Dynamic changes in the localization of thermally unfolded nuclear proteinschaperone-dependent associated with protection

Molecular chaperones are involved in the protection of cells against protei n damage through their ability to hold, disaggregate, and refold damaged pr oteins or their ability to facilitate degradation of damaged proteins. Litt le is known about how these processes are spatially coordinated in cells. U sing a heat-sensitive nuclear model protein luciferase fused to the traceab le, heat-stable enhanced green fluorescent protein (N-luc-EGFP), we now sho w that heat inactivation and insolubilization of luciferase were associated with accumulation of N-luc-EGFP at multiple foci throughout the nucleus. C oexpression of Hsp70, one of the major mammalian chaperones, reduced the fo rmation of these small foci during heat shock. Instead, the heat-unfolded N -luc-EGFP accumulated in large, insoluble foci. Immunofluorescence analysis revealed that these foci colocalized with the nucleoli. Time-lapse analysi s demonstrated that protein translocation to the nucleolus, in contrast to the accumulation at small foci, was fully reversible upon return to the nor mal growth temperature. This reversibility was associated with an increase in the level of active and soluble luciferase. Expression of a carboxyl-ter minal deletion mutant of Hsp70(1-543), which lacked chaperone activity, had no effect on the localization of N-luc-EGFP, which suggests that the Hsp70 chaperone activity is required for the translocation events. Our data show that Hsp70 not only is involved in holding and refolding of heat-unfolded nuclear proteins but also drives them to the nucleolus during stress. This might prevent random aggregation of thermolabile proteins within the nucleu s, thereby allowing their refolding at the permissive conditions and preven ting indirect damage to other nuclear components.