Identification and Characterisation of the α and β Subunits of Succinyl CoA Ligase of Tomato

Abstract

Despite the central importance of the TCA cycle in plant metabolism not all of the genes encoding itsconstituent enzymes have been functionally identified. In yeast, the heterodimeric protein succinyl CoAligase is encoded for by two single-copy genes. Here we report the isolation of two tomato cDNAs codingfor a- and one coding for the b-subunit of succinyl CoA ligase. These three cDNAs were used to complementthe respective Saccharomyces cerevisiae mutants deficient in the a- and b-subunit, demonstratingthat they encode functionally active polypeptides. The genes encoding for the subunits were expressed in alltissues, but most strongly in floral and leaf tissues, with equivalent expression of the two a-subunit genesbeing expressed to equivalent levels in all tissues. In all instances GFP fusion expression studies confirmedan expected mitochondrial location of the proteins encoded. Following the development of a novel assay tomeasure succinyl CoA ligase activity, in the direction of succinate formation, the evaluation of the maximalcatalytic activities of the enzyme in a range of tissues revealed that these paralleled those of mRNA levels.We also utilized this assay to perform a preliminary characterisation of the regulatory properties of theenzyme suggesting allosteric control of this enzyme which may regulate flux through the TCA cycle in amanner consistent with its position therein.