Skp, Cullin, F-box (SCF)-Met30 and SCF-Cdc4-Mediated Proteolysis of CENP-A Prevents Mislocalization of CENP-A for Chromosomal Stability in Budding Yeast
Fig 2
SCF-Cdc4 and SCF-Met30 mutants display SDL with GAL-CSE4.
(A) met30 and cdc4 mutants display SDL with GAL-CSE4. WT (BY4741) or the indicated mutant strains transformed with vector (pMB433), GAL-HA-CSE4 (pMB1597) or GAL-HA-cse4Δ129 (pMB1459) were grown to logarithmic phase, five-fold serial dilutions were prepared and plated on either glucose or galactose plates at 25°C. Complementation of GAL-CSE4 induced SDL of met30-6 and cdc4-1 was performed with met30-6 (TSA948) and cdc4-1 (TSA878) strains with or without GAL-CSE4 transformed with vector or plasmids expressing MET30 (pMB1619) or CDC4 (pMB1617) at 25°C. (B) Mutants of SCF components display SDL with GAL-CSE4. WT (BY4741) or the indicated mutant strains transformed with vector (pMB433) or GAL-HA-CSE4 (pMB1597) were grown to logarithmic phase; five-fold serial dilutions were prepared and plated on either glucose or galactose plates at 25°C except for the cdc53-1 strain, which was incubated at 33°C. Complementation of GAL-CSE4 induced SDL of cdc34-1 was performed with cdc34-1 with or without GAL-CSE4 transformed with vector or plasmid expressing CDC34 (pMB1618) at 25°C. (C) sic1Δ does not rescue the SDL of GAL-CSE4 cdc4-1. Growth assays were performed using cdc4-1 (YMB9571) and cdc4-1 sic1Δ (YMB9713) with or without GAL-CSE4 plated on glucose or galactose plates at 25°C. (D) met32Δ does not rescue the SDL of GAL-CSE4 met30-6 strain. Growth assays were performed using met30-6 (YMB8442) and met32Δ met30-6 (YMB10681) strains with or without GAL-CSE4 (pMB1597) plated on glucose or galactose medium at 25°C. The suppression of temperature sensitivity of met30-6 by met32Δ was tested on glucose medium at the non-permissive temperature of 33°C.