Figure 2

Oaz1 mediates degradation of ODC. (A) Steady-state levels of ODC-ha in strains PMY1 (wt) and PMY2 (oaz1-) grown for 3 h in the absence (-spd) or presence of 100 M spermidine (+spd) were analysed by anti-ha Western blotting. The blot was simultaneously probed with anti-Cdc11 antibodies to control for differences in protein loading. (B) Quantitation of fluorescence signals shown in (A). Values were normalized using the data obtained for Cdc11 and are given in % of the signal detected for oaz1- grown in the absence of spermidine, which was set to 100%. (C) 'Spermidine chase' of ODC-ha. ODC-ha in cell extracts was detected as in (A), but at the indicated time points after adding spermidine to a concentration of 100 M to the media. (D) Pulse-chase analysis of ODC turnover. The same strains as in (A) were grown for 3 h in the absence or presence of 100 M spermidine before labelling. An asterisk marks the position of a nonspecific band. (E) Quantitation of radioactive ODC-ha signals shown in (D), which were normalized using data for nonspecific background bands. (F) Spermidine induction of Oaz1-myc. Extracts from chromosomally tagged wt or spe1- (=odc-) strains (Table I) were analysed by anti-myc Western blotting. Both strains were incubated in the presence of the indicated concentrations of spermidine in the media.