Functional Dissection of Trigger Factor and DnaK: Interactions with Nascent Polypeptides and Thermally Denatured Proteins
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Elke Schaffitzel
Abstract
In Escherichia coli, the ribosomeassociated Trigger Factor (TF) cooperates with the DnaK system in the folding of newly synthesized cytosolic polypeptides. Here we investigated the functional relationship of TF and DnaK by comparing various functional properties of both chaperones. First, we analyzed the ability of TF and DnaK to associate with nascent polypeptides and fulllength proteins released from the ribosome. Toward this end, we established an E. coli based transcription/translation system containing physiological ratios of TF, DnaK and ribosomes. In this system, TF can be crosslinked to nascent polypeptides of σ32. No TF crosslink was found to fulllength σ32, which is known to be a DnaK substrate. In contrast, DnaK crosslinked to both nascent and fulllength σ32. DnaK crosslinks critically depended on the type of chemical crosslinker. Crosslinks represent specific substratechaperone interactions since they relied on the association of the nascent polypeptides with the substrate binding pocket of DnaK. While DnaK is known to be the major chaperone to prevent protein aggregation under heat shock conditions, we found that TF did not prevent aggregation of thermally unfolded proteins in vitro and was not able to complement the heatsensitive phenotype of a δnaK52 mutant in vivo. These data indicate that TF and DnaK show strong differences in their ability to prevent aggregation of denatured proteins and to associate with native like substrates, but share the ability to associate with nascent polypeptides.
Copyright © 2001 by Walter de Gruyter GmbH & Co. KG
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