FIGURE 4.

A, Aup1 is required for efficient up-regulation of Cit2 in response to 3-AT and during mitophagy. Wild type (wt) and aup1Δ cells expressing Cit2-HA (DJY3 and DJY4, top) and harboring either empty vector or a vector expressing wild type Aup1 were grown overnight in SD medium lacking histidine and supplemented with 0.2% glutamate to an A₆₀₀ of 0.5. The cells were then challenged with 50 mm 3-AT for 30 min, and 10 A₆₀₀ units of cells were sampled at each time point and processed for immunoblotting. 20 μg of protein were loaded per lane, and the filters were probed with anti-HA antibodies to detect Cit2 and with anti-Tlg2 antibodies as load control. B, the RTG pathway was induced during stationary phase mitophagy in an Aup1-dependent fashion. Wild type (DJY3) and aup1Δ (DJY4) cells expressing Cit2-HA were grown in lactate as described in the legend to Fig. 3B, and samples (20 μg of protein) were analyzed by SDS-PAGE and immunoblotting with anti-HA antibody at each time point. Anti-Tlg2 immunoblotting was used as load control. C, oxidative damage accumulates in aup1Δ cells between the second and third days of the incubation, relative to wild type cells. Wild type (HAY75) and aup1Δ (HAY809) cells were grown on lactate medium and sampled as in B. Protein extracts were prepared, and 15 μg of each sample were conjugated to DNPH as described under “Experimental Procedures.” Anti-DNP antibody signals (fmol of DNP incorporated/μg of total protein) were quantified by SDS-PAGE followed by Western blotting. Bars, S.E. (n = 3).