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First published online 25 September 2007
doi: 10.1242/jcs.009860

Journal of Cell Science 120, 3613-3624 (2007)
Published by The Company of Biologists 2007
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Research Article

Proteins involved in sterol synthesis interact with Ste20 and regulate cell polarity

Christopher Tiedje*, Daniel G. Holland*, Ursula Just and Thomas Höfken{ddagger}

Institute of Biochemistry, Christian Albrecht University Kiel, Olshausenstrasse 40, 24098 Kiel, Germany

{ddagger} Author for correspondence (e-mail: thoefken{at}biochem.uni-kiel.de)

Accepted 3 August 2007


   Summary
Top
 Summary
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The Saccharomyces cerevisiae p21-activated kinase (PAK) Ste20 regulates various aspects of cell polarity during vegetative growth, mating and filamentous growth. To gain further insight into the mechanisms of Ste20 action, we screened for interactors of Ste20 using the split-ubiquitin system. Among the identified proteins were Erg4, Cbr1 and Ncp1, which are all involved in sterol biosynthesis. The interaction between Ste20 and Erg4, as well as between Ste20 and Cbr1, was confirmed by pull-down experiments. Deletion of either ERG4 or NCP1 resulted in various polarity defects, indicating a role for these proteins in bud site selection, apical bud growth, cell wall assembly, mating and invasive growth. Interestingly, Erg4 was required for the polarized localization of Ste20 during mating. Lack of CBR1 produced no detectable phenotype, whereas the deletion of CBR1 in the absence of NCP1 was lethal. Using a conditional lethal mutant we demonstrate that both proteins have overlapping functions in bud morphology.

Key words: Cell polarity, Ergosterol, Ste20, Filamentous growth, Mating


   Introduction
Top
 Summary
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
The small Rho GTPase Cdc42 plays a central role in establishing and maintaining cell polarity in eukaryotes (Etienne-Manneville, 2004Go; Jaffe and Hall, 2005Go). In the yeast Saccharomyces cerevisiae, Cdc42 regulates different types of polarized growth at several stages of the life cycle. During budding, growth is restricted to a zone adjacent to the previous budding site in haploid cells or at either pole of the cell in diploids. First, a hierarchy of positional signals defines the bud site (Casamayor and Snyder, 2002Go). Haploid cells use an axial budding pattern in which cells bud adjacent to the preceding site of cytokinesis. Diploid cells exhibit a more complex bipolar budding pattern: daughter cells bud opposite to their previous birth pole and mother cells bud at either pole (Casamayor and Snyder, 2002Go). Then, growth is focused at that location and is restricted to the new bud by a diffusion barrier made of septins that is placed as a collar at the neck between mother and daughter cell (Douglas et al., 2005Go; Versele and Thorner, 2005Go). S. cerevisiae also exhibits polarized growth during mating. Binding of a pheromone secreted by cells of the opposite mating type to specific membrane receptors results in the stimulation of a mitogen-activated protein (MAP) kinase signaling cascade and in polarized growth towards the mating partner (Dohlman and Thorner, 2001Go). Cell polarization is also required for the transformation from cellular yeast to a filamentous form. In diploid yeast, the lack of nitrogen causes the transition to pseudohyphal growth, in which cells are elongated and bud in a unipolar fashion (Palecek et al., 2002Go). In haploid cells, nutrient limitation results in a similar developmental switch that allows cells to penetrate the surface of an agar medium in a process called invasive growth (Palecek et al., 2002Go).

Cdc42 promotes polarized growth through multiple pathways, including polarization of the actin cytoskeleton and directed vesicle trafficking. Among the Cdc42 effectors that trigger these pathways are Iqg1, Gic1, Gic2 (Brown et al., 1997Go; Chen et al., 1997Go; Epp and Chant, 1997Go; Osman and Cerione, 1998Go) and members of the p21-activated kinase (PAK) family. S. cerevisiae encodes three of these highly conserved serine/threonine protein kinases: Ste20, Cla4 and Skm1. Whereas little is known about the function of Skm1 (Martin et al., 1997Go), Cla4 has been shown to be involved in septin ring assembly and actin polarization (Cvrckova et al., 1995Go; Holly and Blumer, 1999Go; Longtine et al., 2000Go; Dobbelaere et al., 2003Go; Schmidt et al., 2003Go; Versele and Thorner, 2004Go). In addition, Cla4 has an important role in mitotic entry and exit (Höfken and Schiebel, 2002Go; Sakchaisri et al., 2004Go). The founding member of the PAK family, Ste20, promotes actin polarization via the type I myosins Myo3 and Myo5 (Wu et al., 1996Go; Evangelista et al., 2000Go; Geli et al., 2000Go; Lechler et al., 2000Go), and possibly by activation of the formin homolog Bni1 (Goehring et al., 2003Go). Ste20 also regulates mating, filamentous growth and osmotic stress response by phosphorylation, and activation of the MAP kinase kinase kinase Ste11 (Roberts and Fink, 1994Go; Wu et al., 1995Go; van Drogen et al., 2000Go). Furthermore, Ste20 has an undefined role in mitotic exit (Höfken and Schiebel, 2002Go) and phosphorylates histone H2B during hydrogen peroxide-induced apoptosis (Ahn et al., 2005Go).

To gain further insight into the role of Ste20 in cell polarity, we performed a screen for interactors of Ste20 using the split-ubiquitin system. Three proteins involved in sterol biosynthesis – Erg4, Cbr1 and Ncp1 – were identified in this screen. The phenotypes of the corresponding deletion strains indicate that these proteins share functions with Ste20 in cell polarization. Erg4, Ncp1 and Cbr1 play a role in bud site selection, apical growth, bud morphology, cell wall assembly, mating and haploid invasive growth. Thus, our results imply that sterol synthesis is crucial for cell polarization.


   Results
Top
 Summary
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
Identification of Ste20 interactors
To identify regulators and targets of Ste20, we screened for interactors of Ste20 by employing the split-ubiquitin technique (Johnsson and Varshavsky, 1994Go). The split-ubiquitin method is based on the ability of the N- and C-terminal halves of ubiquitin to assemble into a quasi-native ubiquitin. UBPs, ubiquitin-specific proteases, which are present in all eukaryotic cells, recognize the reconstituted ubiquitin, but not its halves, and cleave the ubiquitin moiety off a reporter protein that had been linked to the C-terminus of ubiquitin (Fig. 1A). A modified version of the enzyme Ura3 that carries an additional arginine at the extreme N-terminus (RUra3) was used as the reporter. Once cleaved off from the C-terminal domain of ubiquitin, RUra3 was rapidly degraded by enzymes of the N- end rule pathway of protein degradation. Therefore, close proximity between two proteins fused to the N- and C-terminal half of ubiquitin, respectively, results in non-growth on medium lacking uracil. Conversely, growth on 5-FOA indicated protein interaction as well, because 5-FOA was converted by Ura3 into 5-fluorouracil, which is toxic for the cell. Using this approach, 22 Ste20-interacting proteins were identified. Among these proteins was Bem1 (Fig. 1B), which acts as a scaffold that links Ste20 with Cdc42 and other factors (Leeuw et al., 1995Go; Butty et al., 1998Go; Drees et al., 2001Go). In this study we focus on Erg4, Cbr1 and Ncp1, because all three proteins are involved in sterol biosynthesis (Reddy et al., 1977Go; Aoyama et al., 1981Go; Yoshida, 1988Go; Aoyama et al., 1989Go; Kelly et al., 1995Go; Lamb et al., 1999Go; Zweytick et al., 2000Go) (Fig. 8) and have not previously been reported to interact with Ste20. The remaining genes will be described elsewhere. As explained above, the interactions between Ste20 and Erg4, Cbr1 as well as Ncp1 were demonstrated by non-growth on medium lacking uracil (Fig. 1B).


Figure 1
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  		Fig. 1. Identification of proteins that interact with Ste20. (A) The split-ubiquitin system. See text for details. Degraded RUra3 is shown on the right. X represents any interactor of Ste20. (B) Ste20 interacts with Erg4, Ncp1 and Cbr1 in vivo using the split-ubiquitin technique. 105 cells of the indicated plasmid combinations were spotted onto media either lacking histidine and leucine to select for the plasmids or lacking histidine, leucine and uracil to monitor protein interactions. The unrelated proteins Rdi1 and Ubc6 served as negative controls. Bem1 is known to interact with Ste20. (C) Ste20 interacts with Erg4 in vitro. Purified GST and GST-Ste20 that were bound to glutathione-sepharose beads were incubated with a yeast lysate of ERG4-9MYC cells. Eluted proteins were analyzed by immunoblotting using anti-Myc antibodies. (D) Ste20 interacts with Cbr1. The experiment was performed as in C using a yeast lysate of 3HA-CBR1 cells. Proteins were detected with anti-HA antibodies.

