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 eukaryotes, but the role of these proteins in thermoprotection remains unclear. To investigate the function of one of these proteins, we disrupted expression of the single-copy hsp30 gene of Neurospora crassa, using repeat-induced point mutagenesis, and we generated and characterized mutant strains that were deficient in hsp30 synthesis. These strains could grow at high temperature and they acquired thermotolerance from a heat shock. However, the hsp30-defective strains proved to be extremely sensitive to the combined stresses of high temperature and carbohydrate limitation, enforced by the addition of a nonmetabolizable glucose analogue. Under these conditions, their survival was reduced by 90% compared with wild-type cells. This sensitive phenotype was reversed by reintroduction of a functional hsp30 gene into the mutant strains. The mutant cells contained mitochondria from which a 22-kDa protein was readily extracted with detergents, in contrast to its retention by the mitochondria of wild-type cells. Antibodies against hsp30 coimmunoprecipitated a protein also of approximately 22 kDa from wild-type cells. Results of this study suggest that hsp30 may be important for efficient carbohydrate utilization during high temperature stress and that it may interact with other mitochondrial membrane proteins and function as a protein chaperone.
Plesofsky-Vig
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Department of Genetics and Cell Biology, University of Minnesota, St. Paul 55108, USA.
Brambl
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R
D003001:Cloning, Molecular; D003433:Crosses, Genetic; D004271:DNA, Fungal / Q000737:chemistry / Q000302:isolation & purification; D004926:Escherichia coli; D005800:Genes, Fungal; D050890:HSP30 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006358:Hot Temperature; D008565:Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009492:Neurospora crassa / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D010641:Phenotype; D010957:Plasmids; D017354:Point Mutation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D013104:Spheroplasts / Q000502:physiology
D004271:DNA, Fungal; D050890:HSP30 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins