TRANSPORT OF LUCIFER YELLOW CH INTO PLANT VACUOLES - EVIDENCE FOR DIRECT ENERGIZATION OF A SULFONATED SUBSTANCE AND IMPLICATIONS FOR THE DESIGN OF NEW MOLECULAR PROBES

Contrasting observations exist which indicate that in plants the fluor escent dye lucifer yellow CH (LYCH) either carl be used as a tracer fo r endocytosis or as a substrate for an anion transporter located at th e vacuolar membrane, in addition, LYCII as a disulphonated substance m ay represent an analogue of sulphonated or sulfated natural compounds like some flavonoids, We performed uptake experiments with LYCH into i solated rye vacuoles and observed saturable (K-m = 0.3-0.6 mM) vacuola r transport and accumulation of the dye against the concentration grad ient only when MgATP was present, GTP and, to a low extent, UTP could substitute for ATP, while the non-hydrolysable ATP analogue AMP-PNP di d not drive LYCH uptake, Vanadate and probenecid, the latter substance is known to inhibit organic anion transport at the liver canalicular membrane, both strongly decreased the vacuolar uptake of LYCH, while b afilomycin A1, a specific inhibitor of the vacuolar H--ATPase, had no effect, Together with the fact that abolishment of the Delta pH via CC CP had only a weak influence on LYCH accumulation, our results indicat e that this compound is taken up into rye vacuoles by a directly energ ized process, Uptake of LYCH was strongly inhibited by other sulfated compounds including sulfobromophthalein and the flavones apigenin 7,3' -disulfate and luteolin 7,4'-disulfate arguing for the presence of a v acuolar transporter for structurally different sulphonated or sulfated compounds, Glucuronates like the rye-specific flavone luteolin 7-O-di glucuronide also strongly decreased uptake of the dye, whereas only a weak effect was observed in the presence of glutathione and a glutathi one conjugate, suggesting that LYCH uptake is not mediated via the vac uolar glutathione conjugate pump. (C) 1997 Federation of European Bioc hemical Societies.