Fig 2: Hsp70 mutants show increased affinity to Ydj1.(A) Purified His6-Ydj1 was immobilized over Co2+-NTA beads and incubated with cellular lysate from indicated strains. The bound proteins were eluted and probed with anti-Hsp70, anti-Hsp90 and anti-Ydj1 antibodies. (B) BLI studies to monitor interaction between Ssa Hsp70 and Ydj1. Ydj1 (0.75µM) was immobilized over the biosensor surface, and Ssa Hsp70 (wt Ssa1 or its mutants) was used as analyte. The binding was monitored in the presence of ATP.

Fig 3: Ssa4-T175N and Ssa4-D158N support cellular growth in absence of Sti1.(A) Strains expressing A1, A2, A3 or A4 (or their respective T175N and D158N mutants) in wt and sti1? background were patched onto FOA plates. Shown here is growth after 8 days of incubation at 30°C. (B) Hsp70 (Ssa1, Ssa2, Ssa3 and Ssa4) and Ydj1 interaction was monitored using BLI. Black, Red, Green and Blue represent Ssa1, Ssa2, Ssa3 and Ssa4 respectively.

Fig 4: Model showing regulation Hsp90 binding to Hsp70 by Ydj1.Misfolded Hsp90 client protein interacts with Ydj1. Ydj1 delivers client proteins to Hsp70. Absence of Hsp90 interaction with Hsp70 mutants due to their higher affinity with Ydj1 results in client aggregation. Red arrow represents lack of Hsp90 interaction with Ssa1 mutants.