Cell cycle arrest in cells lacking DDI1 , DSK2 and the c-terminal UBA (UBA2) domain of Rad23. (A) Western blots showing relative protein levels of Rad23, Ddi1 and mutant forms in yeast extracts. Left two panels show endogenous levels of these proteins and the right panel shows levels produced after expression of constructs from the GAL1 promoter. In each case, the wild type and mutant version were tagged identically at their C-termini – [MYC]6x for Rad23 and [HIS]6x for Ddi1. PSTAIRE is a loading control. Strains expressed (from left to right) – Left panel: RAD23 [MYC]6x, DDI1 [HIS]6x, RAD23ΔUBA2
[HIS]6 x; Middle Panel: no tag control, RAD23[MYC]6x and DDI1[HIS]6x, RAD23ΔUBA2
[MYC]6x and DDI1[HIS]6x, RAD23[MYC]6x and DDI1ΔUBA
[HIS]6 x, RAD23ΔUBA2
[MYC]6x and DDI1ΔUBA
[HIS]6 x; Right Panel: no tag control, GAL1-RAD23[MYC]6x, GAL1-RAD23ΔUBA2
[MYC]6 x, GAL1-DDI1[HIS]6x, GAL1-DDI1ΔUBA
[HIS]6 x. Upper band of Ddi1 is a phosphorylated species (data not shown). (B) Synthetic effect of UBL-UBA mutants on temperature sensitivity. The indicated strains were grown to mid-log phase and serial dilutions were spotted onto YEPD plates and incubated at the indicated temperatures for 48 hrs. Levels of Rad23 and Rad23ΔUBA2 protein expression were determined as shown in A. (C) Kinetics of cell cycle progression in wild type , ddi1Δ dsk2Δ rad23Δ and ddi1Δ dsk2Δ rad23ΔUBA2 cells. (ddi1Δdsk2Δ strains behaved identically to the wild type control; Figure 1 and data not shown.) Cells were arrested in G1 at 30°C with alpha-factor and released in rich medium at 37°C. Cell cycle progression was monitored by bud morphology: unbudded cells (G1, yellow line), cells with small buds (S-phase, blue line) and cells with large buds (G2/M, red line).