1+ overexpression alters the cdc42G12V morphology while nrf1+ + pak1+ co-overexpression results in normal morphology. The indicated cdc42 (A) and pak1 (B) alleles and nrf1+ plasmid constructs were transformed into ED668 cells and selected on EMMS-Leu-Ura + Thi media. Individual transformants were grown in liquid EMMS-Leu-Ura-Thi media at 30° for ~48 hr and examined microscopically. Scale bars, 10 µm.

In (pREP2-nrf1⁺)-containing cells, a lethal phenotype was observed in combination with the wild-type pak1⁺ and kinase-inactive pak1^K415,416R alleles (Table 3). Cells co-overexpressing nrf1⁺ and pak1^K415,416R showed large ellipsoidal cells (which is characteristic of both cell types) and the nrf1⁺ phenotype of large vacuoles was observed. Medium- or low-level expression of nrf1⁺ (pREP42X-nrf1⁺ or pREP82X-nrf1⁺) with the pak1⁺ or pak1^K415,416R alleles led to a synthetic overdose phenotype with the cells showing both nrf1⁺ and pak1⁺ morphological phenotypes (Table 3). High-level co-overexpression of nrf1⁺ and pak1⁺ resulted in ~80% of cells with predominantly normal morphology and ~20% with a pak1⁺ morphology of large, abnormally shaped cells (Fig 4B; Table 3), but these cells were still inviable, reinforcing the separable nature of the abnormal cellular morphology and inviability. This result suggested that co-overexpression of these two proteins counteracted each other's morphological phenotypes, substantiating a negative regulatory role for Nrf1p in polarized cell growth vs. the positive Pak1p role.

Effects of overexpression of Nrf1p on Cdc42p localization: The subcellular localization of GFP-Cdc42p (low-level expression, pREP81X-GFP-A₈-cdc42⁺) was examined in the presence of Nrf1p expressed under the high-strength promoter (pREP1-nrf1⁺). GFP-Cdc42p localized to the periphery of the cell, nucleus, and vacuoles and was strongly associated with the septum of dividing cells (Fig 5A, arrows; A. MERLA and D. I. JOHNSON, unpublished results). Overexpression of Nrf1p altered the GFP-Cdc42p staining, with the appearance of cytoplasmic clearing and an accumulation of GFP-Cdc42p around 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei (Fig 5B, arrowheads and data not shown). This accumulation of GFP-Cdc42p coincided with vacuoles, assayed by carboxy-DCFDA staining, and was observed in 49% of cells (n = 100) overexpressing Nrf1p vs. 3% in nonoverexpressing cells. Cells overexpressing Nrf1p at intermediate and low levels also showed these vacuolar abnormalities but to a lesser extent. These data suggest that Nrf1p overexpression alters vacuolar morphology and, thereby, Cdc42p localization patterns.

View larger version (71K): In this window In a new window Download PPT slide   Figure 5. GFP-Cdc42p localizes correctly in Nrf1p overexpressing cells. (A, B) ED668 cells containing the indicated plasmid-borne genes were grown in EMMS-Leu-Ura-Thi liquid media for 18–24 hr and examined microscopically. The fields shown are collages of individual photos manipulated in Adobe Photoshop 5.0. Arrows indicate GFP-Cdc42p localizing to the septum. Arrowheads indicate vacuole accumulation around the nucleus. Scale bars, 10 µm.

