The folding of polypeptides emerging from ribosomes was analysed in a mammalian translation system using firefly luciferase as a model protein. The growing polypeptide interacts with a specific set of molecular chaperones, including Hsp70, the DnaJ homologue Hsp40 and the chaperonin TRiC. The ordered assembly of these components on the nascent chain forms a high molecular mass complex that allows the cotranslational formation of protein domains and the completion of folding once the chain is released from the ribosome.

T‐complex polypeptide 1 (TCP‐1) was analyzed as a potential chaperonin (GroEL/Hsp60) equivalent of the eukaryotic cytosol. We found TCP‐1 to be part of a hetero‐oligomeric 970 kDa complex containing several structurally related subunits of 52–65 kDa. These members of a new protein family are assembled into a TCP‐1 ring complex (TRiC) which resembles the GroEL double ring. The main function of TRiC appears to be in chaperoning monomeric protein folding: TRiC binds unfolded polypeptides, thereby preventing their aggregation, and mediates the ATP‐dependent renaturation of unfolded firefly luciferase and tubulin. At least in vitro, TRiC appears to function independently of a small co‐chaperonin protein such as GroES. Folding of luciferase is mediated by TRiC but not by GroEL/ES. This suggests that the range of substrate proteins interacting productively with TRiC may differ from that of GroEL. We propose that TRiC mediates the folding of cytosolic proteins by a mechanism distinct from that of the chaperonins in specific aspects.

The role of the abundant stress protein Hsp90 in protecting cells against stress-induced damage is not well understood. The recent discovery that a class of ansamycin antibiotics bind specifically to Hsp90 allowed us to address this problem from a new angle. We find that mammalian Hsp90, in cooperation with Hsp70, p60, and other factors, mediates the ATP-dependent refolding of heatdenatured proteins, such as firef ly luciferase. Failure to refold results in proteolysis. The ansamycins inhibit refolding, both in vivo and in a cell extract, by preventing normal dissociation of Hsp90 from luciferase, causing its enhanced degradation. This mechanism also explains the ansamycin-induced proteolysis of several protooncogenic protein kinases, such as Raf-1, which interact with Hsp90. We propose that Hsp90 is part of a quality control system that facilitates protein refolding or degradation during recovery from stress. This function is used by a limited set of signal transduction molecules for their folding and regulation under nonstress conditions. The ansamycins shift the mode of Hsp90 from refolding to degradation, and this effect is probably amplified for specific Hsp90 substrates.Exposure of prokaryotic and eukaryotic cells to heat and other stresses induces several classes of highly conserved stress proteins, including the members of the Hsp70, Hsp60, and Hsp90 families (1-3). These proteins are generally thought to act as molecular chaperones in preventing the aggregation of nonnative polypeptides and in aiding their correct folding. Although significant progress has been made in understanding the chaperone mechanisms in de novo protein folding, surprisingly little is known about the role of chaperones under stress conditions. This lack of knowledge is particularly apparent for the Hsp90s, the most abundant constitutively expressed stress proteins in the eukaryotic cytosol. Although Hsp90 can prevent protein aggregation in vitro (4-6) and is required for the survival of yeast at elevated temperature (7), its actual role in protein refolding and repair under stress has remained elusive (2, 8). Instead, current thinking views Hsp90 as part of a specific chaperone system for the conformational maturation and regulation of signal transduction molecules, such as several potentially oncogenic protein kinases and the nuclear receptors of steroid hormones (8-11). In the mammalian cytosol, these proteins are found in heterocomplexes containing Hsp90, Hsp70͞Hsc70, the Hsp70 regulator Hip (p48), p60, various immunophilins, and the small acidic protein p23.A recent study proposed that the benzoquinone ansamycins geldanamycin (GA) and herbimycin A (HA), originally classified as tyrosine kinase inhibitors (12), do not execute their biological effects directly by inhibiting kinase activities, but rather indirectly by acting on Hsp90 (13). Thus, these agents provide a powerful tool to explore the physiological role of Hsp90. We show that Hsp90, in cooperation with Hsc70, p60, and other factors, mediates the refolding of t...