[
FEBS Lett,
2004]
Based on the amino acid alignment, Caenorhabditis elegans F32D1.1 was identified to be a homologue of the mammalian fidgetin. We produced and purified the F32D1.1 protein by using a baculovirus-expression system. F32D1.1 has an ATPase activity, which is sensitive to N-ethylmaleimide. Km and Vmax for the ATPase activity of F32D1.1 were estimated to be 0.44 mM and 225 nmol/mg/min, respectively. When the cysteine at the position of 368 was mutated to alanine, the ATPase activity was greatly decreased; Vmax was decreased to one-sixth, while Km remained similar. These results suggest that the unique position of cysteine 368, located immediately downstream of the Walker A motif, plays an important role in the ATP hydrolysis process of C. elegans F32D1.1 protein.
[
International Worm Meeting,
2003]
We have studied Escherichia coli FtsH, which is a membrane-bound, oligomeric ATP-dependent metalloprotease belonging to the AAA (ATPases associated with diverse cellular activities. For details of AAA, see the poster presented by K. Yamanaka et al.) family. C. elegans has three FtsH homologues (Y47G6A.10, Y38F2AR.para and M03C11.5). Their expression and roles are yet unknown. Y47G6A.10 and Y38F2AR.para are highly homologous with human paraplegin, which is a nuclear-encoded mitochondrial protein and whose mutation is responsible for a human genetic disease, hereditary spastic paraplegia (HSP). Progressive weakness and spasticity of the lower limbs due to degeneration of corticospinal axons are the clinical hallmarks of HSP. The marked genetic heterogeneity in HSP, with 20 loci chromosomally mapped and eight genes so far identified, suggests that a number of defective cellular processes may result in the disease. However, little is known about pathogenesis of HSP. Furthermore, no specific treatment is currently available to prevent, cure, or delay progression of symptoms of HSP. In order to establish a model system for HSP, we initiated to analyze paraplegin homologues in C. elegans.The analysis of GFP fusion constructs of Y47G6A.10 and Y38F2AR.para showed that their expression pattern was not identical. In addition, effects of RNAi were also different; mixed phenotype (embryonic lethal, larval lethal or slow growth) for Y47G6A.10, but no obvious effect for Y38F2AR.para. Progressively retarded motility was also observed for Y47G6A.10. Succinate dehydrogenase assay and electron microscopic observation of Y47G6A.10 (RNAi) indicated mitochondrial defects. These are in good agreement with the previous reports of the clinical characteristic of HSP patients and the analyses of muscle biopsy from patients, suggesting that Y47G6A.10 is a functional homologue of paraplegin.C24B5.2 and/or F32D1.1 are homologous with another AAA protein, spastin, which is also related to HSP. Results on these proteins are also discussed.