 

Figure 8
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  		Fig. 8. The ergosterol biosynthetic pathway in budding yeast. Only the ergosterol-specific part of the pathway is shown. Large arrows indicate major regulatory steps. The role of Erg4, Ncp1 and Cbr1, which were isolated in the screen, in the pathway is illustrated. Proteins that depend on Ncp1 and/or Cbr1 are enclosed in a rectangle. Furthermore, it is highlighted whether these reactions depend on Ncp1 and Cbr1 or on Cbr1 alone. The oval denotes Erg4.

 

To confirm these interactions using an independent approach, recombinant GST and GST-Ste20 purified from Escherichia coli were bound to glutathione-sepharose beads, which were then incubated with a yeast extract of ERG4-9MYC cells. Erg4-9myc interacted with GST-Ste20 but not with GST (Fig. 1C). In a similar experiment using a yeast extract of 3HA-CBR1 cells we demonstrated a specific binding between Ste20 and Cbr1 (Fig. 1D). However, we did not observe these interactions by co-immunoprecipitation, possibly owing to the transient or weak nature of these interactions.

In order to find out whether the identified genes share a common function with STE20, we examined their genetic interactions. STE20 deletion is lethal in a cla4{Delta} background because of functional redundancy of these PAK-family kinases (Cvrckova et al., 1995Go) (Fig. 2A). We reasoned that important downstream effectors of Ste20 might show the same negative interaction with cla4{Delta}. To test this hypothesis we individually deleted ERG4, NCP1 and CBR1 in a cla4{Delta} strain. In fact, cells deleted for CLA4 and ERG4 were no longer viable, whereas single deletions of each gene resulted in no difference in growth compared to wild-type cells (Fig. 2A). Deletion of NCP1 alone resulted in slower growth, but in a cla4{Delta} background NCP1 was essential for viability (Fig. 2A). These data indicate that Erg4 and Ncp1 share functions with Cla4, possibly as downstream effectors of Ste20. The deletion of CBR1 in a cla4-null mutant had no growth defect (Fig. 2A).


Figure 2
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  		Fig. 2. Genetic interactions of erg4{Delta}, cbr1{Delta} and ncp1{Delta}. (A) Deletion of CLA4 in erg4{Delta} cells or ncp1{Delta} is lethal. Serial dilutions (1:10) of the indicated strains were spotted on YPD and 5-FOA plates and grown for 2 days at 30°C. Notice that cla4{Delta} erg4{Delta} and cla4{Delta} ncp1{Delta} carried the plasmid pRS316-CLA4, whereas cla4{Delta} ste20{Delta} carried the plasmid pRS316-STE20. 5-FOA selects against these URA3-based pRS316 derivatives. (B) STE20 deletion reduces the growth of ncp1{Delta} cells. Serial dilutions of the indicated strains were spotted on YPD and 5-FOA plates as described in A. (C) Genetic interactions with och1{Delta}. Cells were spotted as in A.

 
We also observed reduced growth for the ste20{Delta} ncp1{Delta} double mutant compared with the single deletion of NCP1 (Fig. 2B). This effect was weak but reproducible and indicates that the situation might be more complex. Therefore, we tested whether single deletions of ERG4, CBR1 and NCP1 are synthetically lethal with other deletions that show negative genetic interactions with ste20{Delta}. It has been reported that STE20 becomes essential in the absence of OCH1, LTE1 or SWE1 (Lee and Elion, 1999Go; Höfken and Schiebel, 2002Go; Goehring et al., 2003Go). Interestingly, deletion of either CLA4 or NCP1 also resulted in lethality in an och1{Delta} background, whereas deletion of CBR1 in these mutants had no effect on growth (Fig. 2C). Unexpectedly, deletion of ERG4 suppressed the slight growth defect of och1{Delta} (Fig. 2C). Because OCH1 encodes an {alpha}-1,6-mannosyltransferase with a role in cell wall biosynthesis (Nakayama et al., 1992Go), STE20, CLA4, NCP1 and ERG4 might also contribute to this process. In contrast to a previous report (Goehring et al., 2003Go), the ste20{Delta} swe1{Delta} double mutant grew normally in our hands and no other genetic interactions were identified with SWE1 (see supplementary material Fig. S1B). In a similar way, no additional phenotypes were observed for the lte1 double mutants (see supplementary material Fig. S1A).

ERG4 has a role in bud site selection and apical growth
The synthetic lethality between cla4{Delta} and erg4{Delta}, as well as between cla4{Delta} and ncp1{Delta}, implies that ERG4 and NCP1, like STE20, have overlapping functions with CLA4 during the cell cycle. Therefore, we tested whether the deletion of ERG4 or NCP1 in addition to that of CBR1 results in phenotypes similar to those described for either ste20{Delta} or cla4{Delta} strains. Ste20 has previously been described to participate in bipolar bud site selection (Sheu et al., 2000Go) and, interestingly, ERG4 was identified in a genome-wide screen for genes involved in bipolar budding (Ni and Snyder, 2001Go). We confirmed the requirement of ERG4 and STE20 for bud site selection in diploids (see supplementary material Fig. S2A). However, whereas the STE20-deletion strain mostly exhibited a unipolar budding pattern, the bud site was chosen randomly in the majority of erg4{Delta}/erg4{Delta} cells (Ni and Snyder, 2001Go) (see supplementary material Fig. S2A). In contrast to the diploid strains, the erg4{Delta} and ste20{Delta} mutants, as well as cbr1{Delta} and ncp1{Delta} cells, displayed a normal axial budding pattern in haploid cells (see supplementary material Fig. S2B).

Next, the role of Erg4, Ncp1 and Cbr1 in apical bud growth was tested. During budding, the cyclin-dependent kinase Cdc28 promotes polarized apical growth when coupled to the G1 cyclins and promotes isotropic growth when associated with mitotic cyclins (Lew and Reed, 1995). The apical growth phase can be prolonged by G1 cyclin overexpression, resulting in hyperelongated buds (Lew and Reed, 1995) (Fig. 3A,B). Cells deleted for genes encoding cell polarity proteins such as Ste20 form no or fewer hyperpolarized buds in response to overexpression of the G1 cyclin CLN1 (Sheu et al., 2000Go). To test whether Erg4 and Cbr1 are involved in apical bud growth, we overexpressed CLN1 in the corresponding deletion strains and scored for the presence of hyperelongated buds. The wild-type strain and cells deleted for CBR1 showed more than 60% of buds were hyperpolarized after 4 hours induction (Fig. 3B). By contrast, in erg4{Delta} and ste20{Delta} cells, the number of hyperelongated buds was reduced to only about 20% (Fig. 3B). Immunoblot analysis revealed that the mutant and wild-type cells expressed comparable levels of galactose-induced CLN1 (Fig. 3C). Unfortunately, this assay could not be performed with ncp1{Delta} cells because of the slow growth of this strain (Fig. 2A). Taken together, the data demonstrate an involvement of ERG4 in bud site selection and apical bud growth.


Figure 3
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  		Fig. 3. Erg4 is involved in apical growth. (A) Morphology of normal and hyperelongated (hyperpolarized) cells. Cells carrying GAL1-CLN1-3HA were induced by the addition of galactose for 4 hours and subsequently fixed with formaldehyde. (B) erg4{Delta} cells have a defect in apical growth. Wild-type, erg4{Delta}, cbr1{Delta} and ste20{Delta} cells were treated as in A. The percentage of cells with a hyperpolarized bud was determined in three independent experiments (n>100 each). (C) Wild-type, erg4{Delta}, cbr1{Delta} and ste20{Delta} cells express comparable amounts of CLN1. Cells from B were analyzed by immunoblotting with anti-HA antibodies. Cdc11 was detected as a loading control.

