Figure 3. Ubiquitin Depletion Is Not Responsible for the Aneuploidy Tolerance Caused by Loss of UBP6 Function.
(A) Wild-type, ubp6Δ, disome V and disome V ubp6Δ cells were grown in −His+G418 medium to an OD600 of 1.0 when 100 μg/ml cycloheximide (time = 0 min) was added. Free ubiquitin and ubiquitin conjugates were analyzed by immunoblotting using an anti-ubiquitin antibody at the indicated times.
(B) Ubiquitin levels in the presence (+) or absence (-) of 100 μg/ml CuSO4.
(C - H) The percentage of cells in co-cultures of strains carrying PGK1 fused to GFP (open squares) and strains harboring a UBP6 deletion (closed triangles) was determined at the indicated times. All strains carry a CUP1-UBI4 multicopy plasmid whose expression was induced with 100 μg/ml CuSO4. The following strains were compared: (C), wild-type and UBP6 deletion cells; (D), disome V PGK1-GFP and disome V ubp6Δ cells; (E), disome XI PGK1-GFP and disome XI ubp6Δ; (F), wild-type and ubp6E256X truncation strains; (G), disome VIII PGK1-GFP and disome VIII ubp6E256X cells; and (H), disome XI PGK1-GFP and disome XI ubp6E256X cells. All strains were grown in −His+G418 medium. See also Figure S6.