FIGURE 9.

Phosphorylated and dephosphorylated forms of Pah1 are not degraded by the 26S proteasome, but dephosphorylation of Pah1 by the Nem1-Spo7 phosphatase complex stimulates degradation by the 20S proteasome. Pah1, which is endogenously phosphorylated in S. cerevisiae (18), was dephosphorylated by the Nem1-Spo7 protein phosphatase. The phosphorylated (left) and dephosphorylated (right) forms of Pah1 (30 nm each) were incubated with 2 nm 26S proteasome plus 1 mm ATP (A) or the 20S proteasome (2 nm) plus SDS (0.02%) (B) with or without 50 μm MG132 for the indicated time intervals. Following the incubations, samples were subjected to SDS-PAGE and Western blotting with anti-Pah1 antibodies. The Western blots shown are representative of three independent experiments. The position of Pah1 is indicated in the figure. B, bottom, the relative amounts of Pah1 in B from incubations without MG132 were quantified using ImageQuant software. The data shown are means ± S.D. (error bars) from triplicate determinations.