 
Cla4, Erg4 and Ncp1 contribute to cell wall organization
Rho GTPases and their downstream effectors play an important role in cell wall assembly. They are required for the polarization of synthetic proteins such as glucan and chitin synthase (Levin, 2005Go). Various mutant strains, which are defective in cell wall integrity, rapidly lyse in distilled water when grown at 37°C (Levin, 2005Go). We observed such a temperature-sensitive cell lysis phenotype for cla4{Delta}, erg4{Delta} and ncp1{Delta} cells, but not for the CBR1- and STE20-deletion strains (data not shown). Cell wall integrity mutants are often also sensitive to compounds like Calcofluor White, which interferes with cell wall assembly (Ram et al., 1994Go). Therefore, we tested whether the deletion of the genes identified in the screen results in an increased Calcofluor White sensitivity. In fact, erg4{Delta} cells grew much slower on Calcofluor White (Fig. 4A). Next we analyzed the role of Erg4, Ncp1 and Cbr1 in cell wall formation using a more reliable and quantitative assay. Here, cells were incubated overnight with various amounts of zymolyase, an enzyme that degrades the principal cell wall component beta-1,3-glucan (Levin, 2005Go), and, subsequently, the survival rate was determined. Hypersensitivity to this zymolyase treatment was observed for erg4{Delta} and ncp1{Delta} cells, and this hypersensitivity was even more pronounced for the cla4{Delta} strain (Fig. 4B). The deletion of CBR1 or STE20 had no effect (Fig. 4B). Together with the genetic interactions of och1{Delta} (Fig. 2C), these results suggest a function for Cla4, Erg4 and Ncp1 in cell wall biosynthesis.


Figure 4
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  		Fig. 4. cla4{Delta}, erg4{Delta} and ncp1{Delta} cells have defects in cell wall assembly. (A) Deletion of ERG4 results in increased sensitivity to Calcofluor White. Serial dilutions (1:10) of wild-type and the indicated deletion strains were spotted on a YPD plate and a YPD plate supplemented with 50 µg/ml Calcofluor White. Cells were cultured for 2 days at 30°C. (B) cla4{Delta}, erg4{Delta} and ncp1{Delta} cells are hypersensitive to zymolyase. The indicated strains were grown for 16 hours at 30°C in the presence of various amounts (0-20 U/ml) of zymolyase, starting with an OD600 of 0.025. Various dilutions of the cell suspensions were then plated on YPD plates to determine the cell titer. Shown here is one of three representative experiments.

 

Ncp1 is required for haploid invasive growth, whereas Erg4 impedes invasion
Ste20 also functions in a signal transduction pathway that governs filamentous growth (Roberts and Fink, 1994Go). As a consequence, cells lacking STE20 fail to invade agar (Roberts and Fink, 1994Go) (Fig. 5A). To analyze the role of ERG4, NCP1 and CBR1 in this pathway, we individually deleted these genes in cells of the {Sigma}1278b strain background, which is necessary for tests of filamentous growth. The CBR1 deletion strain was undistinguishable from wild type (Fig. 5A). ncp1{Delta} cells were unable to invade agar, whereas the deletion of ERG4 resulted in a hyperinvasive phenotype (Fig. 5A). These strains were further examined for filamentous morphology. To this end, cells were spread onto synthetic medium lacking glucose. Under these conditions, wild-type cells undergo few divisions and form small colonies of branched and elongated cells (Cullen and Sprague, 2000Go) (Fig. 5B). By contrast, colonies of the ncp1{Delta} mutant exhibited a reduction in branching and elongation (Fig. 5B). Cells isolated from these colonies were mainly spherical without a bud or with only a small round bud (Fig. 5C). Buds of the few large-budded cells were round rather than elongated (Fig. 5C). Consistent with this result, actin was evenly distributed within the cell and not polarized to the bud, as occurs in wild-type cells (Fig. 5D). Interestingly, we did not observe any morphological difference between wild-type and erg4{Delta} microcolonies (Fig. 5B). Therefore, the mechanisms of hyperinvasive growth in this strain remain unclear.


Figure 5
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  		Fig. 5. Erg4 and Ncp1 affect invasive growth. (A) Agar invasion. 105 cells of the indicated strains were spotted on a YPD plate and incubated for 2 days at 30°C. Pictures were taken before and after gentle rinsing with water. Notice that ncp1{Delta} cells in a {Sigma}1278b strain background, in contrast to the YPH499 background (Fig. 2A), grow like wild-type cells. (B) Filamentous colony morphology. The indicated strains were spread onto SC medium lacking glucose and were incubated for 18 hours at 30°C. Representative microcolonies growing on the agar are shown. (C) Single-cell morphology. Cells were treated as in B and were then scraped off from the plates, fixed with formaldehyde and briefly sonicated. (D) Actin localization. Cells were treated as in C and stained with rhodamine-phalloidin to visualize the actin cytoskeleton.

 

Erg4 has a role in cell polarization during mating
Ste20 was originally identified as a component in the yeast mating pathway (Leberer et al., 1992Go; Ramer and Davis, 1993Go). Upon pheromone stimulation, Ste20 triggers a MAP kinase cascade, which leads to transcriptional activation of mating-specific genes such as FUS1 and cell cycle arrest in G1 (Dohlman and Thorner, 2001Go). Furthermore, the actin cytoskeleton and other factors polarize, resulting in the formation of a mating projection (Dohlman and Thorner, 2001Go). Therefore, we tested whether the proteins identified in the screen are involved in mating. Almost all wild-type cells (96%) treated with pheromone formed a mating projection after 150 minutes and had the actin cytoskeleton polarized towards the tip of that projection (Fig. 6A). Longer incubation of cells with a high concentration of mating pheromone leads to the initiation and termination of projection growth in regular cycles (Bucking-Throm et al., 1973Go). After 6 hours, 90% of the cells had two or more mating projections (Fig. 6A). Cells lacking CBR1 behaved like wild type (Fig. 6A). Again, this assay could not be performed with ncp1{Delta} cells because of their slow growth (Fig. 2A). In contrast to wild type, the erg4{Delta} mutant completely arrested in G1 phase but more than 90% of the cells failed to form a mating projection (Fig. 6A). The actin cytoskeleton in these unpolarized cells was evenly distributed. Even after 6 hours, multiple mating projections were never observed in the erg4{Delta} strain. As a consequence of that lack of polarization, erg4{Delta} cells also had a reduced mating efficiency (Fig. 6B). We further examined other polarity markers, such as the mating-specific protein Fus1, which is restricted to the tip of the mating projection (Fig. 6C). Interestingly, Fus1 was also enriched at a small membrane region in most cells deleted for ERG4. By sharp contrast, Ste20, which also localizes at the tip of the mating projection, was completely mislocalized in erg4{Delta} cells (Fig. 6D). However, ERG4 was not required for the polarized localization of Ste20 at bud tips during vegetative growth (see supplementary material Fig. S3A).


Figure 6
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  		Fig. 6. Pheromone-induced polarization requires ERG4. (A) erg4{Delta} cells do not form a mating projection. Exponentially growing wild-type, cbr1{Delta} and erg4{Delta} cells were incubated with 1 µg/ml {alpha}-factor for 150 and 360 minutes. Subsequently, cells were fixed with formaldehyde and stained with rhodamine-phalloidin to visualize the actin cytoskeleton (right panels). The percentage of cells with the shown phenotype (formation of no, one or multiple mating projections) was determined in two independent experiments (n>100 in each). (B) Deletion of ERG4 results in a reduced mating efficiency. Logarithmically growing cells were mixed together and concentrated onto a nitrocellulose filter at a density of 3x106 cells per parent. The filter was then transferred to a YPD plate and incubated for 4 hours at 30°C. Subsequently, the cells were washed from the filter with water and various dilutions were plated to determine cell titers. The mating efficiency (diploids/total cells) is given as the mean of three independent experiments with s.d. bars (n>100 for each experiment). (C) Erg4 is not required for proper Fus1 localization. Exponentially growing Fus1-GFP and erg4{Delta} Fus1-GFP cells were incubated with 1 µg/ml {alpha}-factor for 150 minutes. Cells were then fixed with formaldehyde and analyzed by fluorescence microscopy. The percentage of cells with polarized Fus1-GFP localization was determined in two independent experiments (n>100 in each). (D) Ste20 is mislocalized in erg4{Delta} cells. Logarithmically growing wild-type and erg4{Delta} cells carrying a plasmid encoding GAL1-STE20-GFP were incubated with 1 µg/ml {alpha}-factor for 150 minutes. GAL1-STE20-GFP was induced for 1 hour by the addition of galactose. The percentage of cells with polarized Ste20-GFP localization in the wild-type background and with cytosolic staining in erg4{Delta} cells was determined in two independent experiments (n>100 in each).

