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Fig. 2. Analysis of the TPR1 interaction with HSP90 and VAP-33 by gel filtration chromatography and surface plasmon resonance. (A) Purified HSP90, TPR1 and VAP-33, lacking its membrane anchor, were incubated as indicated in the presence or absence of ATPS, separated by gel filtration chromatography on a Superose 6 column and fractions analyzed by SDS-PAGE. Elution profiles of marker proteins are shown on top (thyroglobulin, 669 kDa; ferritin, 440 kDa; catalase 232 kDa; bovine serum albumin, 67 kDa). *, a degradation product of the HSP90 preparation. (B) Tpr1unique binds to VAP-33MSP. Tpr1unique was incubated with either VAP-33 lacking its membrane anchor, or VAP-33MSP, and the mixtures were run on a Superdex 75 column. Co-migration indicated complex formation between Tpr1unique and VAP-33 or VAP-33MSP. Marker proteins are shown on top (bovine serum albumin, 67 kDa; ribonuclease A, 14 kDa). (C) 12-mer peptides containing the C-terminal sequence of HSP90 (90C-12) were covalently coupled to a BiaCore chip. The binding kinetics of TPR1 in the concentration range of 0.1 to 60 µM were tested, and the relative response units during the equilibrium phase of binding to 90C-12 were plotted against TPR1 concentrations. (D) Increasing concentrations (0.1 to 100 µM) of 90C-12 in solution were used to compete for binding of TPR1 to immobilized 90C-12. Binding of TPR1 to HSP90 peptides could be competed using free 12mer peptides from HSP90 (90C-12) but not the control peptide SKL. (E) Cow brain lysate was incubated with or without an HSP90-specific antibody. After pulldown with ProteinG beads, immunoprecipitates were analyzed with antibodies against HSP90 and VAP-33. Antibody reactivity using 1% or 10% of the input (brain lysate) is shown as a standard.

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