FIGURE 1:

Phosphorylation of Tom40 by yeast PKA. (A) Yeast cells from rho⁺ wild-type (WT), rho⁻ wild type, and bcy1Δ were grown on sucrose-containing medium at 30°C. Protein extracts were prepared by postalkaline lysis and analyzed by SDS–PAGE and Western blotting. Alo1, d-arabinono-1,4-lactone oxidase; Pgk1, 3-phosphoglycerate kinase. (B) Yeast PKA (Bcy1-Tpk) was purified by affinity chromatography using a Bcy1_ProtA yeast strain as described in Materials and Methods. Total (yeast lysate) and TEV eluate were analyzed by SDS–PAGE and Western blotting. Total, 10%; eluate, 100%. Cka1, Cka2, catalytic subunits of CK2; Ssa1, cytosolic member of heat shock protein 70 family. (C) Kemptide, the cytosolic domain of Tom70, and recombinant Tom40 (WT and S54E mutant form) were incubated with purified yeast PKA (Bcy1-Tpk) and [γ-³³P]ATP in the presence of 8Br-cAMP as indicated. The samples were analyzed by SDS–PAGE, digital autoradiography, and staining with Coomassie brilliant blue R-250. Arrowhead, bovine serum albumin. (D) Recombinant Tom40^WT and Tom40^S54E were incubated with affinity-purified yeast Tpk1 as indicated and [γ-³³P]ATP. The samples were analyzed as described for C.