Mutations in the Cu/Zn-superoxide dismutase (SOD-I) gene are responsible for a familial form of amyotrophic lateral sclerosis, In humans and experimental models, death of motor neurons is preceded by formation of cytoplasmic aggregates containing mutant SOD-l protein. In our previous studies, heat shock protein 70 (HSP70) prolonged viability of cultured motor neurons expressing mutant human SOD-l and reduced formation of aggregates. in this paper, we report that mutant SOD-l proteins have altered solubility in cells relative to wild-type SOD-l and can form a direct association with HSP70 and other stress proteins. Whereas wild-type human and endogenous mouse SOD-1 were detergent-soluble, a portion of mutant SOD-l was detergent-insoluble in protein extracts of NIH3T3 transfected with SOD-l gene constructs, spinal cord cultures established from G93A SOD-1 transgenic mouse embryos, and lumbar spinal cord from adult G93A transgenic mice. A direct association of HSP70, HSP40, and alphaB-crystallin with mutant SOD-1 (G93A or G41S), but not wild-type or endogenous mouse SOD-1, was demonstrated by coimmunoprecipitation, Mutant SOD-1 HSP70 complexes were predominantly in the detergent-insoluble fraction. However, only a small percentage of total cellular mutant SOD-l was detergent-insoluble, suggesting that mutation-induced alteration of protein conformation may not in itself be sufficient for direct interaction with heat shock proteins.
