Degradation of citrate synthase lacking the mitochondrial targeting sequence is inhibited in cells defective in Hsp70/Hsp40 chaperones under heat stress conditions

Most nucleus-encoded mitochondrial precursor proteins are synthesized in the cytosol and imported into mitochondria in a post-translational manner. In recent years, the quality control mechanisms of nonimported mitochondrial proteins have been intensively studied. In a previous study, we established that in budding yeast a mutant form of citrate synthase 1 (N increment Cit1) that lacks the N-terminal mitochondrial targeting sequence, and therefore mislocalizes to the cytosol is targeted for proteasomal degradation by the SCFUcc1 ubiquitin ligase complex. Here, we show that Hsp70 and Hsp40 chaperones (Ssa1 and Ydj1 in yeast, respectively) are required for N increment Cit1 degradation under heat stress conditions. In the absence of Hsp70 function, a portion of N increment Cit1-GFP formed insoluble aggregates and cytosolic foci. However, the extent of ubiquitination of N increment Cit1 was unaffected, implying that Hsp70/Hsp40 chaperones are involved in the postubiquitination step of N increment Cit1 degradation. Intriguingly, degradation of cytosolic/peroxisomal gluconeogenic citrate synthase (Cit2), an endogenous substrate for SCFUcc1-mediated proteasomal degradation, was not highly dependent on Hsp70 even under heat stress conditions. These results suggest that mitochondrial citrate synthase is thermally vulnerable in the cytosol, where Hsp70/Hsp40 chaperones are required to facilitate its degradation. Hayashi et al. report that Hsp70/Hsp40 chaperones are required for the degradation of citrate synthase lacking the mitochondrial targeting sequence under heat stress conditions.