Figure 5. Rpa190 Degradation Is Not Nutrient or DNA-Damage Sensitive, but Is Cold Sensitive.

(A) Cycloheximide-chase degradation assays of UBP10 or ubp10Δ cells expressing Rpa190-3HA. Cells were subject to rapamycin treatment (200 nM), amino acid depletion (media with no tryptophan), or glucose depletion (media with no glucose) for 2 hr prior to addition of cycloheximide. Time after cycloheximide addition is indicated above each lane. Western analysis was performed using anti-HA antibodies.

(B) Growth of UBP10 and ubp10Δ cells (left panels) with or without UV exposure. Tenfold serial dilutions of cells were spotted onto the appropriate media and exposed to the indicated amount of UV irradiation. rad6Δ cells were used as a positive control. Plates were subsequently incubated at 30°C for 3 days.

(C) Cycloheximide-chase degradation assays of UBP10 or ubp10Δ cells expressing Rpa190-3HA after UV exposure. Cells were exposed to the indicated amount of UV irradiation, and cycloheximide was subsequently added. Time after cycloheximide addition is indicated above each lane. Western analysis was performed using anti-HA antibodies.

(D) Cycloheximide-chase degradation assays of UBP10 or ubp10Δ cells expressing Rpa190-3HA. Cells were grown at the indicated temperature. Time after cycloheximide addition is indicated above each lane. Western analysis was performed using anti-HA antibodies. Growth of UBP10 and ubp10Δ cells (bottom panels). Tenfold serial dilutions of cells were spotted onto the appropriate media and incubated at the indicated temperature for 3 days (30°C, 33°C, or 38°C) or 5 days (25°C).

(E) Ubiquitin proteomes from UBP10 (wt) or ubp10Δ (Δ) cells incubated at the indicated temperatures were isolated by metal affinity purification. Levels of Rpa190-3HA in lysates (bottom panel) and eluates (top panel) were determined by western analysis using anti-HA antibodies.

(F) Cycloheximide-chase degradation assays of cells expressing two San1 substrates (Tdh3* and Hom2*) were performed as in (B).