High-molecular-weight FK506-binding proteins are components of heat-shock protein 90 heterocomplexes in wheat germ lysate

Abstract

In animal cell lysates the multiprotein heat-shock protein 90 (hsp90)-based chaperone complexes consist of hsp70, hsp40, and p60. These complexes act to convert steroid hormone receptors to their steroid-binding state by assembling them into heterocomplexes with hsp90, p23, and one of several immunophilins. Wheat germ lysate also contains a hsp90-based chaperone system that can assemble the glucocorticoid receptor into a functional heterocomplex with hsp90. However, only two components of the heterocomplex-assembly system, hsp90 and hsp70, have thus far been identified. Recently, purified mammalian p23 preadsorbed with JJ3 antibody-protein A-Sepharose pellets was used to isolate a mammalian p23-wheat hsp90 heterocomplex from wheat germ lysate (J.K. Owens-Grillo, L.F. Stancato, K. Hoffmann, W.B. Pratt, and P. Krishna [1996] Biochemistry 35: 15249-15255). This heterocomplex was found to contain an immunophilin(s) of the FK506-binding class, as judged by binding of the radiolabeled immunosuppressant drug [3H]FK506 to the immune pellets in a specific manner. In the present study we identified the immunophilin components of this heterocomplex as FKBP73 and FKBP77, the two recently described high-molecular-weight FKBPs of wheat. In addition, we present evidence that the two FKBPs bind hsp90 via tetratricopeptide repeat domains. Our results demonstrate that binding of immunophilins to hsp90 via tetratricopeptide repeat domains is a conserved protein interaction in plants. Conservation of this protein-to-protein interaction in both plant and animal cells suggests that it is important for the biological action of the high-molecular-weight immunophilins.

Figure 4

Inhibition of p23-hsp90 heterocomplex formation by geldanamycin in a concentration-dependent manner. A, Protein-antibody complex visualized using an enhanced chemiluminescence detection system. Lane 1, Commercial wheat germ lysate (0.25 μL). Lanes 2 through 7, Wheat germ lysate (30 μL) preincubated for 10 min at 30°C with 6 μL of DMSO (lane 2) or with geldanamycin at a concentration of 0.5 μg/mL (lane 3), 1.0 μg/mL (lane 4), 1.5 μg/mL (lane 5), 2.0 μg/mL (lane 6), or 2.5 μg/mL (lane 7) in a final volume of 6 μL of DMSO. The samples were then incubated with immunoadsorbed p23 for 30 min at 30°C. After the pellets were washed, proteins were extracted in 60 μL of SDS sample buffer, and an aliquot of 20 μL was separated on a 11% SDS-polyacrylamide gel. Following transfer to nitrocellulose membrane, hsp90 and FKBP73 were detected by western blotting. Lanes 8 and 9 are results of a similar experiment in which incubation of immunoadsorbed p23 with geldanamycin (10 μg/mL) containing wheat germ lysate was at either 30°C (lane 8) or 0°C (lane 9). B, Proteins visualized by silver staining. Lanes 1 and 2 are same as lanes 8 and 9, respectively, in A.

Figure 5

FKBP77 is also a component of the mammalian p23-plant hsp90 heterocomplex. Protein A-Sepharose (50 μL) was coated with 10 μL of JJ3 ascites followed by adsorption of p23. Immunoadsorbed p23 was incubated with 50 μL of wheat germ lysate. After the pellets were washed, proteins were extracted in SDS sample buffer and separated on a 7.5% SDS-polyacrylamide gel. FKBP77 was detected by western blotting. Lane 1, Wheat germ lysate; lane 2, nonimmune IgG-bound protein A-Sepharose incubated first with p23 and then with wheat germ lysate; lane 3, JJ3-bound protein A-Sepharose incubated directly with wheat germ lsyate; lane 4, JJ3-bound-protein A-Sepharose incubated first with p23 and then with wheat germ lysate; lane 5, immunoadsorbed p23 incubated with wheat germ lysate mixed with 15 μL of insect cell lysate without the TPR domain; lane 6, immunoadsorbed p23 incubated with wheat germ lysate mixed with 15 μL of insect cell lysate containing the TPR domain; lane 7, immunoadsorbed p23 incubated with wheat germ lysate in the presence of geldanamycin (10 μg/mL) at 30°C; lane 8, same as lane 7 but incubation was at 0°C.

