Co-translational trimerization of the reovirus cell attachment protein.

Abstract

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.

Hsp90 Phosphorylation Is Linked to Its Chaperoning Function

“…To this end, 1 mRNA was translated only long enough to generate immature trimers. These molecules have been previously characterized as the earliest posttranslational folding intermediate of 1 and possess loosely wound trimeric N termini and unfolded C termini (previously designated the unstable hydra form) (20,21). At least three chaperones are associated with these structures: Hsp90, p23, and Hsp70 (21).…”

“…This has provided yet another piece of supportive evidence for the re- 35 S]methionine for 10 min. The reaction was then centrifuged to pellet the ribosomes, and the supernatant was incubated further at 37°C in the presence and the absence of 7 M GA. At the various times indicated, the aliquots were taken and analyzed by SDS-PAGE under nondissociating conditions that allowed for the identification of all three 1 forms previously characterized (20,21). B, FL s1 transcripts were translated in vitro at 37°C for 8 min.…”

“…During 1 biogenesis, assembly of three neighboring nascent chains occurs cotranslationally (i.e. on the polysome) at the N terminus (19,20). This process does not involve any chaperones or ATP and results in the generation of a loose triple coiled-coil.…”

Cotranslational Folding of Globin

“…However, the possibility of globin tetramer assembly on the ribosome in those experiments could not be excluded. Cotranslational trimerization of the retrovirus cell attachment protein, 1, has been demonstrated recently (32), and thus the assembly of the nascent globin chains could as well be the case. If this was the case, the previously obtained data could be alternatively explained by the presence of complete globin chains (with labeled hemin) associated with the nascent chain.…”

The Cellular Chaperone Hsc70 Is Specifically Recruited to Reovirus Viral Factories Independently of Its Chaperone Function

“…This ␦ fragment of 1 is removed from core particles in a process dependent on Hsc70 (19). Furthermore, the folding of the 1 attachment protein is dependent on Hsp70 and Hsp90 (16,21).…”