 
The observed polarization defect of erg4{Delta} cells could be attributed to the defect in ergosterol synthesis or might be caused by another activity of the Erg4 protein. To distinguish these two possibilities, the erg4 deletion mutant was grown anaerobically in the presence of {alpha}-factor and ergosterol. Under these conditions cells take up sterol from the medium (Raychaudhuri and Prinz, 2006Go) and both wild type and the erg4{Delta} strain polarized to the same extent (see supplementary material Fig. S3A). Thus, efficient pheromone-induced polarization requires Erg4 enzyme activity.

Ncp1 and Cbr1 have redundant functions in bud morphology
Whereas loss of either ERG4 or NCP1 resulted in various polarity defects, no phenotypes were observed for cbr1{Delta} cells in the assays described above. We reasoned that this lack of mutant phenotype in the cbr1{Delta} strain might be due to redundancy. In fact, Ncp1 and Cbr1 have overlapping functions in sterol synthesis. They both transfer electrons to proteins of the sterol biosynthetic pathway, such as cytochromes P450 (see Discussion for details). Therefore, we deleted both genes and characterized the cbr1{Delta} ncp1{Delta} double mutant. As described above, the deletion of the NCP1 gene alone resulted in slower growth, whereas cbr1{Delta} cells behaved like the wild-type strain (Fig. 2A, Fig. 7A). Interestingly, cbr1{Delta} ncp1{Delta} cells were unable to grow. To examine the terminal phenotype of this double mutant, we generated a conditional lethal strain using a temperature-sensitive degron (td). The degron is added to the N-terminus of a protein, causing it to be degraded by a specific ubiquitin-mediated pathway when cells are shifted from 23°C to 37°C (Labib et al., 2000Go). As expected, cbr1{Delta} ncp1-td cells could not be distinguished from wild type at the permissive temperature but were inviable when grown at 37°C (Fig. 7B). We were now able to study the phenotypic consequences of the simultaneous loss of CBR1 and NCP1 function. At 3 hours after Ncp1 depletion was induced, the large majority of cells (around 70%) showed abnormal bud morphology (Fig. 7C,D). Most of these cells had hyperelongated buds, some of them with a constriction, but pointed or bent buds were observed as well. By contrast, only a negligible percentage (2-3%) of wild type, cbr1{Delta} and ncp1-td had similar defects (Fig. 7D). This clearly indicates that Ncp1 and Cbr1 have overlapping functions in bud morphology. Because these morphological defects are often due to an aberrant localization or organization of septins, we examined a GFP-tagged version of the septin Cdc12 in cbr1{Delta} ncp1-td cells. However, Cdc12-GFP was normally localized and organized in these cells (see supplementary material Fig. S4A). Many Cdc42 effectors play a role in exit from mitosis and the corresponding mutants arrest as large-budded cells with an elongated mitotic spindle and separated nuclei (Höfken and Schiebel, 2002Go; Jensen et al., 2002Go; Seshan et al., 2002Go; Höfken and Schiebel, 2004Go). Therefore, we examined whether the large-budded cbr1{Delta} ncp1-td cells have a defect in mitotic exit as well. However, we did not observe a significant increase in cells with two separated DAPI-staining regions in these cells (see supplementary material Fig. S4B). This indicates that the cbr1{Delta} ncp1-td strain has a morphological and not a mitotic exit defect.


Figure 7
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  		Fig. 7. Inactivation of CBR1 together with NCP1 results in aberrant bud morphology. (A) Deletion of NCP1 in cbr1{Delta} is lethal. Serial dilutions (1:10) of the indicated strains were spotted on YPD and 5-FOA plates and grown for 2 days at 30°C. Notice that ncp1{Delta} and cbr1{Delta} ncp1{Delta} carried the plasmid pRS316-NCP1. 5-FOA selects against this URA3-based plasmid. (B) Construction of a conditional lethal cbr1{Delta} ncp1-td strain. The NCP1 degron fusion gene is under the control of the CUP1 promoter. Because rapid and conditional degradation requires recognition of the degron cassette by the Ubr1 protein, which is associated with a ubiquitin-conjugating enzyme, all strains have the UBR1 gene placed under the control of the GAL1 promoter. Serial dilutions (1:10) of the indicated strains were grown on glucose supplemented with 100 µM CuSO4 (YPDCu) at 23°C (permissive condition) and on galactose medium without the addition of CuSO4 (YPG) at 37°C (restrictive condition) for 3 days. The suffix `td' denotes temperature-sensitive degron. (C) cbr1{Delta} ncp1-td cells display bud morphology defects. Cells were grown in liquid culture at 23°C. To induce Ncp1 depletion, galactose was added for 1 hour, then cells were shifted to 37°C and after 3 hours were fixed with formaldehyde. Shown are representative phenotypes of the cbr1{Delta} ncp1-td strain. (D) Quantification of morphological defects. The indicated strains were treated as described in C. The percentage of cells with an abnormal bud was determined in three independent experiments (n>100 in each).

 

We also tested whether the cbr1{Delta} ncp1-td mutant had other defects. These cells formed a normal mating projection, as occurred in wild-type cells and in the corresponding single mutants (see supplementary material Fig. S5A). The actin cytoskeleton and Ste20-GFP were highly polarized in all strains (see supplementary material Fig. S5A). Interestingly, when cells were stained with Calcofluor White to examine the budding pattern, we observed a diffused chitin staining in the cbr1{Delta} ncp1-td double mutant (see supplementary material Fig. S5B). Notably, such a phenotype has also been described for other cell polarity mutants (Novick and Botstein, 1985Go).


   Discussion
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 Summary
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
Erg4, Ncp1 and Cbr1 have overlapping functions with Ste20 and Cla4
The Cdc42 effector Ste20 promotes the establishment and maintenance of cell polarity during vegetative growth, mating and filamentous growth. Although some downstream targets of Ste20 have been described, the underlying molecular mechanisms of cell polarization are poorly understood. Here, we describe the identification and characterization of three novel interactors: Erg4, Cbr1 and Ncp1. The fact that Bem1, a well-characterized interactor of Ste20, was found in this screen as well, demonstrates that the split-ubiquitin technique used in this study is suitable for the identification of proteins that bind to Ste20 under physiological conditions. Notably, we can confirm the interaction between Ste20 and Erg4, as well as between Ste20 and Cbr1, by pull-down assays. We further show negative genetic interactions, which are consistent with the observed protein interactions. The deletion of either ERG4 or NCP1 is lethal in a cla4{Delta} background. Such a negative interaction with CLA4 has been described for the STE20 deletion previously (Cvrckova et al., 1995Go). Therefore, Erg4 and Ncp1 might act in the same pathway as Ste20 – either upstream or downstream. The synthetic lethality also indicates that Erg4 and Ncp1 have overlapping functions with Cla4 during vegetative growth. In fact, in line with previous reports, we demonstrated that the deletion of either ERG4 or NCP1 results in defects similar to those described for either ste20{Delta} or cla4{Delta} strains (Ni and Snyder, 2001Go; Keniry et al., 2004Go).

The first polarized process in the cell cycle is the selection of a new bud site. As previously reported (Sheu et al., 2000Go; Ni and Snyder, 2001Go) and shown by us here, both Erg4 and Ste20 contribute to bud site selection in diploid but not haploid cells. Thus, Ste20 and Erg4 might act together to regulate bud site selection in diploids.

Once a bud site has been selected, the actin cytoskeleton orients towards this site and guides secretory vesicles to the cell surface, where they fuse. As a bud forms, this apical growth continues. During the G2-M transition, cells switch to isotropic growth, which results in the formation of an ellipsoid-shaped bud. Our data demonstrate that Erg4 has a role in apical growth, as previously described for Ste20 (Sheu et al., 2000Go). Therefore, both proteins might cooperate in this process.

