Vacuolar transport of the glutathione conjugate of trans-cinnamic acid

Abstract

Red beet ( Beta vulgaris L.) tonoplast membrane vesicles and [ 14C] trans-cinnamic acid-glutathione were used to study the vacuolar transport of phenylpropanoid-glutathione conjugates which are formed in peroxidase-mediated reactions. It was determined that the uptake of [ 14C] trans-cinnamic acid-glutathione into the tonoplast membrane vesicles was MgATP dependent and was 10-fold faster than the uptake of non-conjugated [ 14C] trans-cinnamic acid. Uptake of the conjugate in the presence of MgATP was not dependent on a trans-tonoplast H +-electrochemical gradient, because uptake was not affected by the addition of NH 4Cl (1 mM; 0% inhibition) and was only slightly affected by gramicidin-D (5 μM; 14% inhibition). Uptake of the conjugate was inhibited 92% by the addition of vanadate (1 mM) and 71% by the addition of the model substrate S-(2,4-dinitrophenyl) glutathione (500 μM). Uptake did not occur when a nonhydrolyzable analog of ATP was used in place of MgATP. The calculated K m and V max values for uptake were 142 μM and 5.95 nmol mg −1 min −1, respectively. Based on these results, phenylpropanoid-glutathione conjugates formed in peroxidase-mediated reactions appear to be transported into the vacuole by the glutathione S-conjugate pump(s) located in the tonoplast membrane.