THERMOSTABILITY OF A NUCLEAR-TARGETED LUCIFERASE EXPRESSED IN MAMMALIAN-CELLS - DESTABILIZING INFLUENCE OF THE INTRANUCLEAR MICROENVIRONMENT

Protein denaturation and aggregation are most likely the cause for the noxious effects of heat shock, There are some indications that the nu cleus is one of the most sensitive cellular compartments, To test the possibility that the intranuclear microenvironment might be detrimenta l to the heat stability of proteins, we compared the in situ thermal s tability of a reporter protein localized in the nucleus or in the cyto plasm. A recombinant firefly (Photynus pyralis) luciferase carrying a point mutation in the C-terminal domain remains in the cytoplasm (cyt- luciferase). A nuclear localization sequence was fused to the N-termin al domain of cyt-luciferase; the resulting nuc-luciferase was efficien tly targeted to the cell nucleus. In both cases, decreased luciferase activity and solubility were found in lysates from heat-shocked cells. These characteristics were taken as an indication of thermal denatura tion in situ, The heat-inactivated luciferases were partially reactiva ted during recovery after stress, indicating the capacity of both the cytoplasmic and nuclear compartments to reassemble proteins from an ag gregated state, Although both the nuc- and the cyt-luciferases were he at inactivated at similar rates in vitro, nuc-luciferase was more susc eptible to thermal denaturation in situ compared to cyt-luciferase. Th is observation suggests that the microenvironment of an intracellular compartment may modulate the thermal stability of proteins. The local concentration might be one element of this microenvironment affecting the heat-stability of proteins. In cells made thermotolerant by a prim ing shack, the thermal inactivation of the recombinant luciferases occ urred at a slower rate during a second challenging stress. However, th is decreased thermal sensitivity was less pronounced for the nuc-lucif erase (threefold) than for the cyt-luciferase (sevenfold). The nuclear luciferase might become a useful tool to investigate the action of mo lecular chaperones in the nucleus.