In vivo newly translated polypeptides are sequestered in a protected folding environment

Molecular chaperones play a fundamental role in cellular protein folding. U sing intact mammalian cells we examined the contribution of cytosolic chape rones to de novo folding. A large fraction of newly translated polypeptides associate transiently with Hsc70 and the chaperonin TRiC/CCT during their biogenesis, The substrate repertoire observed for Hsc70 and TRiC is not ide ntical: Hsc70 interacts with a wide spectrum of polypeptides larger than 20 kDa, while TRiC associates with a diverse set of proteins between 30 and 6 0 kDa. Overexpression of a bacterial chaperonin 'trap' that irreversibly ca ptures unfolded polypeptides did not interrupt the productive folding pathw ay. The trap was unable to bind newly translated polypeptides, indicating t hat folding in mammalian cells occurs without the release of non-native fol ding intermediates into the bulk cytosol. We conclude that ne novo protein folding occurs in a protected environment created by a highly processive ch aperone machinery and is directly coupled to translation.