Abstract
Glutathione synthetase predicted from the reported gene sequence from Schizosaccharomyces pombe is substantially smaller than the equivalent protein predicted from the cDNAs sequenced from Arabidopsis thaliana, Saccharomyces cerevisiae and other eukaryotes. Sequence alignments of the proteins encoded by the cDNA clones for glutathione synthetase from Arabidopsis and S. pombe show that the Arabidopsis protein contains 200 extra amino acids at the N-terminus. In order to test if this sequence is essential in the function of the protein, the full-length Arabidopsis protein and as two N-terminal deletions (Delta67-71 and Delta67-200) were expressed in S. pombe mutant MN101, which lacks endogenous glutathione synthetase activity. Although the wild-type plant cDNA could complement the yeast mutation, neither deletion mutant was able to restore glutathione-dependent cadmium resistance. When the three proteins were expressed as fusion proteins in Escherichia coli, they accumulated to the same level, but only the plasmid containing the full-length cDNA, pFLAG222, produced detectable enzyme activity in vitro. These results suggested that the N-terminus of the Arabidopsis glutathione synthetase is essential for its function and opened up the possibility that there was a sequencing error in the reported S. pombe sequence. Therefore the gsh2 sequence from wild-type S. pombe and the mutant strain MN101 were determined. The wild-type S. pombe gsh2 encodes a protein that is about the same length as that found in Arabidopsis, and the MN101 mutation involves a frameshift mutation early in the glutathione synthetase reading frame.
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- Forsburg S. L. Comparison of Schizosaccharomyces pombe expression systems. Nucleic Acids Res. 1993 Jun 25;21(12):2955–2956. doi: 10.1093/nar/21.12.2955. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gali R. R., Board P. G. Sequencing and expression of a cDNA for human glutathione synthetase. Biochem J. 1995 Aug 15;310(Pt 1):353–358. doi: 10.1042/bj3100353. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Habenicht A., Hille S., Knöchel W. Molecular cloning of the large subunit of glutathione synthetase from Xenopus laevis embryos. Biochim Biophys Acta. 1993 Sep 23;1174(3):295–298. doi: 10.1016/0167-4781(93)90202-o. [DOI] [PubMed] [Google Scholar]
- Howden R., Andersen C. R., Goldsbrough P. B., Cobbett C. S. A cadmium-sensitive, glutathione-deficient mutant of Arabidopsis thaliana. Plant Physiol. 1995 Apr;107(4):1067–1073. doi: 10.1104/pp.107.4.1067. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Howden R., Goldsbrough P. B., Andersen C. R., Cobbett C. S. Cadmium-sensitive, cad1 mutants of Arabidopsis thaliana are phytochelatin deficient. Plant Physiol. 1995 Apr;107(4):1059–1066. doi: 10.1104/pp.107.4.1059. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Huang C. S., He W., Meister A., Anderson M. E. Amino acid sequence of rat kidney glutathione synthetase. Proc Natl Acad Sci U S A. 1995 Feb 14;92(4):1232–1236. doi: 10.1073/pnas.92.4.1232. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mutoh N., Hayashi Y. Isolation of mutants of Schizosaccharomyces pombe unable to synthesize cadystin, small cadmium-binding peptides. Biochem Biophys Res Commun. 1988 Feb 29;151(1):32–39. doi: 10.1016/0006-291x(88)90555-4. [DOI] [PubMed] [Google Scholar]
- Mutoh N., Nakagawa C. W., Ando S., Tanabe K., Hayashi Y. Cloning and sequencing of the gene encoding the large subunit of glutathione synthetase of Schizosaccharomyces pombe. Biochem Biophys Res Commun. 1991 Nov 27;181(1):430–436. doi: 10.1016/s0006-291x(05)81437-8. [DOI] [PubMed] [Google Scholar]
- Nakagawa C. W., Mutoh N., Hayashi Y. Glutathione synthetase from the fission yeast. Purification and its unique heteromeric subunit structure. Biochem Cell Biol. 1993 Sep-Oct;71(9-10):447–453. doi: 10.1139/o93-066. [DOI] [PubMed] [Google Scholar]
- Rauser W. E. Phytochelatins and related peptides. Structure, biosynthesis, and function. Plant Physiol. 1995 Dec;109(4):1141–1149. doi: 10.1104/pp.109.4.1141. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rawlins M. R., Leaver C. J., May M. J. Characterisation of an Arabidopsis thaliana cDNA encoding glutathione synthetase. FEBS Lett. 1995 Nov 27;376(1-2):81–86. doi: 10.1016/0014-5793(95)01253-1. [DOI] [PubMed] [Google Scholar]
- Ullmann P., Gondet L., Potier S., Bach T. J. Cloning of Arabidopsis thaliana glutathione synthetase (GSH2) by functional complementation of a yeast gsh2 mutant. Eur J Biochem. 1996 Mar 1;236(2):662–669. doi: 10.1111/j.1432-1033.1996.00662.x. [DOI] [PubMed] [Google Scholar]
- Wang C. L., Oliver D. J. Cloning of the cDNA and genomic clones for glutathione synthetase from Arabidopsis thaliana and complementation of a gsh2 mutant in fission yeast. Plant Mol Biol. 1996 Sep;31(6):1093–1104. doi: 10.1007/BF00040827. [DOI] [PubMed] [Google Scholar]
- Wang C. L., Oliver D. J. Identification of a putative flexible loop in Arabidopsis glutathione synthetase. Biochem J. 1997 Feb 15;322(Pt 1):241–244. doi: 10.1042/bj3220241. [DOI] [PMC free article] [PubMed] [Google Scholar]