Deletion mapping of the cyclophilin 40 (CyP40) binding domain for heat shock cognate (Hsc)70. (A) The domain structure of CyP40 is shown schematically and includes the acidic domain, the tetratricopeptide repeat domain (shaded) and putative calmodulin (CaM) binding site (black). Also shown are glutathione S-transferase (GST)-bCyP40 WT and CyP40 deletion mutants (Ratajczak and Carrello 1996). GST was fused in-frame to the 5′ end of CyP40 complementary deoxyribonucleic acid fragments. Numbers refer only to CyP40 amino acids. (B) Wild-type bCyP40 and its deletion mutants were immobilized as GST fusion proteins on glutathione-agarose and assayed for Hsc70 binding as described in Figure 1. In each lane, the corresponding bCyP40 construct is identified by the major protein band

 Alignment of tetratricopeptide repeat (TPR) domains of heat shock cognate (Hsc)70–interacting proteins and binding of wild-type and mutant GST-CyP40 185–370 to full-length Hsc70 and heat shock protein (Hsp)90β 527–724. (A) Sequence alignment of the Hsp70-binding TPR1 domain of Hop (residues 4–105), SGT (residues 89–190), and CHIP (residues 26–127) with the TPR units of CyP40 (residues 223–340). All sequences are of human origin. Residues of the 34 amino acid TPR consensus sequence (Sikorski et al 1990) are indicated by motif numbering above the alignment and are shown in bold. Residues that determine chaperone specificity are indicated in white on a gray background. CyP40 residues that were mutated for this study are denoted beneath the alignments and correspond to those associated with the 2-carboxylate clamp (filled circles) and chaperone specificity (open circles). The consensus sequence for Hsc70-interacting proteins was determined on the basis of 3 of 4 residues being identical or conserved in charge (− for Glu or Asp; + for Lys or Arg) or hydrophobic residue (Φ). The consensus for Hsp90-interacting proteins was derived from an alignment of Hsp90-binding TPR domains (TPR2A of Hop, PP5, FKBP51, FKBP52, CyP40, TOM34, TOM70, and CNS1, see Figure 3 Scheufler et al 2000) and was determined on the basis of 5 of 8 conforming residues. (B) Wild-type or mutant GST-hCyP40 was incubated with full-length Hsc70 or hHsp90β 527–724 bound to the wells of a microtiter plate, and the amount of bound CyP40 was detected by incubating with goat anti-GST antibody followed by rabbit anti-goat IgG antibody conjugated to horseradish peroxidase and 3,3′,5,5′-tetramethylbenzidine as substrate. Binding is expressed as a percentage relative to that of wild-type CyP40 185–370 and represents the mean (bars, ±SE) for at least 3 separate assays, each assay being performed in triplicate. Asterisks denote a significant difference (P < 0.05) in percent relative binding of mutants to Hsc70 and hHsp90β 527–724. CyP40, cyclophilin 40; GST, glutathione S-transferase; SGT, small glutamine-rich TPR protein

 Determination of a mutually exclusive interaction of heat shock protein (Hsp)90 and heat shock cognate (Hsc)70 with cyclophilin 40 (CyP40). Ni-NTA agarose was charged with His-tagged Hsp90β and mixed 1:1 with Sepharose 4B. Bacterial lysate (150 μL) for wild-type bCyP40 was added to the diluted gel (40 μL), and after dilution to 500 μL total volume with binding buffer containing 35 mM imidazole and 0.2% Triton X-100, the mixture was rotated at 4°C for 3 hours. After removal of unbound proteins by successive washing, the gel containing Hsp90-bound CyP40 was exposed to 0, 100, 200, and 400 μL of induced Hsc70 bacterial lysate. Reaction volumes were brought to 500 μL with binding buffer containing imidazole and Triton X-100, and incubation was continued with rotation at 4°C for 3 hours. Gel-retained proteins were recovered with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and analyzed by SDS-PAGE with Coomassie blue staining. Glutathione-agarose containing immobilized GST-bCyP40 was mixed 1:5 with Sepharose 4B. The diluted gel (40 μL) was incubated as described above with 400 μL of Hsc70 lysate and gel-retained proteins were analyzed by SDS-PAGE. GST, glutathione S-transferase

 Interaction of cytosolic heat shock protein (Hsp)70 and recombinant heat shock cognate (Hsc)70 with the C-terminal domain of cyclophilin 40 (CyP40). (A) Glutathione-agarose (40 μL) charged with glutathione S-transferase (GST)-bCyP40 185–370 (see Fig 2A) was incubated with rotation for 6 hours at 4°C with bovine myometrial cytosol (200 μL) prepared in binding buffer. After centrifugation, the gel was washed eight times with binding buffer, the first 4 washes containing 0.2% vol/vol Triton X-100. Retained proteins (lane 2) were extracted from the gel with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and were analyzed by SDS-PAGE on a 10% wt/vol polyacrylamide gel followed by Coomassie blue staining. Fusion protein-charged gel not exposed to cytosol was used as control (lane 1). (B) A parallel study was performed as in (A) except that proteins separated by SDS-PAGE were electrotransferred to nitrocellulose membrane (Amersham Boisciences) and immunoblotted with N27 (Hsp70) monoclonal antibody. (C) GST and GST-bCyP40 185–370 were immobilized on glutathione-agarose and the protein-charged gels (40 μL) were incubated as described in (A) with bacterial lysate (400 μL) containing recombinant Hsc70. Contaminating proteins were removed by washing, and retained proteins were analyzed by SDS-PAGE on a 12.5% polyacrylamide gel followed by Coomassie blue staining. Proteins recovered with GST (control) are shown in lane 2. Protein molecular weight markers (Pharmacia) are shown on the left (panel A) and on the right (panel C). BPB, bromophenol blue

