Abstract
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.
Highlights
It is known that the folding of nascent proteins in the cytosol is mediated by a group of proteins known as chaperones
Protein kinases associated with Hsp90 have included receptor tyrosine kinases such as erbB2 [4], nonreceptor tyrosine kinases such as Wee 1 [5] and v-src [6, 7], ser/thr kinases such as Raf-1, Mek, and Cdk4 (8 –10) as well as the heme-regulated eukaryotic initiation factor kinase (HRI)1 [11, 12]
Association of Hsp90 with Immature but Not Mature 1—We previously reported that Hsp70 is associated with 1 intermediates and is actively involved in 1 biogenesis [35]
Summary
It is known that the folding of nascent proteins in the cytosol is mediated by a group of proteins known as chaperones. Further ATP-dependent release and rebinding of Hsp-70 and other proposed chaperones leads to global assembly and folding of the C terminus, generating mature 1 with the characteristic “lollipop”shaped structure, which migrates as an unretarded trimer in SDS-PAGE under nondissociating conditions [34, 35].
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