Molecular cloning and expression analyses of mitochondrial and plastidic isoforms of cysteine synthase (O-acetylserine(thiol)lyase) from Arabidopsis thaliana.

Abstract

Cysteine synthase, the key enzyme for fixation of inorganic sulfide, catalyses the formation of cysteine from O-acetylserine and inorganic sulfide. Here we report the cloning of cDNAs encoding cysteine synthase isoforms from Arabidopsis thaliana. The isolated cDNA clones encode for a mitochondrial and a plastidic isoform of cysteine synthase (O-acetylserine (thiol)-lyase, EC 4.2.99.8), designated cysteine synthase C (AtCS-C, CSase C) and B (AtCS-B; CSase B), respectively. AtCS-C and AtCS-B, having lengths of 1569-bp and 1421-bp, respectively, encode polypeptides of 430 amino acids (approximately 45.8 kD) and of 392 amino acids (approximately 41.8 kD), respectively. The deduced amino acid sequences of the mitochondrial and plastidic isoforms exhibit high homology even with respect to the presequences. The predicted presequence of AtCS-C has a N-terminal extension of 33 amino acids when compared to the plastidic isoform. Northern blot analysis showed that AtCS-C is higher expressed in roots than in leaves whereas the expression of AtCS-B is stronger in leaves. Furthermore, gene expression of both genes was enhanced by sulfur limitation which in turn led to an increase in enzyme activity in crude extracts of plants. Expression of the AtCS-B gene is regulated by light. The mitochondrial, plastidic and cytosolic (Hesse and Altmann, 1995) isoforms of cysteine synthase of Arabidopsis are able to complement a cysteine synthase-deficient mutant of Escherichia coli unable to grow on minimal medium without cysteine, indicating synthesis of functional plant proteins in the bacterium. Two lines of evidence proved that AtCS-C encodes a mitochondrial form of cysteine synthase; first, import of in vitro translation products derived from AtCS-C in isolated intact mitochondria and second, Western blot analysis of mitochondria isolated from transgenic tobacco plants expressing AtCS-C cDNA/c-myc DNA fusion protein.