TRANSIENT INTERACTION OF HSP90 WITH EARLY UNFOLDING INTERMEDIATES OF CITRATE SYNTHASE - IMPLICATIONS FOR HEAT-SHOCK IN-VIVO

At normal temperatures, Hsp90 is one of the most abundant proteins in the cytosol of various eucaryotic cells, Upon heat shock, the level of Hsp90 is increased even more, suggesting that it is important for hel ping cells to survive under these conditions, However, studies so far have been almost exclusively concerned with the function of Hsp90 unde r non-stress conditions, and therefore only little is known about the role of Hsp90 during heat shock, As a model for heat shock in vitro, w e have monitored the inactivation and subsequent aggregation of dimeri c citrate synthase (CS) at elevated temperatures, Hsp90 effectively '' stabilized'' CS under conditions where the enzyme is normally inactiva ted and finally aggregates very rapidly, A kinetic dissection of the u nfolding pathway of CS succeeded in revealing two intermediates which form and subsequently undergo irreversible aggregation reactions, Hsp9 0 apparently interacts transiently with these highly structured early unfolding intermediates, finding and subsequent release of the interme diates favorably influences the kinetic partitioning between two compe ting processes, the further unfolding of CS and the productive refoldi ng to the native state, As a consequence, CS is effectively stabilized in the presence of Hsp90. The significance of this interaction is esp ecially evident in the suppression of aggregation, the major end resul t of thermal unfolding events in vivo and in vitro. These effects, whi ch are ATP-independent, seem to be a general function of members of th e Hsp90 family, since yeast and bovine Hsp90 as well as the Hsp90 homo logue from Escherichia coil gave similar results, It seems likely that this function also reflects the role of Hsp90 under heat shock condit ions in vivo, We therefore propose that members of the Hsp90 family co nvey thermotolerance by transiently binding to highly structured early unfolding intermediates, thereby preventing their irreversible aggreg ation and stabilizing the active species.