Nrf1p vacuolar membrane localization depended on Scd1p:
In cell fractionation experiments, HA-tagged Nrf1p was found in the 100,000 x g pellet under all expression levels tested (data not shown). GFP-Nrf1p (medium-level expression, pREP41X-GFP-A₈-nrf1⁺) localized to the periphery of the cell, nucleus, and vacuoles and was also associated with 66% of the septa during cytokinesis and septation (Fig 6A, arrows), indicating that GFP-Nrf1p and GFP-Cdc42p colocalize during the cell cycle. In addition, large patches of GFP-Nrf1p were observed along the plasma and nuclear membranes, which were not observed in GFP-Cdc42p expressing cells. There was also an accumulation of GFP-Nrf1p around the nucleus (Fig 6A, arrowheads). Identical localization patterns were observed when GFP-Nrf1p was expressed in the nrf1 strain (data not shown). Extremely faint vacuolar GFP-Nrf1p staining was observed in scd1 cells, and although carboxy DCFDA-stained vacuoles were present, no accumulation of vacuoles around the nucleus was observed (Fig 6B). No accumulation of vacuoles was observed in scd1 cells overexpressing Nrf1p at the highest level (pREP1-nrf1⁺), but these cells were still inviable, suggesting that the inviability associated with Nrf1p overexpression was not due to the vacuolar abnormalities. These data are consistent with Scd1p being necessary both for efficient localization of Nrf1p to the vacuole and the subsequent accumulation of vacuoles around the nucleus.

View larger version (98K): In this window In a new window Download PPT slide   Figure 6. GFP-Nrf1p localizes to cellular membranes and induces vacuolar accumulation around the nucleus. (A) The GFP-nrf1+ plasmid was transformed into ED668 cells and selected on EMMS-Leu+Thi media. Cells were grown and observed as in Fig 5. Arrows indicate GFP-Nrf1p localizing to the septum; arrowheads indicate vacuolar accumulation around the nucleus. (B) SPSCD1U (scd1) cells containing the indicated plasmids were grown in EMMS-Leu-Thi media for 24 hr at which time GFP fluorescence was documented (left and right) or cells were stained with carboxy-DCFDA to observe vacuolar morphology (middle and right). Arrowheads indicate vacuole staining. Scale bars, 10 µm.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In a cross-species genetic screen for negative regulators of S. pombe Cdc42p, we identified 32 cDNAs that were able to reverse the S. pombe cdc42⁺ suppression of the S. cerevisiae cdc24-4^ts mutant. There are a number of mechanisms by which overexpression of an heterologous gene could reverse the Cdc42p suppression, including the overproduced protein either (i) shifting Cdc42-GTP to a Cdc42-GDP state as the S. cerevisiae Rga1p and Bem3p GAPs presumably do in control experiments; (ii) inhibiting the proper subcellular localization of Cdc42p; (iii) reducing cdc42⁺ expression or affecting cdc24-4 expression, thereby shifting the cellular stoichiometry of these proteins; (iv) sequestering Cdc42p into a specific effector complex where it cannot properly function; (v) sequestering a downstream component leading to inhibition of the pathway; or (vi) causing a Ts^- phenotype of its own in S. cerevisiae or enhancing the cdc24-4 Ts^- phenotype (control experiments with the 32 cDNAs did not support this mechanism). We did not obtain a cDNA that displayed amino-acid sequence similarity to known Cdc42p-GAPs, although several potential S. pombe Cdc42p-GAPs have recently been identified (E. BARFOD, J. M. MURRAY, and D. I. JOHNSON, unpublished results). A similar screen in S. cerevisiae also did not identify the three known GAPs (BI and PRINGLE 1996 ), and presumably, the cDNA library used in the screen did not contain appreciable amounts of these long open reading frames. The analysis of nrf1⁺ function described herein suggested that Nrf1p reversal of the Cdc42p-dependent suppression in S. cerevisiae was not through mechanisms (i) and (vi), and Nrf1p localization to intracellular membranes and septa in S. pombe suggested that mechanism (iii) was unlikely. Overexpression of nrf1⁺ did affect the GFP-Cdc42p localization pattern in vacuoles, but it did not affect the Cdc42p subcellular fractionation pattern (J. M. MURRAY and D. I. JOHNSON, unpublished results). Hence, we cannot completely rule out mechanism (ii). However, Nrf1p is most likely involved in the sequestration of either Cdc42p or a downstream effector complex, thereby turning off the pathway.

