THE TONOPLAST-ASSOCIATED CITRATE BINDING-PROTEIN (CBP) OF HEVEA-BRASILIENSIS - PHOTOAFFINITY-LABELING, PURIFICATION, AND CLONING OF THE CORRESPONDING GENE

A detailed comparison of citrate uptake into the vacuole like lutoids of rubber tree (Hevea brasiliensis Muell. Arg,) and of malate and citr ate transport into barley (Hordeum vulgare L.) vacuoles revealed very similar transport specificities, In order to identify proteins mediati ng the transport, two photoreactive analogues 2-hydroxy-5-azido)-diazo -N-3,5-benzenedicarboxylic acid and 5-azidoisophthalic acid) of malate /citrate were synthesized and found to efficiently inhibit citrate upt ake into barley vacuoles (K-i = 18 mu M) and Hevea lutoid vesicles (K- i = 27 mu M). In vacuoles from both plant species, these photoaffinity probes specifically labeled a single protein with a molecular mass of 23.6 kDa. This citrate binding protein (CBP) was purified to homogene ity from Hevea lutoids, and amino acid sequences were determined for N H2-terminal and tryptic peptides, Using degenerate oligonucleotides of the NH2-terminal sequence, a cDNA coding for the CBP protein of Hevea was isolated. The cDNA codes for a precursor protein of 238 amino aci ds, containing an NH2-terminal 31-amino acid signal sequence for endop lasmic reticulum targeting, a prerequisite for vacuolar localization. The mature CBP does not show significant sequence similarities to any known primary protein structure and thus represents a member of a nove l class of proteins.