Here, we also show that Erg4, Ncp1 and Cla4 are involved in cell wall assembly. The corresponding deletion mutants not only interact with OCH1, which encodes a {alpha}-1,6-mannosyltransferase and contributes to cell wall biosynthesis, but also exhibit a zymolyase hypersensitivity. Furthermore, we demonstrate that chitin deposition is not restricted to bud scars, but is evenly distributed in the cell wall of cells lacking CBR1 and NCP1. Proper cell wall biogenesis requires polarization of synthetic enzymes such as chitin and glucan synthase. Rho1 is considered a master regulator in this process (Levin, 2005Go). By contrast, little is known about the role of Cdc42 and its downstream targets in cell wall maintenance. Various genes involved in the synthesis of the cell wall components chitin and beta-1,3-glucan become essential in the absence of CLA4 (Goehring et al., 2003Go; Lesage et al., 2005Go). Together with our data, this suggests that Cla4, like Erg4 and Ncp1, regulates cell wall assembly by a hitherto unknown mechanism.

Ste20 and other Cdc42 effectors trigger exit from mitosis (Höfken and Schiebel, 2002Go; Jensen et al., 2002Go; Seshan et al., 2002Go; Höfken and Schiebel, 2004Go). In contrast to Cla4 and Gic1, the molecular mechanisms of Ste20 action in this process remain unclear. Recently, Ncp1 has also been described to contribute to mitotic exit (Keniry et al., 2004Go). Because our data show that Ncp1 binds to Ste20, it is tempting to speculate that Ncp1 and Ste20 act together in mitotic exit.

Like Ste20, Erg4 and Ncp1 not only have various functions during vegetative growth but also play a role in mating and filamentous growth. Ste20 is an essential component of the yeast mating pathway (Leberer et al., 1992Go; Ramer and Davis, 1993Go). Upon pheromone stimulation, Ste20 activates a MAP kinase cascade, which results in a cell cycle arrest in G1 phase. Via actin polarization, these cells then form a mating projection and fuse with a cell of the opposite mating type (Dohlman and Thorner, 2001Go). Here, we show that cells in which ERG4 is deleted undergo a cell cycle arrest but fail to polarize the actin cytoskeleton. Consequently, a reduced mating efficiency was observed for these cells. These overlapping phenotypes, together with the protein interaction data, suggest that Ste20 and Erg4 cooperate in cell polarization during mating.

Finally, we demonstrate that Erg4 and Ncp1 contribute to haploid invasive growth, although with opposing functions. The ERG4 deletion strain displayed hyperinvasive growth. Thus, Erg4 is a negative regulator of filamentous growth. By contrast, loss of NCP1 resulted in a complete loss of invasive growth due to a polarization defect. This is in accordance with the phenotype described for ste20{Delta} cells.

Due to redundancy (see below), the CBR1 deletion alone has no phenotypic effects. By contrast, the inactivation of CBR1 and NCP1 is lethal and results in bud morphology defects, which demonstrates that Cbr1, like Erg4 and Ncp1, plays a role in cell polarity

Sterol synthesis and cell polarity
Erg4, Cbr1 and Ncp1 are all involved in the biosynthesis of ergosterol (Fig. 8), the predominant sterol in budding yeast. The biosynthesis of sterols in yeast can be divided into two stages. The early sterol biosynthetic pathway starts with acetyl CoA and provides essential cellular constituents, such as isopentenyl diphosphate, quinones and dolichol (Daum et al., 1998Go). It also provides squalene, the first specific intermediate of the ergosterol biosynthetic pathway. The proteins of the later sterol pathway, including Erg4, Ncp1 and Cbr1, catalyze the transformation of squalene to ergosterol (Daum et al., 1998Go) (Fig. 8). Whereas Erg4 catalyzes the final step in this pathway (Zweytick et al., 2000Go), Ncp1 and Cbr1 have a more indirect role. Ncp1 transfers electrons from NADPH to two cytochrome P450 enzymes involved in sterol synthesis, Erg5 (sterol 22-desaturase; CYP61) and Erg11 (sterol 14{alpha}-demethylase; CYP51) (Aoyama et al., 1989Go; Kelly et al., 1995Go) (Figs 8, 9). Erg1 (squalene epoxidase/squalene monooxygenase), another enzyme in the sterol biosynthetic pathway, also requires Ncp1 (Yoshida, 1988Go). Cbr1 (NADH cytochrome b5 reductase) acts in a very similar way to Ncp1 and has overlapping electron acceptors. It transports electrons from NADH via Cyb5 (cytochrome b5) to Erg11, and very probably to Erg1 and Erg5 as well (Lamb et al., 1999Go) (Figs 8, 9). Additionally, the Cbr1-Cyb5 electron transport system participates in other sterol biosynthetic reactions – such as C5-desaturation, catalyzed by Erg3, and C4-demethylation, catalyzed by Erg25, Erg26 and Erg27 (Reddy et al., 1977Go; Aoyama et al., 1981Go). The redundant functions of Cbr1 and Ncp1 explains why no phenotypes were observed for the CBR1-deletion strain. Furthermore, the lethality of the double mutant cbr1{Delta} ncp1{Delta} is in accordance with the biochemical data. These cells presumably die because the ergosterol biosynthesis pathway is perturbed at several steps.


Figure 9
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  		Fig. 9. Model for the function of Ste20. Proposed model for the regulation of cell polarity by Ste20 via sterol synthesis. For simplification, proteins that require only Cbr1, as described in Fig. 8, were omitted. See Discussion for details

 

In addition to our results, other groups have reported the involvement of ergosterol-synthesizing proteins in homotypic vacuole fusion and endocytosis (Kato and Wickner, 2001Go; Seeley et al., 2002Go; Pichler and Riezman, 2004Go). Cdc42 and its effectors also play a role in these processes. Ste20 and Cla4 promote actin patch assembly and, as a consequence of that, endocytosis via type I myosin phosphorylation (Wu et al., 1996Go; Evangelista et al., 2000Go; Geli et al., 2000Go; Lechler et al., 2000Go). By contrast, the role of Cdc42 and other polarity proteins in vacuole maintenance is poorly understood (Wickner, 2002Go). Recently, homologs of oxysterol-binding proteins, a family of proteins that regulate the synthesis and transport of sterols, were found to participate in Cdc42-dependant polarity (Kozminski et al., 2006Go). All these observations, together with our data, demonstrate the requirement of sterol synthesis for cell polarization and possibly for other functions involving Cdc42. It is important to notice that the major regulatory steps in the post-squalene biosynthetic pathway, catalyzed by Erg1 and Erg11 (Veen et al., 2003Go) (Fig. 8), both depend on electron transfer from Ncp1 and Cbr1. Therefore, the whole pathway might be regulated through Ste20. This is of particular interest because homologs of Ste20, Ncp1, Cbr1 and Cyb5 have been found in mammals, and four of the enzyme activities depending on Ncp1 and/or Cbr1, including the major regulatory steps of the pathway, are conserved throughout evolution (Fig. 8). Thus, this proposed regulatory module (Fig. 9) might be conserved from yeast to humans. Interestingly, emb-8, the NCP1 homolog in Caenorhabditis elegans, has a role in polarization during early embryonic development (Rappleye et al., 2003Go). Furthermore, it has recently been reported that, in Arabidopsis thaliana, squalene epoxidase (Erg1 in budding yeast), a major enzyme of the sterol biosynthetic pathway that depends on Ncp1 and Cbr1 (Fig. 8), regulates the polarized growth of roots and hypocotyl (Rasbery et al., 2007Go).

Although these observations support a conserved regulatory link between Ste20 and ergosterol pathway genes such as ERG4, CBR1 and NCP1, neither these findings nor our results indicate whether Erg4, Cbr1 and Ncp1 lie upstream or downstream of Ste20. Although the delocalization of Ste20-GFP in mating projections of erg4{Delta} cells supports the ergosterol pathway lying upstream of Ste20, we find it tempting to also speculate that Ste20 might activate these proteins. This important question will be addressed in the future.

Whereas Ste20 is enriched at sites of polarized growth at the plasma membrane (Peter et al., 1996Go), Erg4, Cbr1 and Ncp1 associate with the endoplasmic reticulum (ER) (Kreibich et al., 1983Go; Zweytick et al., 2000Go). This raises the issue of where and how these proteins interact. Although Ste20 is activated by Cdc42 at the plasma membrane, it is also present in the cytosol (Peter et al., 1996Go). Importantly, Cbr1 and Ncp1 face the cytoplasmic side of the ER (Kreibich et al., 1983Go) and could, therefore, be easily accessible for cytosolic Ste20. Alternatively, plasma membrane-bound Ste20 could interact with Erg4, Cbr1 and Ncp1 in the ER. It has been reported that a subfraction of the ER closely associates with the plasma membrane (Pichler et al., 2001Go). Interestingly, enzymes of sterol synthesis are enriched at these contact sites (Pichler et al., 2001Go).

