Cycloheximide- and puromycin-induced heat resistance: different effects oncytoplasmic and nuclear luciferases

Inhibition of translation can result in cytoprotection against heat shock. The mechanism of this protection has remained elusive so far. Here, the the rmoprotective effects of the translation inhibitor cycloheximide (CHX) and puromycin were investigated, using as reporter firefly luciferase localized either in the nucleus or in the cytoplasm, A short preincubation of O23 ce lls with either translation inhibitor was found to attenuate the heat inact ivation of a luciferase directed into the cytoplasm, whereas the heat sensi tivity of a nuclear-targeted luciferase remained unaffected. After a long-t erm CHX pretreatment, both luciferases were more heat resistant. Both the c ytoplasmic and the nuclear luciferase are protected against heat-induced in activation in thermotolerant cells and in cells overexpressing heat shock p rotein (Hsp)70. CHX incubations further attenuated cytoplasmic luciferase i nactivation in thermotolerant and in Hsp70 overexpressing cells, even when Hsp70-mediated protection was saturated. It is concluded that protection by translation inhibition is unlikely due to an increase in the pool of free Hsps normally engaged in translation and released from the nascent polypept ide chains on the ribosomes. Rather, a decrease in nascent chains and therm olabile polypeptides may account for the heat resistance promoted by inhibi tors of translation.