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

Analysis of theArabidopsis O-Acetylserine(thiol)lyase Gene Family Demonstrates Compartment-Specific Differences in the Regulation of Cysteine Synthesis

“…The same was true for the oastlAB mutant. The abundance of the five OAS-TL proteins that bound to SAT correlated well with the relative amounts of mRNAs in leaves and roots at this developmental stage in the literature (Hell et al, 1994;Hesse et al, 1999;Hatzfeld et al, 2000;Yamaguchi et al, 2000) and in a public microarray database (Zimmermann et al, 2004).…”

“…Thus, CAS might be able to replace OAS-TL in mitochondria, at least in spinach. While the presence of other OAS-TL-like proteins in spinach mitochondria cannot be ruled out (Yamaguchi et al,1998), enzymatically defined mitochondrial OAS-TLs have been demonstrated in D. innoxia and Arabidopsis, suggesting that CAS probably has no function for Cys synthesis in these species (Kuske et al, 1994(Kuske et al, , 1996Hesse et al, 1999;Jost et al, 2000).…”

Functional analysis of the cysteine synthase protein complex from plants: Structural, biochemical and regulatory properties

“…The cytosolic localization of OAS-TL A1 is evident from its apparent lack of any transit peptide (Hell et al, 1994). The compartmentation of OAS-TL B and C was confirmed using isolated chloroplasts and mitochondria (Hesse et al, 1999). CAS, AtCysD1 and AtCysD2D localizations were shown by GFP fusions and transient expression (Yamaguchi et al, 2000).…”

“…OAS-TL-like proteins can be involved in the synthesis of secondary plant products (Noji et al, 1993;Saito et al, 1994) and indeed the E. coli CysM protein has been engineered for the fermentative production of unnatural amino acids (Maier, 2003). Three OAS-TL isoforms A1 (cytosolic), B (plastidic) and C (mitochondrial) and a structurally related b-cyanoalanine synthase (CAS, AtCysC1) (Hatzfeld et al, 2000;Yamaguchi et al, 2000) give rise to the dominating OAS-like mRNAs according to the GeneInvestigator database and individual studies (Hell et al, 1994;Hesse et al, 1999;Hatzfeld et al, 2000). CAS (EC4.4.1.9) catalyzes a partial backward reaction of cysteine synthesis that consumes cysteine and releases sulfide Hawkesford, 1998, 2000, Fig.…”

Improving the levels of essential amino acids and sulfur metabolites in plants

“…However, their single contributions to net Cys synthesis and any functional interactions that may occur between these subcellular locations are unknown. Interestingly, only Arabidopsis seems to possess a mitochondrial localised OASTL (Hesse et al, 1999), while in other plants, such as spinach, b-cyanoalanine synthase (CAS) is responsible for this function (Saito et al, 1994;Hawkesford, 1998, 2000;Hatzfeld et al, 2000). In this context, it is important to note that CAS exists in addition to OASTL in Arabidopsis mitochondria (Hatzfeld et al, 2000).…”