In vivo reactivation of heat-denatured protein in the endoplasmic reticulum of yeast

Abstract

Saccharomyces cerevisiae cells grown at 24°C acquire thermotolerance and survive exposure to 50°C, but only if they are first incubated at 37°C, the temperature where heat shock genes are activated. We show here that the enzymatic activity of a secretory β-lactamase fusion protein, pre-accumulated at 37°C in the endoplasmic reticulum, was abolished by exposure of the cells to 50°C. When the cells were returned to 24°C, β-lactamase activity was resumed. Reactivation occurred in the endoplasmic reticulum, but not in the Golgi apparatus. It was dependent on metabolic energy, but did not require de novo protein synthesis. According to co-immunoprecipitation experiments, immunoglobulin-binding protein (BiP/Kar2p) was associated with the fusion protein. We suggest that recovery from thermal insult involves, in addition to cytoplasmic and nuclear events, refolding of heat-damaged proteins in the endoplasmic reticulum by a heat-resistant machinery, which forms part of a fundamental survival mechanism.