Abstract
Hop is a 60-kDa protein characterized by its ability to bind the two chaperones, hsp70 and hsp90. We have tested the function of Hop using an assay for the refolding of denatured firefly luciferase. We show that Hop is involved in the process of refolding thermally denatured firefly luciferase in rabbit reticulocyte lysate. Hop also stimulates refolding by hsp70 and Ydj-1 in a purified refolding system. Hsp90 can also stimulate refolding, and optimal refolding is observed in the presence of both Hop and hsp90. Similar stimulation was observed when Hop was replaced by its yeast homolog Sti1. In assays of the binding of Hop to hsp70 and hsp90, Hop preferentially forms a complex with ADP-bound hsp70, and this process is unaffected by the presence of hsp90. Hop does not alter the ATPase activity or the rate of ADP dissociation of hsp70. Hop also appears to bind to the ADP-bound form of hsp90, blocking the ATP-dependent conversion of hsp90 to a form capable of interacting with p23. Conversely, once p23 is bound to hsp90, Hop binding is diminished. These results confirm that Hop provides a physical link between hsp70 and hsp90 and also indicate that Hop modulates the activities of both of these chaperone proteins.
Highlights
The molecular chaperones hsp701 and hsp90 are two of the most prominent heat shock proteins in the eukaryotic cytosol
Refolding—To determine whether Hop is involved in the process of refolding firefly luciferase in rabbit reticulocyte lysate, we added thermally denatured luciferase to a refolding mixture containing reticulocyte lysate dialyzed in TB, with ATP, and an ATP-regenerating system and incubated the reaction at 25 °C to promote refolding
These results show that the refolding of luciferase in reticulocyte lysate is influenced by Hop, but Hop is clearly not an essential component of the refolding machinery
Summary
The molecular chaperones hsp701 and hsp90 are two of the most prominent heat shock proteins in the eukaryotic cytosol. 4 shows the results of an ATPase assay in which the hydrolysis of ATP by hsp70 is determined by measuring free phosphate release over a time course in the presence or absence of Hop. Ydj, which is known to stimulate the ATPase activity of hsp70 [5, 6], was used as the positive control.
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