Lipid rafts and cell polarity
Our data suggest that sterol composition of membranes is crucial for the establishment of cell polarity. It has been suggested that membrane microdomains rich in sterol and sphingolipids known as lipid rafts compartmentalize the plasma membrane and might play an important role in cell polarization (Rajendran and Simons, 2005Go). In budding yeast, sterol-rich membranes have been observed to localize in a polarized manner towards the tip of the mating projection (Bagnat and Simons, 2002Go). Thus, there they might contribute to the polarized localization of proteins involved in the mating process. However, it is not clear whether ergosterol molecules at the mating projection really form lipid rafts. Another open question is whether asymmetric lipid distribution is sufficient for polarized protein localization. Furthermore, the existence of lipid rafts is controversial and is still being debated (Munro, 2003Go). In addition, it has been questioned whether sterols are polarized in budding yeast (Valdez-Taubas and Pelham, 2003Go). Because endocytosis requires sterol (Pichler and Riezman, 2004Go) and the endocytic pathway is necessary and sufficient for cell polarization (Valdez-Taubas and Pelham, 2003Go), sterols could establish polarity via endocytosis. Further work will determine the role of sterols in cell polarity and whether Ste20 regulates sterol synthesis.


   Materials and Methods
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 Summary
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
Yeast strains and constructs
All yeast strains are listed in Table 1. The strains used in this study were in the YPH499 background with the exception of strains used for invasive growth and mating assays. For invasive growth experiments, strains in the {Sigma}1278b background were used (MBY15, MBY16, MBY22, PPY966 and PPY1209). Strains for quantitative mating assays (ESM356, ESM357, THY580, THY581, THY595 and THY597) are derivatives of FY1679. All constructs used in this work are listed in Table 2.


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  		Table 1. Yeast strains used in this work

 

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  		Table 2. Plasmids used in this work

 

Split-ubiquitin technique
For the split-ubiquitin screen, ste20{Delta} cells carrying pMET25-STE20-CUBI-RURA3 were transformed with a split-ubiquitin library and grown on 5-FOA plates for 3 days at 30°C. The interaction assays were performed as described previously (Johnsson and Varshavsky, 1994Go).

Genes were subcloned after PCR amplification to confirm the interaction.

Pull-down assays and antibodies
GST and GST-Ste20 were expressed in E. coli BL21 (DE3) and purified using glutathione-sepharose (Amersham Biosciences). The immobilized GST and GST-Ste20 proteins were presented to a yeast lysate of 3HA-CBR1 and ERG4-9myc, respectively, for 9 minutes at 4°C in IP buffer (20 mM Tris pH 7.5, 100 mM NaCl, 10 mM EDTA, 1 mM EGTA, 5% glycerol, 1% NP-40, 1% BSA). After five washes with IP buffer, the associated proteins were eluted with sample buffer and analyzed by immunoblotting. Monoclonal mouse anti-HA (12CA5) was obtained from Roche. Mouse monoclonal anti-Myc (9E10) and rabbit polyclonal anti-Cdc11 were from Santa Cruz Biotechnology. Secondary antibodies were from Jackson Research Laboratories.

Apical growth assays
Cells carrying the plasmid pMT485 (GAL1-CLN1-3HA) were grown overnight in selective medium. Exponentially growing cells were induced with galactose for 4 hours, fixed for microscopic examination and prepared for immunoblot analysis.

Zymolyase-sensitivity assays
Cells were inoculated to an OD600 of 0.025 in YPD supplemented with various amounts of zymolyase 20T (Seikagaku) and grown overnight at 30°C. Various dilutions of the cell suspensions were then plated on YPD medium and grown for 2 days at 30°C to determine the number of colony forming units. Zymolyase sensitivity was expressed as percentage of growth compared with growth in YPD without zymolyase.

Invasive growth assays
For agar invasion assays, 105 cells of an overnight culture were spotted on YPD and grown for 2 days at 30°C. Plates were photographed before and after being rinsed under a gentle stream of deionized water. For analysis of filamentous colony morphology, cells were grown to stationary phase in synthetic complete medium, washed twice with water and spread onto synthetic complete medium lacking glucose at a concentration of 105 cells/plate. Plates were incubated at 30°C for 18 hours and analyzed microscopically. Single cells of these microcolonies were analyzed by scraping off the colonies from the plates using a cell scraper and subsequent fixation with formaldehyde for morphological examination and actin staining.

Pheromone-response assays
Logarithmically growing cells were incubated with 1 µg/ml {alpha}-factor for 150 and 360 minutes. These cells were fixed with formaldehyde for morphological examination, analysis of GFP-tagged proteins and actin staining. For quantitative mating assays 3x106 cells of each mating type were mixed and collected on nitrocellulose filters. The filters were placed on YPD plates for 4 hours. Filters were then suspended in water and serial dilutions were plated on double-selective SC plates to determine the number of diploids. Mating efficiency was calculated as the percentage of input cells that formed diploids.

Actin staining and fluorescence microscopy
F-actin of yeast cells was stained with rhodamine-phalloidin as described previously (Höfken and Schiebel, 2002Go). Cells were examined with a Zeiss Axiovert 200M fluorescence microscope equipped with a 100x Plan oil-immersion objective and images were captured using a Zeiss AxioCam MRm CCD camera.


   Acknowledgments
 
We are very grateful to Nils Johnsson for providing us with the split-ubiquitin library and plasmids. We thank C. David Allis, Kathryn Ayscough, Peter Pryciak, Elmar Schiebel, Kazuma Tanaka and Mike Tyers for strains and plasmids. We thank Melanie Boß for excellent technical support. This work is part of the doctoral thesis of C.T. and the diploma thesis of D.H. The project was supported by the Deutsche Forschungsgemeinschaft (HO 2098/3-1).


   Footnotes
 
Supplementary material available online at http://jcs.biologists.org/cgi/content/full/120/20/3613/DC1

* These authors contributed equally to this work Back


   References
Top
 Summary
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

Ahn, S. H., Cheung, W. L., Hsu, J. Y., Diaz, R. L., Smith, M. M. and Allis, C. D. (2005). Sterile 20 kinase phosphorylates histone H2B at serine 10 during hydrogen peroxide-induced apoptosis in S. cerevisiae. Cell 120, 25-36.[CrossRef][Medline]

Aoyama, Y., Yoshida, Y., Sato, R., Susani, M. and Ruis, H. (1981). Involvement of cytochrome b5 and a cyanide-sensitive monooxygenase in the 4-demethylation of 4,4-dimethylzymosterol by yeast microsomes. Biochim. Biophys. Acta 663, 194-202.[Medline]

Aoyama, Y., Yoshida, Y., Sonoda, Y. and Sato, Y. (1989). Deformylation of 32-oxo-24,25-dihydrolanosterol by the purified cytochrome P-45014DM (lanosterol 14 alpha-demethylase) from yeast evidence confirming the intermediate step of lanosterol 14 alpha-demethylation. J. Biol. Chem. 264, 18502-18505.[Abstract/Free Full Text]

Bagnat, M. and Simons, K. (2002). Cell surface polarization during yeast mating. Proc. Natl. Acad. Sci. USA. 99, 14183-14188.[Abstract/Free Full Text]

Brown, J. L., Jaquenoud, M., Gulli, M. P., Chant, J. and Peter, M. (1997). Novel Cdc42-binding proteins Gic1 and Gic2 control cell polarity in yeast. Genes Dev. 11, 2972-2982.[Abstract/Free Full Text]

Bucking-Throm, E., Duntze, W., Hartwell, L. H. and Manney, T. R. (1973). Reversible arrest of haploid yeast cells in the initiation of DNA synthesis by a diffusible sex factor. Exp. Cell Res. 76, 99-110.[CrossRef][Medline]

Butty, A. C., Pryciak, P. M., Huang, L. S., Herskowitz, I. and Peter, M. (1998). The role of Far1p in linking the heterotrimeric G protein to polarity establishment proteins during yeast mating. Science 282, 1511-1516.[Abstract/Free Full Text]

Casamayor, A. and Snyder, M. (2002). Bud-site selection and cell polarity in budding yeast. Curr. Opin. Microbiol. 5, 179-186.[CrossRef][Medline]

Chen, G. C., Kim, Y. J. and Chan, C. S. (1997). The Cdc42 GTPase-associated proteins Gic1 and Gic2 are required for polarized cell growth in Saccharomyces cerevisiae. Genes Dev. 11, 2958-2971.[Abstract/Free Full Text]

