Exploring the Functional Complementation between Grp94 and Hsp90
Fig 5
Grp94/Hsp90 chimeras interact with Hsp90 co-chaperones.
(A) Cartoon summary of Hsp90:cochaperone binding loci. (B) Ni-NTA pull-down assay shows that His6-Cdc37 interacts with hspNM1-grpM2C and Hsp90 but not grpN-hspMC; His6-Hop interacts with grpN-hspMC and Hsp90 but not hspNM1-grpM2C; His6-Aha1 interacts with hspNM1-grpM2C, Hsp90, and partially with grpN-hspMC. Equal amounts of untagged chaperone and His-tagged cochaperone were mixed together and incubated for 1 h at 4°C followed by overnight incubation with Ni-NTA resin. The Load sample was removed prior to the addition of Ni-NTA resin. Unbound protein was removed by sequential washes of buffer containing 20 mM imidazole. Bound proteins and protein complexes were eluted with buffer containing 300 mM imidazole and resolved in parallel with the Load sample on SDS-PAGE gels. (C) hspNM1-grpM2C ATPase activity is stimulated 5-fold by Aha1. Wild-type Hsp90 is stimulated 10-fold whereas grp-hspMC is stimulated a modest 1.5-fold (D). ATPase activity was monitored by a NADH coupled enzymatic assay system, which measures the consumption of NADH as a function of ADP released by Hsp90s. Assays were measured with or without the addition of cochaperone Aha1 at a 1:2 molar ratio of hsp90:Aha1. Wild-type Aha1 alone did not exhibit ATPase activity (not shown).
