Identification and characterisation of the alpha and beta subunits of succinyl CoA ligase of tomato.

STUDART-GUIMARÃES C, GIBON Y, FRANKEL N, WOOD CC, ZANOR MI, FERNIE AR, CARRARI F.

PLANT MOLECULAR BIOLOGY

Springer Netherlands

Año: 2005 p. 781 - 791

0167-4412

Despite the central importance of the TCA cycle in plant metabolism not all of the genes encoding its constituent enzymes have been functionally identified. In yeast the heterodimeric protein succinyl CoA ligase is encoded for by two single-copy genes. Here we report the isolation of two tomato cDNAs coding for a- and one coding for the b-subunit of succinyl CoA ligase. These three cDNAs were used to complement the respective S. cerevisiae mutants deficient in the a- and b-subunit, demonstrating that they encode functionally active polypeptides. The genes encoding for the subunits were expressed in all plant organs, but most strongly in flowers and leaves, with equivalent expression of the two a-subunit genes being expressed to equivalent levels in all plant organs. In all instances GFP fusion expression studies confirmed an expected mitochondrial location of the proteins encoded. Following the development of a novel assay to measure succinyl CoA ligase activity, in the direction of succinate formation, the evaluation of the maximal catalytic activities of the enzyme in a range of plant organs revealed that these paralleled those of mRNA levels. We also utilized this assay to perform a preliminary characterisation of the regulatory properties of the enzyme suggesting allosteric control of this enzyme may regulate flux through the TCA cycle in a manner consistent with its position therein.