Cullen, P. J. and Sprague, G. F. (2000). Glucose depletion causes haploid invasive growth in yeast. Proc. Natl. Acad. Sci. USA 97, 13619-13624.[Abstract/Free Full Text]

Cvrckova, F., De Virgilio, C., Manser, E., Pringle, J. R. and Nasmyth, K. (1995). Ste20-like protein kinases are required for normal localization of cell growth and for cytokinesis in budding yeast. Genes Dev. 9, 1817-1830.[Abstract/Free Full Text]

Daum, G., Lees, N. D., Bard, M. and Dickson, R. (1998). Biochemistry, cell biology and molecular biology of lipids of Saccharomyces cerevisiae. Yeast 14, 1471-1510.[CrossRef][Medline]

Dobbelaere, J., Gentry, M. S., Hallberg, R. L. and Barral, Y. (2003). Phosphorylation-dependent regulation of septin dynamics during the cell cycle. Dev. Cell 4, 345-357.[CrossRef][Medline]

Dohlman, H. G. and Thorner, J. W. (2001). Regulation of G protein-initiated signal transduction in yeast: paradigms and principles. Annu. Rev. Biochem. 70, 703-754.[CrossRef][Medline]

Douglas, L. M., Alvarez, F. J., McCreary, C. and Konopka, J. B. (2005). Septin function in yeast model systems and pathogenic fungi. Eukaryot. Cell 4, 1503-1512.

Drees, B. L., Sundin, B., Brazeau, E., Caviston, J. P., Chen, G. C., Guo, W., Kozminski, K. G., Lau, M. W., Moskow, J. J., Tong, A. et al. (2001). A protein interaction map for cell polarity development. J. Cell Biol. 154, 549-571.[Abstract/Free Full Text]

Epp, J. A. and Chant, J. (1997). An IQGAP-related protein controls actin-ring formation and cytokinesis in yeast. Curr. Biol. 7, 921-929.[CrossRef][Medline]

Etienne-Manneville, S. (2004). Cdc42-the centre of polarity. J. Cell Sci. 117, 1291-1300.[Abstract/Free Full Text]

Evangelista, M., Klebl, B. M., Tong, A. H., Webb, B. A., Leeuw, T., Leberer, E., Whiteway, M., Thomas, D. Y. and Boone, C. (2000). A role for myosin-I in actin assembly through interactions with Vrp1p, Bee1p, and the Arp2/3 complex. J. Cell Biol. 148, 353-362.[Abstract/Free Full Text]

Geli, M. I., Lombardi, R., Schmelzl, B. and Riezman, H. (2000). An intact SH3 domain is required for myosin I-induced actin polymerization. EMBO J. 19, 4281-4291.[CrossRef][Medline]

Goehring, A. S., Mitchell, D. A., Tong, A. H., Keniry, M. E., Boone, C. and Sprague, G. F. (2003). Synthetic lethal analysis implicates Ste20p, a p21-activated potein kinase, in polarisome activation. Mol. Biol. Cell 14, 1501-1516.[Abstract/Free Full Text]

Höfken, T. and Schiebel, E. (2002). A role for cell polarity proteins in mitotic exit. EMBO J. 21, 4851-4862.[CrossRef][Medline]

Höfken, T. and Schiebel, E. (2004). Novel regulation of mitotic exit by the Cdc42 effectors Gic1 and Gic2. J. Cell Biol. 164, 219-231.[Abstract/Free Full Text]

Holly, S. P. and Blumer, K. J. (1999). PAK-family kinases regulate cell and actin polarization throughout the cell cycle of Saccharomyces cerevisiae. J. Cell Biol. 147, 845-856.[Abstract/Free Full Text]

Jaffe, A. B. and Hall, A. (2005). Rho GTPases: biochemistry and biology. Annu. Rev. Cell Dev. Biol. 21, 247-269.[CrossRef][Medline]

Jensen, S., Geymonat, M., Johnson, A. L., Segal, M. and Johnston, L. H. (2002). Spatial regulation of the guanine nucleotide exchange factor Lte1 in Saccharomyces cerevisiae. J. Cell Sci. 115, 4977-4991.[CrossRef][Medline]

Johnsson, N. and Varshavsky, A. (1994). Split ubiquitin as a sensor of protein interactions in vivo. Proc. Natl. Acad. Sci. USA 91, 10340-10344.[Abstract/Free Full Text]

Kato, M. and Wickner, W. (2001). Ergosterol is required for the Sec18/ATP-dependent priming step of homotypic vacuole fusion. EMBO J. 20, 4035-4040.[CrossRef][Medline]

Kelly, S. L., Lamb, D. C., Corran, A. J., Baldwin, B. C., Parks, L. W. and Kelly, D. E. (1995). Purification and reconstitution of activity of Saccharomyces cerevisiae P450 61, a sterol delta 22-desaturase. FEBS Lett. 377, 217-220.[CrossRef][Medline]

Keniry, M. E., Kemp, H. A., Rivers, D. M. and Sprague, G. F. (2004). The identification of Pcl1-interacting proteins that genetically interact with Cla4 may indicate a link between G1 progression and mitotic exit. Genetics 166, 1177-1186.[Abstract/Free Full Text]

Kozminski, K. G., Alfaro, G., Dighe, S. and Beh, C. T. (2006). Homologues of oxysterol-binding proteins affect Cdc42p- and Rho1p-mediated cell polarization in Saccharomyces cerevisiae. Traffic 7, 1224-1242.[CrossRef][Medline]

Kreibich, G., Sabatini, D. D. and Adesnik, M. (1983). Biosynthesis of hepatocyte endoplasmic reticulum proteins. Meth. Enzymol. 96, 530-542.[Medline]

Labib, K., Tercero, J. A. and Diffley, J. F. (2000). Uninterrupted MCM2-7 function required for DNA replication fork progression. Science 288, 1643-1647.[Abstract/Free Full Text]

Lamb, D. C., Kelly, D. E., Manning, N. J., Kaderbhai, M. A. and Kelly, S. L. (1999). Biodiversity of the P450 catalytic cycle: yeast cytochrome b5/NADH cytochrome b5 reductase complex efficiently drives the entire sterol 14-demethylation (CYP51) reaction. FEBS Lett. 462, 283-288.[CrossRef][Medline]

Leberer, E., Dignard, D., Harcus, D., Thomas, D. Y. and Whiteway, M. (1992). The protein kinase homologue Ste20p is required to link the yeast pheromone response G-protein beta gamma subunits to downstream signalling components. EMBO J. 11, 4815-4824.[Medline]

Lechler, T., Shevchenko, A. and Li, R. (2000). Direct involvement of yeast type I myosins in Cdc42-dependent actin polymerization. J. Cell Biol. 148, 363-373.[Abstract/Free Full Text]

Lee, B. N. and Elion, E. A. (1999). The MAPKKK Ste11 regulates vegetative growth through a kinase cascade of shared signaling components. Proc. Natl. Acad. Sci. USA 96, 12679-12684.[Abstract/Free Full Text]

Leeuw, T., Fourest-Lieuvin, A., Wu, C., Chenevert, J., Clark, K., Whiteway, M., Thomas, D. Y. and Leberer, E. (1995). Pheromone response in yeast: association of Bem1p with proteins of the MAP kinase cascade and actin. Science 270, 1210-1213.[Abstract/Free Full Text]

Lesage, G., Sdicu, A. M., Menard, P., Shapiro, J., Hussein, S. and Bussey, H. (2005). Analysis of beta-1,3-glucan assembly in Saccharomyces cerevisiae using a synthetic interaction network and altered sensitivity to caspofungin. Genetics 167, 35-49.

