CCB2 and CCB4 interaction in a yeast split ubiquitin two-hybrid assay. Constructs CCB2-N or 4-N expressing CCB2 (or 4) mature protein fused to the mutated N-terminal half of ubiquitin NubG were cotransformed in yeast with constructs CCB2- (or 4)-C expressing CCB2 (or 4) protein fused the C-terminal half of ubiquitin Cub; tests of these protein interactions are shown on the left side of the broken line. Constructs expressing yeast protein Alg5, not expected to interact with CCBs, fused to Cub or NubG, were cotransformed, respectively with the CCB2- (or 4)-N or CCB2- (or 4)-C constructs; results of these controls are shown on the right side of the broken line. The interaction between two proteins at the membrane of yeast allows ubiquitin reconstitution and results in the activation of auxotrophic growth markers ADE2 and HIS3 and reporter gene lacZ. (a) To monitor expression of auxotrophic growth markers ADE2 and HIS3, several cotransformant clones were resuspended in water to an OD_600nm of 1, 0.1, and 0.01; plated on SD medium lacking leucine, tryptophan, histidine, and adenine with 10 mM 3-aminotriazole; and allowed to grow for 30 h. (b) Expression of lacZ was followed by measuring at OD_417nm the accumulation of the product metabolized by β-galactosidase. Liquid SD medium lacking leucine and tryptophan was inoculated with several cotransformant clones, and cultures were allowed to grow for 16 h. After cell lysis, β-galactosidase activity was measured as described in Materials and methods and quantified according to the following formula: Activity = 1,000 × DO_417nm/V × t × DO_600nm, were V is the volume of assay and t is the time of incubation. All assays were repeated three times and the resulting error bars are shown.