AIR SYNTHETASE IN COWPEA NODULES - A SINGLE-GENE PRODUCT TARGETED TO 2 ORGANELLES

A cDNA (VUpur5) encoding phosphoribosyl aminoimidazole (AIR) synthetas e, the fifth enzyme of the de novo purine biosynthesis pathway has bee n isolated from a cowpea nodule cDNA library. It encodes a 388 amino a cid protein with a predicted molecular mass of 40.4 kDa. The deduced a mino acid sequence has significant homology with AIR synthetase from o ther organisms. AIR synthetase is present in both mitochondria and pla stids of cowpea nodules [7]. A signal sequence encoded by the VUpur5 c DNA has properties associated with plastid transit sequences but there is no consensus cleavage site as would be expected for a plastid targ eted protein. Although the signal sequence does not have the structura l features of a mitochondrial targeted protein, it has a mitochondrial cleavage site motif (RX/XS) close to the predicted N-terminus of the mature protein. Southern analysis suggests that AIR synthetase is enco ded by a single gene raising questions as to how the product of this g ene is targeted to the two organelles. VUpur5 is expressed at much hig her levels in nodules compared to other cowpea tissues and the gene is active before nitrogen fixation begins. These results suggest that pr oducts of nitrogen fixation do not play a role in the initial inductio n of gene expression. VUpur5 was expressed in Escherichia coli and the recombinant protein used to raise antibodies. These antibodies recogn ize two forms of AIR synthetase which differ in molecular size. Both f orms are present in mitochondria, although the larger protein is more abundant. Only the smaller protein was detected in plastids.