Figure 1

Mammalian p23-plant hsp90-plant FKBP73 heterocomplex formation. Protein A-Sepharose (40 μL) was coated with 8 μL of JJ3 ascites followed by immunoadsorption of p23. The immunoadsorbed p23 was incubated with 30 μL of wheat germ lysate for 30 min at 30°C. After the pellets were washed, proteins were extracted in 50 μL of SDS sample buffer. Proteins (30 μL of the sample) were separated on a 7.5% SDS-polyacrylamide gel and then transferred to a nitrocellulose membrane by electroblotting. hsp90 and FKBP73 were detected using R₂ and anti-wheat FKBP73 antisera, respectively. An aliquot (7 μL) of the sample containing extracted proteins was run on a 12.5% SDS-polyacrylamide gel. After transfer to a nitrocellulose membrane, p23 was detected using the JJ3 antibody. Lane 1, Wheat germ lysate; lane 2, nonimmune IgG-bound protein A-Sepharose incubated first with p23 and then with wheat germ lysate; lane 3, JJ3-bound protein A-Sepharose incubated directly with wheat germ lsyate; lane 4, JJ3-bound-protein A-Sepharose incubated first with p23 and then with wheat germ lysate.

Figure 2

Rat PP5 TPR domain competes for wheat FKBP73 binding to wheat hsp90. A, Protein A-Sepharose beads (40 μL) were coated with 8 μL of JJ3 ascites followed by adsorption of p23. The immunoadsorbed p23 was incubated with wheat germ lysate either without addition of insect cell lysate or with addition of insect cell lysate containing the expressed rat PP5 TPR domain. After the pellets were washed, proteins were extracted in SDS sample buffer, resolved on a 7.5% polyacrylamide gel, transferred to nitrocellulose membrane, and analyzed for hsp90 and FKBP73 by western blotting. Lane 1, Wheat germ lysate; lane 2, immunoadsorbed p23 incubated with 30 μL of wheat germ lysate; lanes 3 to 7, immunoadsorbed p23 incubated with wheat germ lysate mixed with 2, 4, 6, 8, and 10 μL, respectively, of insect cell lysate containing the expressed TPR domain; lane 8, immunoadsorbed p23 incubated with 50 μL of wheat germ lysate mixed with 20 μL of wild-type insect cell lysate without TPR domain; lane 9, immunoadsorbed p23 incubated with wheat germ lysate mixed with 20 μL of insect cell lysate containing the TPR domain. B, Insect cell lysates (0.5, 1.0, 2.0, and 3.0 μL) without the TPR domain (wild type [WT]) and with the TPR domain were subjected to electrophoresis on a 12.5% SDS-polyacrylamide gel. Proteins were visualized by Coomassie blue staining. The arrow indicates the expressed TPR domain of PP5 only in the lysates of cells infected with the recombinant baculovirus.

Figure 3

Wheat proteins coimmunoadsorbed with human p23. Samples were purified from 50 μL of wheat germ lysate using p23 immunoadsorbed with JJ3 IgG prebound to 60 μL of protein A-Sepharose. Proteins were extracted in SDS sample buffer, separated on a 11% SDS-polyacrylamide gel, and visualized by silver staining of the gel. Lane 1, One microliter of wheat germ lysate; lane 2, nonimmune IgG-bound protein A-Sepharose incubated with p23 and then with wheat germ lysate; lane 3, JJ3-bound protein A-Sepharose incubated directly with wheat germ lysate; lane 4, JJ3-bound protein A-Sepharose incubated first with p23 and then with wheat germ lysate; lanes 5 and 6 are the same as lane 4 with the exception that the wheat germ lysate was mixed with 20 μL of insect cell lysate without PP5 TPR domain (lane 5) or with lysate containing the expressed TPR domain (lane 6). HC, Heavy chain; LC, light chain.