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

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

Christopher Tiedje^*, Daniel G. Holland^*, Ursula Just and Thomas Höfken

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

View larger version (31K): [in this window] [in a new window]   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.

View larger version (39K): [in this window] [in a new window]   Fig. 2. Genetic interactions of erg4, cbr1 and ncp1. (A) Deletion of CLA4 in erg4 cells or ncp1 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 erg4 and cla4 ncp1 carried the plasmid pRS316-CLA4, whereas cla4 ste20 carried the plasmid pRS316-STE20. 5-FOA selects against these URA3-based pRS316 derivatives. (B) STE20 deletion reduces the growth of ncp1 cells. Serial dilutions of the indicated strains were spotted on YPD and 5-FOA plates as described in A. (C) Genetic interactions with och1. Cells were spotted as in A.

View larger version (23K): [in this window] [in a new window]   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 cells have a defect in apical growth. Wild-type, erg4, cbr1 and ste20 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, cbr1 and ste20 cells express comparable amounts of CLN1. Cells from B were analyzed by immunoblotting with anti-HA antibodies. Cdc11 was detected as a loading control.

View larger version (35K): [in this window] [in a new window]   Fig. 4. cla4, erg4 and ncp1 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, erg4 and ncp1 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.

View larger version (35K): [in this window] [in a new window]   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 cells in a 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.

View larger version (42K): [in this window] [in a new window]   Fig. 6. Pheromone-induced polarization requires ERG4. (A) erg4 cells do not form a mating projection. Exponentially growing wild-type, cbr1 and erg4 cells were incubated with 1 µg/ml -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 Fus1-GFP cells were incubated with 1 µg/ml -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 cells. Logarithmically growing wild-type and erg4 cells carrying a plasmid encoding GAL1-STE20-GFP were incubated with 1 µg/ml -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 cells was determined in two independent experiments (n>100 in each).

View larger version (31K): [in this window] [in a new window]   Fig. 7. Inactivation of CBR1 together with NCP1 results in aberrant bud morphology. (A) Deletion of NCP1 in cbr1 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 and cbr1 ncp1 carried the plasmid pRS316-NCP1. 5-FOA selects against this URA3-based plasmid. (B) Construction of a conditional lethal cbr1 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 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 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).

View larger version (16K): [in this window] [in a new window]   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.

View larger version (29K): [in this window] [in a new window]   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