Fig 1.
Domains and functional regions of hsp90 chaperones.
The structure of Grp94 (PDB 2o1v) is shown. A) Schematic organization. B) Cartoon representation of the Grp94 dimer. C) surface representation of the Grp94 monomer.
Fig 2.
Chimeric constructs used in this study.
Schematic representation showing the domain composition of Hsp90, Grp94, and the fragments used to create chimeras of Hsp90 and Grp94. Boundaries are shown with residue numbering for yeast Hsp90 and canine Grp94. The N-terminal chimeras referred to as grpN-hspMC (4), hspN-grpMC (3) and hspNM-grpM2C (9) were previously described in Ref. [20].
Fig 3.
Select Grp94 domains support yeast viability.
(A) Plasmid shuffle experiments demonstrate grpN-hspMC and hspNM1grpM2C are able to support yeast viability when expressed as the sole Hsp90. Cells were plated on FOA plates as 5-fold serial dilutions and were grown at the indicated temperatures for 3–9 days. Images are from day 4. Green highlights indicate positive complementation. (B) Western blot of yeast whole cell lysates. 20 μg of total protein extracted from the lysis of plasmid-transformed yeast cells grown in SD–Ura -Trp was separated by SDS-PAGE. The expression level of plasmid-expressed hsp90 chimeras was evaluated by Western blotting against the N-terminal His6 epitope tag. Lysate from Chimera 1 (yeast Hsp90) was included on each blot as a transfer and antibody control. Coomassie blue stained PVDF membranes used for blotting are displayed below their respective luminographs to allow comparison of total protein levels.
Table 1.
Relative ATPase rates of Grp94/Hsp90 chimeras.
Fig 4.
Growth in liquid media of ECU82a expressing yeast Hsp90, grpN-hspMC, human Hsp90a, hspNM1grpM2C, and hspNM1grpM2C-hspCx as its sole Hsp90.
Cultures were started at a cell density of 3.5 x 106 cells per mL in YPD medium (time 0) and incubated with shaking at 30°C. Data represents an average OD600 from two yeast clones.
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).
Fig 6.
GRP94 client expression with Grp94/Hsp90 chimeras.
A) Schematic representation of constructs tested. All constructs contained the Grp94 signal sequence (ss), the preN-domain, and the KDEL ER retention sequence found in the Grp94 C-terminal extension (Cx). The FLAG tag is shown as a red bar. B) Detail of the FLAG tag incorporated into each tested construct. C) EGFP gating and intracellular staining of Grp94-null E4.126 cells virally transduced with Grp94 constructs. Top panel: dot plot of the side scattering intensity (SSC-A, Y-axis) of analyzed cells (a measure of cell size and clustering) vs. the fluorescence of the co-expressed EGFP (X-axis). The number of events is represented by the color of the dots. The dots in the magenta box correspond to EGFP positive cells and the numbers show the percentage of all cells that were assigned to this gate. Lower panel: Histogram of the normalized cell count vs. conjugated anti-FLAG (blue) or isotype control (red) fluorescence. Fluorescence intensity is proportional to the copy number of the antibody target. Numbers represent the mean fluorescence intensity of the blue histograms. D) Cell surface expression staining of Grp94 clients as a function of virally transduced Grp94 construct. Cells were gated for EGFP expression as in C). Red = isotype control antibody histogram, blue = anti-client antibody histogram. The mean fluorescence intensity of the blue histogram is indicated.
Fig 7.
The M1 domain of Grp94 and Hsp90 differ in their surface residues.
A) Alignment of the M1 lobe of the M domains of Grp94, yeast Hsp90, and human Hsp90. Grp94 residues highlighted in yellow differ from the yeast and human Hsp90 consensus. Purple boxes indicate residues shown to be mutagenically sensitive in yeast Hsp90 for client maturation. Green boxes indicate residues of human Hsp90 that interact with Cdc37 in the cryo-EM structure of Cdk4-Cdc37-Hsp90 (PDB code 5fwl). Asterisks and numbers (Grp94 numbering) highlight Cdc37-interacting residues that differ between Hsp90 and Grp94. B) Mapping the unique residues onto the structure of the Grp94 dimer. PDB code 2o1v was used. Residues highlighted in yellow in A) are shown as sticks or spheres on the on protomers A and B, respectively. Green colored residues correspond to residues highlighted with asterisks in panel A.
Table 2.
Solvent Accessible Surface Area of Domain Interfaces.