IDENTIFICATION WITH A PHOTOAFFINITY REAGENT OF A TONOPLAST PROTEIN INVOLVED IN VACUOLAR MALATE TRANSPORT OF CATHARANTHUS-ROSEUS

The effect of N(4-azido-salicylyl) aspartic acid (AzSA), a photolysabl e analogue of malate, was tested on the malate transport activity of t onoplast vesicles isolated from Catharanthus roseus cell suspension cu ltures. AzSA inhibited malate uptake in a competitive manner with a K- i of 1.7 millimolar. When iodinated, the malate analogue was found to be still photolysable and a competitive inhibitor of malate uptake. Ph otolysis of I-125-labelled AzSA in the presence of purified tonoplast vesicles led to label incorporation into several polypeptides after an alysis by gel electrophoresis. Only one polypeptide, with an apparent molecular mass of 37 kDa, was totally protected by the inclusion of 50 millimolar malate, the original substrate, in the photolysis medium. The labelled polypeptide is therefore apparently a specific malate-bin ding protein. Diethylpyrocarbonate (DEPC), a very potent inhibitor of malate transport acting at the active site of the transporter, also pr otected the 37 kDa polypeptide from labelling. Citrate and, to a lesse r extent, quinate afforded protection from labelling whilst other orga nic acids or aspartic acid (100 millimolar) did not. These photoprotec tion results are in good agreement with the data concerning the specif icity of malate transport across the tonoplast. Polyclonal antibodies against the 37 kDa polypeptide strongly inhibited malate uptake both i n tonoplast vesicles and in isolated vacuoles. These results suggest t he involvement of the 37 kDa polypeptide in vacuolar malate transport.