Molecular genetics of metal detoxification: Prospects for phytoremediation. 1997 annual progress report

'The authors proposed to characterize a number of fission yeast mutants that are hypersensitivity to cadmium and deficient in the production of metal-peptide complexes. For each of the mutants the authors sought to clone the gene responsible for the mutant phenotype and more importantly to define the gene function. They summarize the progress made thus far for each of the mutants. Mutants that hypoproduce phytochelatins are: (1) DS12--The gene has been cloned, but a full length cDNA remains to be isolated. They believe the longest clone is short at the 5 feet end by less than 100 bp. This gene encodes sulfite reductase and its function is needed for Pb-induced sulfide production, but not Cd-induced sulfide production. Since this enzyme acts upstream of cysteine biosynthesis, the likely reason that this mutant hypoproduces phytochelatins is that it fails to produce sufficient cysteine during Cd stress. (2) JS563--The gene has been cloned and found to encode a sulfide dehydrogenase. In vitro, the authors found that the protein binds FAD, converts S{sup 2-} to S{sup 0} while reducing quinone. The protein is membrane associated and has been localized to the mitochondria. Its likely function is to detoxify sulfide in the mitochondria resulting from cadmium-induced sulfide production. The sulfide electrons are likely used for the electron transport chain. Cells that have a defect in this enzyme cannot oxidize mitochondrial S{sup 2-} resulting in high toxic levels of S{sup 2-} during Cd stress. In addition, the high S{sup 2-} level precipitates Cd{sup 2-} to form CdS, and the lack of free Cd{sup 2+} fails to induce phytochelatin synthase activity to produce phytochelatin peptides. (3) JS282--The genomic clone that restores Cd tolerance to JS282 has been isolated. Surprisingly, this genomic clone when present in a multicopy vector in a wild type background causes hypersensitivity to Cd and selenium. The cDNA corresponding to the genomic clone has been isolated and its sequence is being determined. (4) JS246--A genomic clone that restores Cd tolerance has been isolated but its corresponding cDNA remains to be cloned. Wild type strains harboring the genomic clone become hypertolerant to cadmium, Extensive efforts are being made to find the cDNA clone, including the construction of new cDNA libraries. Mutant that fails to accumulate a high molecular weight phytochelatin-cadmium-sulfide complex. (5) JS237--This mutant has a mRNA splicing defect in a gene homologous to the human Wiskott-Aldrich Syndrome protein (WASP). The human disease symptom is associated with defects in cytoskeletal organization. Likewise, mutant JS237 exhibits aberrant arrangement of polymerized actin and the fission yeast WASP has been found to co-localize with cortical actin and to interact with actin associated proteins. The genomic clone complemented the defect associated with the polymerized actin organization, but did not fully restore Cd tolerance. Hence, they entertained the possibility of another gene encoded by the genomic clone. Indeed, they found that the mutation appears to affect more than the WASP mRNA. The data thus far suggest an upstream mRNA overlaps the WASP coding region. When the upstream DNA was sequenced, they found an open reading frame encoding a P-type ATPase which may be a cation transporter. Where this transporter resides within the cell and what role it plays in Cd tolerance are currently under investigation.