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).