CYTOSOLIC ACONITASE PARTICIPATES IN THE GLYOXYLATE CYCLE IN ETIOLATEDPUMPKIN COTYLEDONS

Two different aconitases are known to be expressed after the germinati on of oil-seed plants. One is a mitochondrial aconitase that is involv ed in the tricarboxylic acid cycle. The other participates in the glyo xylate cycle, playing a role in gluconeogenesis from stored oil. We is olated and characterized a cDNA for an aconitase from etiolated pumpki n cotyledons. The cDNA was 3,145 bp long and capable of encoding a pro tein of 98 kDa. N-terminal and C-terminal amino acid sequences deduced from the cDNA did not contain mitochondrial or glyoxysomal targeting signals. A search of protein databases suggested that the cDNA encoded a cytosolic aconitase. Immunoblotting analysis with a specific antibo dy against the aconitase expressed in Escherichia coli revealed that d evelopmental changes in the amount of the aconitase were correlated wi th changes in levels of other enzymes of the glyoxylate cycle during g rowth of seedlings. Further analysis by subcellular fractionation and immunofluorescence microscopy revealed that aconitase was present only in the cytosol and mitochondria. No glyoxysomal aconitase was found i n etiolated cotyledons even though all the other enzymes of the glyoxy late cycle are known to be localized in glyoxysomes. Taken together, t he data suggest that the cytosolic aconitase participates in the glyox ylate cycle with four glyoxysomal enzymes.