JCS009860 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Genetic interactions of erg4Δ, cbr1Δ and ncp1Δ. (A) swe1Δ is not synthetically lethal with erg4Δ, cbr1Δ and ncp1Δ. Serial dilutions (1:10) of the indicated strains were spotted on YPD and 5-FOA plates and grown for 2 days at 30°C. Because cla4Δ, cbr1Δ and erg4Δ cells grow like wild type (Fig. 2A), these strains are not shown here again. (B) The lte1Δ mutant is not synthetically lethal with erg4Δ, cbr1Δ and ncp1Δ. The indicated strains were spotted as described in (A).

• Supplemental Figure S2 -

  Fig. S2. Erg4 has a role in bud site selection of diploid but not haploid cells. (A) Budding pattern of diploid strains. Cells of the indicated strains were stained with Calcofluor White to visualize bud scars. The percentage of the indicated budding pattern was determined in two independent experiments (n>100). (B) Budding patterns of haploid deletion strains.

• Supplemental Figure S3 -

  Fig. S3. Polarization of erg4Δ cells. (A) Ste20-GFP is localized to the bud cortex in erg4Δ cells. Logarithmically growing wild-type and erg4Δ cells carrying a plasmid encoding for GAL1-STE20-GFP were incubated with galactose for 1 hour to induce GAL1-STE20-GFP. The percentage of cells with polarized Ste20-GFP localization was determined in two independent experiments (n>100). (B) erg4Δ cells do not have a polarization defect in response to pheromone when grown under anaerobic conditions. Exponentially growing cells were incubated with 1 μg/ml α-factor for 150 minutes in the presence of 80 μg/ml in a N₂ atmosphere. Subsequently, cells were fixed with formaldehyde and stained with rhodamine-phalloidin to visualize the actin cytoskeleton. The percentage of cells with a polarized actin cytoskeleton was determined in two independent experiments (n>100).

• Supplemental Figure S4 -

  Fig. S4. cbr1Δ ncp1-td cells do not have a defect in septin organization or mitotic exit. (A) cbr1Δ ncp1-td cells exhibit normal Cdc12-GFP localization. Cells were grown in liquid culture at 23°C, then galactose was added for 1 hour, cells were shifted to 37°C and after 3 hours the cells were fixed with formaldehyde and analyzed by fluorescence microscopy. Representative cells before (left panel) and after (right panel) splitting of the septin ring are shown. Given is the percentage of cells with normal Cdc12-GFP localization at the bud neck (n>100). (B) cbr1Δ ncp1-td cells do not arrest in late anaphase. Cells were grown as in A, fixed with formaldehyde and stained with DAPI. Shown is the percentage of cells with two separated DAPI staining regions among large-budded cells (n>100).

• Supplemental Figure S5 -

  Fig. S5. Characterization of the cbr1Δ ncp1-td double mutant. (A) The formation of a mating projection is not disturbed in the cbr1Δ ncp1-td background. The indicated strains were first grown at 23°C, then galactose was added for 1 hour and cells were shifted to 37°C. After 1 hour, α-factor was added and cells were incubated for another 3 hours, fixed with formaldehyde and analyzed by fluorescence microscopy. Shown is the percentage of cells with a polarized actin cytoskeleton (n>100). (B) Chitin is not restricted to bud scars in the cbr1Δ ncp1-td strain. Cells were grown at 23°C, then galactose was added for 1 hour. Subsequently, cells were shifted to 37°C for 4 hours and stained with Calcofluor White to visualize chitin. Given is the percentage of cells with the shown phenotype (axial budding and diffused chitin).