Abstract
We have shown recently that the immunophilins CyP-40 and FKBP52/hsp56 bind to a common site on hsp90 and that they exist in separate heterocomplexes with the glucocorticoid receptor (GR). FKBP52/hsp56 binds to hsp90 via its tetratricopeptide repeat (TPR) domains, it is not required for GR.hsp90 heterocomplex assembly, and it is thought to play a role in targeted movement of the GR. In this work we examine the hsp90 binding of four proteins (FKBP52/hsp56, CyP-40, p50, Mas70p) thought to be involved in targeted protein trafficking. FKBP52/hsp56 and CyP-40 (each with three TPRs), localize to the nucleus and nucleoli, respectively, and form relatively weak complexes with hsp90 that are competed by a CyP-40 fragment containing its three TPRs. The p50 component of the Src.hsp90 and Raf.hsp90 heterocomplexes localizes to cytoskeletal fibers extending from the perinuclear region to the plasma membrane and forming a rim under the plasma membrane of endothelial cells. p50, Mas70p (seven TPRs), which is a receptor for mitochondrial import, and the p60 (six to eight TPRs) component of the steroid receptor.hsp90 heterocomplex assembly system bind very tightly to hsp90 in a manner that is not competed by the CyP-40 fragment. However, bacterially expressed p60 blocks the binding of p50, Mas70p, FKBP52/hsp56, and CyP-40 to purified hsp90. The data are consistent with binding of all of these proteins to a site on hsp90 that is a general TPR domain acceptor. Our localization and binding data are used to develop a model in which proteins that are chaperoned by hsp90 move as dynamic complexes to their cellular sites of action, with the TPR-containing protein participating in targeting the movement of the complexes.
Highlights
Little is known about how proteins that are not conveyed by a vesicle-based protein trafficking system move through the cytoplasm to arrive at their sites of action in organelles, such as the nucleus or mitochondria, or at a cellular locus like the
We have shown recently that the immunophilins CyP-40 and FKBP52/hsp56 bind to a common site on hsp90 and that they exist in separate heterocomplexes with the glucocorticoid receptor (GR)
In 1992, we proposed that the receptors shuttle through the cytoplasm in the heterocomplex form, with hsp90 and the immunophilin acting as a protein transport unit or transportosome [15]
Summary
Untreated rabbit reticulocyte lysate was from Green Hectares (Oregon, WI). 125I-Conjugated goat anti-mouse and anti-rabbit IgGs were from DuPont NEN. 125I-Conjugated goat anti-mouse and anti-rabbit IgGs were from DuPont NEN. Trypsin, powdered Dulbecco’s modified Eagle’s medium (high glucose), goat anti-mouse IgG-horseradish peroxidase conjugate, monoclonal nonimmune IgG and IgM, nonimmune rabbit serum, TUB2.1 monoclonal anti--tubulin IgG, antinucleolar antibody (nucleolar positive control) and the fluorescein isothiocyanate (FITC)-conjugated antihuman IgG were from Sigma. Goat anti-mouse IgM, donkey anti-rabbit IgG-horseradish peroxidase conjugate, and protein A-agarose were from Pierce. The anti-cyclophilin 40 (COOH-terminal peptide) antibody and the 3G3 monoclonal anti-hsp IgM were from Affinity BioReagents (Golden, CO). FITC-conjugated donkey anti-mouse IgG and IgM and rhodamine-conjugated donkey anti-rabbit IgG were from Jackson ImmunoResearch Laboratories (West Grove, PA). The UPJ56 rabbit antiserum against FKBP52/hsp56 [58] was a gift from Drs Karen Leach and Martin Deibel (The Upjohn Co.). 4059 is a bacterially expressed human CyP-40 COOH-terminal fragment containing the FKBP52-like TPR domain, but not the CyP-18-like domain, and it was purified by Ni2ϩ affinity chromatography and thrombin cleavage.
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