DIRECTLY ENERGIZED UPTAKE OF BETA-ESTRADIOL 17-(BETA-D-GLUCURONIDE) IN PLANT VACUOLES IS STRONGLY STIMULATED BY GLUTATHIONE CONJUGATES

A directly energized vacuolar pump for glutathione (GS) conjugates has been described for several plant species, Since glucuronate conjugate s also occur in plants, we addressed the question whether plant vacuol es take up the abiotic glucuronate conjugate estradiol 17-(beta-glucur onide) (E(2)17G) via a GS conjugate pump, which in some cases has been reported to accept various organic anions as substrate, or via a dist inct glucuronate transporter, Uptake studies into vacuoles from rye an d barley were performed with E(2)17G and metolachlor-GS (MOC-GS), a su bstrate of the GS conjugate ATPase, to compare glucuronate conjugate t ransport into vacuoles containing endogenous flavone glucuronides with those lacking specific glucuronate conjugates, respectively, Our resu lts indicate that E(2)17G and MOC-GS are taken up into vacuoles of bot h plants via a directly energized mechanism since transport was (i) st rictly ATP-dependent; (ii) inhibited by vanadate but not by bafilomyci n Al, azide, verapamil, nor by dissipation of the vacuolar Delta pH or Delta Psi; (iii) E(2)17G uptake into rye vacuoles was partially drive n by other nucleotides in the following order of efficiency: ATP > GTP > UTP congruent to CTP, whereas the non-hydrolyzable ATP analogue 5'- adenylyl-beta,gamma-imidodiphosphate, ADP, or PP; did not energize upt ake, E(2)17G transport into rye vacuoles was saturable (K-m approximat e to 0.2 mM), The rye-specific luteolin glucuronides decreased uptake rates of E(2)17G and MOC-GS into rye and barley vacuoles to comparable degrees with the mono-and diglucuronidated derivatives (40-60% inhibi tion) being more effective than the triglucuronide, Inhibition of E(2) 17G uptake by luteolin 7-O-diglucuronide was competitive (K-i = 120 mu M) Taurocholate had no effect on E(2)17G transport, and uptake of MOC -GS was not inhibited by E(2)17G, Although GS conjugates and oxidized GS decreased MOC-GS transport, E(2)17G uptake into rye and barley vacu oles was stimulated up to 7-fold in a concentration-dependent manner b y these substances, with dinitrobenzene-GS being most effective, The s timulation of the GS conjugates was not due to detergent or redox effe cts and was specific for the E(2)17G pump, GS conjugate stimulation of glucuronate uptake was unique for plants as E(2)17G uptake into yeast microsomal vesicles was not affected, By comparison with a Delta YCF1 yeast mutant, defective in vacuolar transport of GS conjugates mediat ed by YCF1, it was shown that E(2)17G was taken up into yeast vesicles via a YCF1-independent directly energized pump, These results indicat e that E(2)17G as a glucuronate conjugate is transported across the va cuolar membranes of plants and yeast by a carrier distinct from the GS conjugate ATPase.