Levin, D. E. (2005). Cell wall integrity signaling in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 69, 262-291.[Abstract/Free Full Text]

Lew, D. J. and Reed, S. I. (1993). Morphogenesis in the yeast cell cycle: regulation by Cdc28 and cyclins. J. Cell Biol. 120, 1305-1320.[Abstract/Free Full Text]

Longtine, M. S., Theesfeld, C. L., McMillan, J. N., Weaver, E., Pringle, J. R. and Lew, D. J. (2000). Septin-dependent assembly of a cell cycle-regulatory module in Saccharomyces cerevisiae. Mol. Cell. Biol. 20, 4049-4061.[Abstract/Free Full Text]

Martin, H., Mendoza, A., Rodriguez-Pachon, J. M., Molina, M. and Nombela, C. (1997) Characterization of SKM1, a Saccharomyces cerevisiae gene encoding a novel Ste20/PAK-like protein kinase. Mol. Microbiol. 23, 431-444.[CrossRef][Medline]

Munro, S. (2003). Lipid rafts: elusive or illusive? Cell 115, 377-388.[CrossRef][Medline]

Nakayama, K., Nagasu, T., Shimma, Y., Kuromitsu, J. and Jigami, Y. (1992). OCH1 encodes a novel membrane bound mannosyltransferase: outer chain elongation of asparagine-linked oligosaccharides. EMBO J. 11, 2511-2519.[Medline]

Ni, L. and Snyder, M. (2001) A genomic study of the bipolar bud site selection pattern in Saccharomyces cerevisiae. Mol. Biol. Cell 12, 2147-2170.[Abstract/Free Full Text]

Novick, P. and Botstein, D. (1985). Phenotypic analysis of temperature-sensitive yeast actin mutants. Cell 40, 405-416.[CrossRef][Medline]

Osman, M. A. and Cerione, R. A. (1998). Iqg1p, a yeast homologue of the mammalian IQGAPs, mediates cdc42p effects on the actin cytoskeleton. J. Cell Biol. 142, 443-455.[Abstract/Free Full Text]

Palecek, S. P., Parikh, A. S. and Kron, S. J. (2002). Sensing, signalling and integrating physical processes during Saccharomyces cerevisiae invasive and filamentous growth. Microbiology 148, 893-907.[Free Full Text]

Peter, M., Neiman, A. M., Park, H. O., van Lohuizen, M. and Herskowitz, I. (1996). Functional analysis of the interaction between the small GTP binding protein Cdc42 and the Ste20 protein kinase in yeast. EMBO J. 15, 7046-7059.[Medline]

Pichler, H. and Riezman, H. (2004). Where sterols are required for endocytosis. Biochim. Biophys. Acta 1666, 51-61.[Medline]

Pichler, H., Gaigg, B., Hrastnik, C., Achleitner, G., Kohlwein, S. D., Zellnig, G., Perktold, A. and Daum, G. (2001). A subfraction of the yeast endoplasmic reticulum associates with the plasma membrane and has a high capacity to synthesize lipids. Eur. J. Biochem. 268, 2351-2361.[Medline]

Rajendran, L. and Simons, K. (2005). Lipid rafts and membrane dynamics. J. Cell Sci. 118, 1099-1102.[Free Full Text]

Ram, A. F., Wolters, A., Ten Hoopen, R. and Klis, F. M. (1994). A new approach for isolating cell wall mutants in Saccharomyces cerevisiae by screening for hypersensitivity to calcofluor white. Yeast 10, 1019-1030.[CrossRef][Medline]

Ramer, S. W. and Davis, R. W. (1993). A dominant truncation allele identifies a gene, STE20, that encodes a putative protein kinase necessary for mating in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 90, 452-456.[Abstract/Free Full Text]

Rappleye, C. A., Tagawa, A., Le Bot, N., Ahringer, J. and Aroian, R. V. (2003). Involvement of fatty acid pathways and cortical interaction of the pronuclear complex in Caenorhabditis elegans embryonic polarity. BMC Dev. Biol. 3, 8.[CrossRef][Medline]

Rasbery, J. M., Shan, H., LeClair, R. J., Norman, M., Matsuda, S. P. and Bartel, B. (2007). Arabidopsis thaliana squalene epoxidase 1 is essential for root and seed development. J. Biol. Chem. 282, 17002-17013.[Abstract/Free Full Text]

Raychaudhuri, S. and Prinz, W. A. (2006). Uptake and trafficking of exogenous sterols in Saccharomyces cerevisiae. Biochem. Soc. Trans. 34, 359-362.[CrossRef][Medline]

Reddy, V. V., Kupfer, D. and Caspi, E. (1977). Mechanism of C-5 double bond introduction in the biosynthesis of cholesterol by rat liver microsomes. J. Biol. Chem. 252, 2797-2801.[Abstract/Free Full Text]

Roberts, R. L. and Fink, G. R. (1994). Elements of a single MAP kinase cascade in Saccharomyces cerevisiae mediate two developmental programs in the same cell type: mating and invasive growth. Genes Dev. 8, 2974-2985.[Abstract/Free Full Text]

Sakchaisri, K., Asano, S., Yu, L. R., Shulewitz, M. J., Park, C. J., Park, J. E., Cho, Y. W., Veenstra, T. D., Thorner, J. and Lee, K. S. (2004). Coupling morphogenesis to mitotic entry. Proc. Natl. Acad. Sci. USA 101, 4124-4129.[Abstract/Free Full Text]

Schmidt, M., Varma, A., Drgon, T., Bowers, B. and Cabib, E. (2003). Septins, under Cla4p regulation, and the chitin ring are required for neck integrity in budding yeast. Mol. Biol. Cell 14, 2128-2141.[Abstract/Free Full Text]

Seeley, E. S., Kato, M., Margolis, N., Wickner, W. and Eitzen, G. (2002). Genomic analysis of homotypic vacuole fusion. Mol. Biol. Cell 13, 782-794.[Abstract/Free Full Text]

Seshan, A., Bardin, A. J. and Amon, A. (2002). Control of Lte1 localization by cell polarity determinants and Cdc14. Curr. Biol. 12, 2098-2110.[CrossRef][Medline]

Sheu, Y. J., Barral, Y. and Snyder, M. (2000). Polarized growth controls cell shape and bipolar bud site selection in Saccharomyces cerevisiae. Mol. Cell. Biol. 20, 5235-5247.[Abstract/Free Full Text]

Sikorski, R. S. and Hieter, P. (1989). A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 19-27.[Abstract/Free Full Text]

Valdez-Taubas, J. and Pelham, H. R. (2003). Slow diffusion of proteins in the yeast plasma membrane allows polarity to be maintained by endocytic cycling. Curr. Biol. 13, 1636-1640.[CrossRef][Medline]

van Drogen, F., O'Rourke, S. M., Stucke, V. M., Jaquenoud, M., Neiman, A. M. and Peter, M. (2000). Phosphorylation of the MEKK Ste11p by the PAK-like kinase Ste20p is required for MAP kinase signaling in vivo. Curr. Biol. 10, 630-639.[CrossRef][Medline]

Veen, M., Stahl, U. and Lang, C. (2003). Combined overexpression of genes of the ergosterol biosynthetic pathway leads to accumulation of sterols in Saccharomyces cerevisiae. FEMS Yeast Res. 4, 87-95.[CrossRef][Medline]

Versele, M. and Thorner, J. (2004). Septin collar formation in budding yeast requires GTP binding and direct phosphorylation by the PAK, Cla4. J. Cell Biol. 164, 701-715.[Abstract/Free Full Text]

Versele, M. and Thorner, J. (2005). Some assembly required: yeast septins provide the instruction manual. Trends Cell Biol. 15, 414-424.[CrossRef][Medline]

Wickner, W. (2002). Yeast vacuoles and membrane fusion pathways. EMBO J. 21, 1241-1247.[CrossRef][Medline]

Wu, C., Whiteway, M., Thomas, D. Y. and Leberer, E. (1995). Molecular characterization of Ste20p, a potential mitogen-activated protein or extracellular signal-regulated kinase kinase (MEK) kinase kinase from Saccharomyces cerevisiae. J. Biol. Chem. 270, 15984-15992.[Abstract/Free Full Text]

Wu, C., Lee, S. F., Furmaniak-Kazmierczak, E., Cote, G. P., Thomas, D. Y. and Leberer, E. (1996). Activation of myosin-I by members of the Ste20p protein kinase family. J. Biol. Chem. 271, 31787-31790.[Abstract/Free Full Text]

Yoshida, Y. (1988). Cytochrome P-450 of fungi: primary target for azole antifungal agents. In Current Topics in Medical Mycology. Vol. 2 (ed. M. R. McGinnis), pp. 388-418. New York: Springer-Verlag.[Medline]

Zweytick, D., Hrastnik, C., Kohlwein, S. D. and Daum, G. (2000). Biochemical characterization and subcellular localization of the sterol C-24(28) reductase, Erg4p, from the yeast Saccharomyces cerevisiae. FEBS Lett. 470, 83-87.[CrossRef][Medline]


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