DISRUPTION OF THE GENE FOR HSP30, AN ALPHA-CRYSTALLIN-RELATED HEAT-SHOCK PROTEIN OF NEUROSPORA-CRASSA, CAUSES DEFECTS IN THERMOTOLERANCE

The alpha-crystallin-related heat shock proteins are produced by all e ukaryotes, but the role of these proteins in thermoprotection remains unclear. To investigate the function of one of these proteins, we disr upted expression of the single-copy hsp30 gene of Neurospora crassa, u sing repeat-induced point mutagenesis, and we generated and characteri zed mutant strains that were deficient in hsp30 synthesis. These strai ns could grow at high temperature and they acquired thermotolerance fr om a heat shock. However, the hsp30-defective strains proved to be ext remely sensitive to the combined stresses of high temperature and carb ohydrate limitation, enforced by the addition of a nonmetabolizable gl ucose analogue. Under these conditions, their survival was reduced by 90% compared with wild-type cells. This sensitive phenotype was revers ed by reintroduction of a functional hsp30 gene into the mutant strain s. The mutant cells contained mitochondria from which a 22-kDa protein was readily extracted with detergents, in contrast to its retention b y the mitochondria of wild-type cells. Antibodies against hsp30 coimmu noprecipitated a protein also of approximate to 22 kDa from wildtype c ells. Results of this study suggest that hsp30 may be important for ef ficient carbohydrate utilization during high temperature stress and th at it may interact with other mitochondrial membrane proteins and func tion as a protein chaperone.