Figure 3.

Effect of Ydj1 point mutations on His-Ste11ΔN-K444R interaction. Strain JJ160 expressing indicated ydj1 mutant was transformed with a plasmid constitutively expressing His-Ste11ΔN-K444R or a control plasmid. (A) Extracts were prepared from cells that lacked a His-tagged protein (−) or from those expressing His-Ste11ΔN-K444R (+). Equal amounts of cell lysates were separated by SDS-PAGE followed by immunoblot analysis to detect His-Ste11ΔN-K444R and Ydj1. (B) Cell extracts were incubated with nickel resin for 1 h at 4°C. In general, His-Ste11ΔN-K444R complexes were isolated as described for Figure 2. The exception was that in the experiment with C159T and C143SC162S, complexes bound to nickel resin were eluted with 200 mM imidazole (three times, 2-min washes) and then precipitated with TCA to reduce background binding to the nickel resin. Samples were analyzed by immunoblot as described in Figure 2.