FIGURE 6.

Enforced N-domain proximity of Hsp90 is compatible with essential function in yeast. A, plasmid swap experiments demonstrate that coilNMC is able to support yeast viability as the sole Hsp90 when the wild-type copy is selected against using FOA. Cells were plated in 5-fold dilutions grown at 25 °C for 3 days. B, Western blotting demonstrates that coilNMC expresses to similar levels as NMC in yeast. To control for loading a separate gel was run and stained with Coomassie and a region corresponding to about 60–80 kDa is shown. C, GR activity in yeast cells induced with the GR agonist deoxycorticosterone with NMC or coilNMC as the sole Hsp90. The coilNMC construct supports GR activity that is reduced relative to NMC. D, maturation of the v-Src kinase in yeast cells with NMC or coilNMC as the sole Hsp90. The coilNMC construct supports v-Src activity albeit at reduced levels relative to NMC. The Hsp90 inhibitor radicicol blocks both GR and v-Src activation demonstrating that these processes are Hsp90-dependent.