Nrf1p overexpression in S. pombe resulted in three phenotypes: dosage-dependent lethality, aberrant cellular morphologies, and abnormal vacuole morphology, including an accumulation of vacuoles around the nucleus. These phenotypes could be separated in the sense that cells displaying aberrant morphologies and/or abnormal vacuoles were not necessarily inviable and vice versa, suggesting that Nrf1p overexpression affects multiple pathways within the cell, including the cell polarity pathway. High-level overexpression of Rho2p or Sts5p in S. pombe, which are both involved in regulating polarized cell growth, resulted in similar aberrant cellular morphologies and inviability (TODA et al. 1996 ; HIRATA et al. 1998 ), reinforcing a role for Nrf1p in the polarity pathway.

The colocalization of Nrf1p and Cdc42p to vacuolar membranes highlights a growing connection between Cdc42p, its regulators and downstream effectors, and vacuolar function. Both S. cerevisiae and S. pombe GFP-Cdc42p show localization to the vacuolar membrane (M. SAWYER, A. MERLA and D. I. JOHNSON, unpublished results). In addition, S. cerevisiae Cdc24p has been implicated in vacuolar function and/or morphology (WHITE and JOHNSON 1997 ), and S. pombe Scd1p is necessary for Nrf1p vacuolar localization and the accumulation of vacuoles around the nucleus (Fig 6). Vacuole accumulation around the nucleus in cells expressing Nrf1p appears to be a prelude to the formation of large vacuoles, a largely uncharacterized process to date. The Nrf1p large vacuolar phenotype is similar to that observed in a S. pombe vps34 mutant (TAKEGAWA et al. 1995 ). Vps34p is a phosphotidylinositol 3-kinase (PI 3-kinase) with 28% identity to the p110 catalytic subunit of the mammalian PI 3-kinase. The p85/p110 PI 3-kinase regulates several cellular processes, including cell proliferation, membrane ruffling, glucose uptake, and growth factor receptor endocytosis, and the p85 subunit binds to Cdc42p, which stimulates PI 3-kinase activity in vitro (ZHENG et al. 1994 ). A mutation within S. cerevisiae VMA5, which encodes a component of the vacuolar H⁺-ATPase as well as a csl3 mutant, displayed a synthetic-lethal phenotype with the cdc24-4^ts allele, and the csl3 allele also conferred a fragmented vacuole phenotype (WHITE and JOHNSON 1997 ). In S. cerevisiae, actin and the Myo2 myosin function in vacuolar inheritance (HILL et al. 1996 ) and a mutation in VMA4, which also encodes a subunit of the vacuolar H⁺-ATPase, caused morphological abnormalities coupled with delocalized actin and chitin, suggesting a connection between the vacuolar H⁺-ATPase and polarized growth (ZHANG et al. 1998 ). Finally, our screen also identified thioredoxin (nrf5⁺), which is necessary for vacuolar inheritance in S. cerevisiae (XU and WICKNER 1996 ; XU et al. 1997 ). Taken together, these data raise the possibility that the Cdc42p signaling pathway also functions in regulating some aspects of vacuolar function and/or morphology.

For Cdc42p to maintain its normal cellular functions, it must be able to interact with multiple regulators that modulate its guanine-nucleotide-bound state and with a myriad of effectors to activate downstream cellular processes (JOHNSON 1999 ). These interactions must take place at precise subcellular locations and at precise times within the cell cycle and are sensitive to Cdc42p levels within the cell (MILLER and JOHNSON 1997 ). The characterization of Nrf1p described herein is consistent with Nrf1p playing a negative role in regulating the cell polarity pathway in S. pombe, possibly by sequestering Cdc42p or downstream components of the pathway. Future studies will investigate the mechanisms by which Nrf1p and the other proteins identified in this genetic screen regulate Cdc42p function, subcellular localization, interactions with regulators/effectors, and/or expression levels.

	ACKNOWLEDGMENTS

We thank E. Chang, J. Cooper, P. Fantes, A. Merla, and I. Rainville for S. pombe strains and reagents. We also thank members of the Johnson lab for thoughtful discussions and critical reading of this manuscript. This research was supported by the American Cancer Society grant RPG-89-012-08 and a Predoctoral Fellowship from National Science Foundation-VT EPSCoR (J.M.M.)

Manuscript received June 22, 1999; Accepted for publication October 15, 1999.

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