Active participation of Hsp90 in the biogenesis of the trimeric reovirus cell attachment protein σ1

The reovirus cell attachment protein, σ1, 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 σ1 biogenesis, we analyzed σ1 synthesized in rabbit reticulocyte lysate. Coprecipitation experiments using anti-Hsp90 antibodies revealed that Hsp90 was associated with immature σ1 trimers (hydra-like intermediates with assembled N termini and unassembled C termini) but not with mature trimers. The use of truncated σ1 further demonstrated that only the C-terminal half of σ1 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 σ1 but prevented the subsequent release of Hsp90 from the immature σ1 complex. We also examined the status of p23, an Hsp90-associated co-chaperone. Like Hsp90, p23 only associated with immature σ1 trimers, and this association was mapped to the C-terminal half of σ1. However, unlike Hsp90, p23 was released from the σ1 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.