| Chaperonins are multisubunit, toroidal-shaped complexes that assist the cellular folding of a subset of newly synthesized cellular polypeptides. In eukaryotes, little is known about the identity of these diverse client proteins, and likewise, the structural features of substrate proteins that confer recognition by chaperonins are as yet poorly defined. We have identified the von Hippel-Lindau (VHL) tumor suppressor protein as a novel substrate of the eukaryotic cytosolic chaperonin 1, TRiC (T&barbelow;CP-1 containing ring c&barbelow;omplex). TRiC is required for the formation of correctly folded VHL protein as well as the incorporation of VHL into a larger holocomplex with its physiological partner proteins, Elongin B and Elongin C. Interaction of VHL with TRiC is directly mediated by discrete binding determinants, each enriched in hydrophobic residues, present within a larger, 55-amino acid region in VHL that is both necessary and sufficient for engaging the chaperonin. Many naturally-occurring, tumorigenic mutations are located adjacent to or within the chaperonin binding sites. We found that some tumor-derived point mutations disrupt the normal association of VHL with TRiC, resulting in misfolding and diminished incorporation of VHL into the Elongin BC complex. Interestingly, several tumorigenic missense alleles can refold to the native state upon dilution from a denaturant. Hence the misfolding observed in these VHL alleles upon cellular translation may arise from the loss of a critical chaperone-substrate interaction operative in the de novo folding pathway of VHL. Collectively, these findings define the structural basis for the interaction of a novel substrate with the cytosolic chaperonin. Further, these findings demonstrate that loss of protein function may arise through the impairment of a critical chaperone-substrate interaction essential for the correct cellular folding of a nascent polypeptide. |
