A membrane-potential dependent ABC-like transporter mediates the vacuolar uptake of rye flavone glucuronides: regulation of glucuronide uptake by glutathione and its conjugates

In this paper we present results on the vacuolar uptake mechanism for two f lavone glucuronides present in rye mesophyll vacuoles. In contrast to barle y flavone glucosides (Klein et al. (1996) J. Biol. Chem. 271, 29666-29671), the flavones luteolin 7-O-diglucuronyl-4'-O-glucuronide (R1) and luteolin 7-O-diglucuronide (R2) were taken up into vacuoles isolated from rye via a directly energised mechanism. Kinetic studies suggested that the vacuolar g lucuronide transport system is constitutively expressed throughout rye prim ary leaf development. Competition experiments argued for the existence of a plant MRP-like transporter for plant-specific and non-plant glucuronides s uch as beta-estradiol 17-(beta-d-glucuronide) (E(2)17G). The interaction of ATP-dependent vacuolar glucuronide uptake with glutathione and its conjuga tes turned out to be complex: R1 transport was stimulated by dinitrobenzene -GS and reduced glutathione but was inhibited by oxidized glutathione in a concentration-dependent manner. In contrast, R2 uptake was not increased in the presence of reduced glutathione. Thus, the transport system for plant- derived glucuronides differed from the characteristic stimulation of vacuol ar E(2)17G uptake by glutathione conjugates but not by reduced glutathione (Klein et al. (1998) J. Biol. Chem. 273, 262-270). Using tonoplast vesicles isolated with an artificial K+ gradient, we demonstrate for the first time for plant MRPs that the ATP-dependent uptake of R1 is membrane-potential d ependent. We discuss the kinetic capacity of the ABC-type glucuronide trans porter to explain net vacuolar flavone glucuronide accumulation in planta d uring rye primary leaf development and the possibility of an interaction of potential substrates at both the substrate binding and allosteric sites of the MRP transporter regulating the activity towards a certain substrate.