 Determination of preferential binding of cyclophilin 40 (CyP40) for heat shock protein (Hsp)90 over heat shock cognate (Hsc)70. Bacterial lysate containing recombinant Hsc70 was supplemented with chelate-purified, His-tagged Hsp90β to give extracts with Hsp90 to Hsc70 ratios of 1:1, 1:5, and 5:1. Aliquots (50 μL) of glutathione-agarose containing immobilized GST-bCyP40 185–370 fusion protein were incubated with the mixed Hsp90-Hsc70 preparations and analyzed for Hsp90 and Hsc70 retention by SDS-PAGE as described in Figure 1. Hsp90-Hsc70 ratios were: lane 1, 1:1; lane 2, 1:5; lane 3, 5:1; lane 4, neat Hsc70; lane 5, neat Hsp90. GST, glutathione S-transferase; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis

 Competition of FKBP52 and Hop for heat shock cognate (Hsc)70 and heat shock protein (Hsp)90 binding by cyclophilin 40 (CyP40). (A) Hsc70 bacterial lysate (100 μL) was incubated at 4°C for 3 hours in the presence and absence of purified recombinant FKBP52. Glutathione-agarose charged with glutathione S-transferase (GST)-bCyP40 185–370 fusion protein was added to each mixture and after rotation for additional 5 hours at 4°C retained proteins were determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis as described. Binding to Hsp90 in the presence of FKBP52 was investigated under identical conditions. Lane 1, neat Hsc70; lane 2, Hsc70 plus FKBP52; lane 3, neat Hsp90; lane 4, Hsp90 plus FKBP52. (B) Lysates (2 μL) from uninduced and isopropyl b-d-thiogalactoside–induced bacterial cultures overexpressing recombinant human Hop were submitted to SDS-PAGE and proteins were observed by Coomassie blue staining. (C) Induced Hop lysate was added in 0, 10, 20, 50, 100, and 200 μL aliquots to separate tubes containing Hsc70 bacterial lysate (200 μL), and all volumes were adjusted to 500 μL with binding buffer. After brief mixing, the extracts were allowed to stand at 4°C for 3 hours. Glutathione-agarose (50 μL) containing immobilized GST-bCyP40 fusion protein was added to each tube and the mixtures were incubated with rotation for an additional 5 hours at 4°C. Gel-retained proteins were extracted with SDS-PAGE sample buffer and analyzed by SDS-PAGE with Coomassie blue staining. A parallel study with 200 μL of uninduced Hop lysate in Hsc70 bacterial lysate was conducted as control. (D) Induced Hop bacterial lysate was added in 0, 0.02, 0.05, 0.1, 0.2, 0.5, and 1.0 mL aliquots to tubes containing extracts (150 μL) of purified, recombinant Hsp90. Total volumes were adjusted to 1.2 mL and after preincubation for 3 hours at 4°C the mixtures were rotated with glutathione-agarose (50 μL) charged with GST-bCyP40 fusion protein. Gel-retained proteins were determined by SDS-PAGE as described. Results from a parallel study using 1.0 mL of uninduced Hop bacterial lysate served as control. (E) After quantitation of Hsc70 and GST-bCyP40 protein in (C) by densitometric scanning, the amount of Hsc70 bound to gel-immobilized GST-bCyP40 was expressed relative to that observed before the addition of induced Hop lysate as control (100%). (F) Hsp90 and GST-bCyP40 proteins in (D) were quantitated by densitometry, and retained Hsp90 was expressed relative to that bound in the absence of induced Hop lysate as control (100%)

 Influence of cyclophilin 40 (CyP40) on heat shock protein (Hsp)70 adenosine triphosphatase (ATPase) function. The hydrolysis of ³²P-labeled adenosine triphosphate (ATP) by Hsp70 (2 μM) was determined in the absence (−) or presence (+) of Ydj-1 (200 nM), with or without purified human CyP40 at molar ratios equivalent to one, two, or four times the heat shock protein (Hsp)70 concentration. After thin-layer chromatography of the reaction mixture, the ratio of adenosine 5′ diphosphate and ATP was quantitated by phosphorimaging and used to calculate the level of ATP hydrolysis. ATPase activity is expressed in micromoles per minute. The data represent the means (bars, ±SE) of results from 3 separate experiments