Search

Studies on Hsp90 have mainly focused on its involvement in the activation of several families of protein kinases and of steroid hormone receptors. Little is known regarding the role of Hsp90 in the folding of nascent proteins. We previously reported that Hsp90 plays an active role in the posttranslational assembly of the C-terminal globular head of the reovirus attachment protein ς1. We show here that Hsp90 becomes phosphorylated in this process. However, only the unphosphorylated form of Hsp90 is complexed with ς1, suggesting that Hsp90 phosphorylation is coupled to the release of the chaperone from the target protein. Geldanamycin, which blocks ς1 maturation by preventing the release of Hsp90 from ς1, also inhibits Hsp90 phosphorylation. Taken together, these results demonstrate that Hsp90 phosphorylation is linked to its chaperoning function.

The reovirus cell attachment protein, sigma1, is a lollipop-shaped homotrimer with an N-terminal fibrous tail and a C-terminal globular head. Biogenesis of this protein involves two trimerization events: N-terminal trimerization, which occurs cotranslationally and is Hsp70/ATP-independent, and C-terminal trimerization, which occurs posttranslationally and is Hsp70/ATP-dependent. To determine if Hsp90 also plays a role in sigma1 biogenesis, we analyzed sigma1 synthesized in rabbit reticulocyte lysate. Coprecipitation experiments using anti-Hsp90 antibodies revealed that Hsp90 was associated with immature sigma1 trimers (hydra-like intermediates with assembled N termini and unassembled C termini) but not with mature trimers. The use of truncated sigma1 further demonstrated that only the C-terminal half of sigma1 associated with Hsp90. In the presence of the Hsp90 binding drug geldanamycin, N-terminal trimerization proceeded normally, but C-terminal trimerization was blocked. Geldanamycin did not inhibit the association of Hsp90 with sigma 1 but prevented the subsequent release of Hsp90 from the immature sigma1 complex. We also examined the status of p23, an Hsp90-associated cochaperone. Like Hsp90, p23 only associated with immature sigma1 trimers, and this association was mapped to the C-terminal half of sigma1. However, unlike Hsp90, p23 was released from the sigma1 complex upon the addition of geldanamycin. These results highlight an all-or-none concept of chaperone involvement in different oligomerization domains within a single protein and suggest a possible common usage of chaperones in the regulation of general protein folding and of steroid receptor activation.

The C-terminal globular head of the lollipop-shaped final sigma1 protein of reovirus is responsible for interaction with the host cell receptor. Like the N-terminal fibrous tail, it has its own trimerization domain. Whereas N-terminal trimerization (formation of a triple alpha-helical coiled coil) occurs at the level of polysomes (i.e. cotranslationally) and is ATP-independent, C-terminal trimerization is a posttranslational event that requires ATP. Coprecipitation experiments using anti-Hsp70 antibodies and truncated final sigma1 proteins synthesized in vitro revealed that only regions downstream of the N-terminal alpha-helical coiled coil were associated with Hsp70. Hsp70 was also found to be associated with nascent final sigma1 chains on polysomes as well as with immature postribosomal final sigma1 trimers (hydra-like intermediates with assembled N termini and unassembled C termini). These latter structures were true intermediates in the final sigma1 biogenetic pathway since they could be chased into mature final sigma1 trimers with the release of Hsp70. Thus, unlike N-terminal trimerization, C-terminal trimerization is Hsp70- and ATP-dependent. The involvement of two mechanistically distinct oligomerization events for the same molecule, one cotranslational and one posttranslational, may represent a common approach to the generation of oligomeric proteins in the cytosol.

The reovirus cell attachment protein, sigma1, is a trimer with a 'lollipop' structure. Recent findings indicate that the N-terminal fibrous tail and the C-terminal globular head each possess a distinct trimerization domain. The region responsible for N-terminal trimerization (formation of a triple alpha-helical coiled-coil) is located at the N-terminal one-third of sigma1. In this study, we investigated the temporality and ATP requirement of this trimerization event in the context of sigma1 biogenesis. In vitro co-synthesis of the full-length (FL) and a C-terminally truncated (d44) sigma1 protein revealed a preference for homotrimer over heterotrimer formation, suggesting that assembly at the N-terminus occurs co-translationally. This was corroborated by the observation that polysome-associated sigma1 chains were trimeric as well as monomeric. Truncated proteins (d234 and d294) with C-terminal deletions exceeding half the length of sigma1 were found to trimerize post-translationally. This trimerization did not require ATP since it proceeded normally in the presence of apyrase. In contrast, formation of stable FL sigma1 trimers was inhibited by apyrase treatment. Collectively, our data suggest that assembly of nascent sigma1 chains at the N-terminus is intrinsically ATP independent, and occurs co-translationally when the ribosomes have traversed past the midpoint of the mRNA.