Accumulation of alkaloids in plant vacuoles does not involve an ion-trap mechanism

Abstract

Alkaloid uptake into vacuoles isolated from a Fumaria capreolata L. cell suspension culture was investigated. The uptake is carrier-mediated as shown by its substrate saturation, its sensitivity to metabolic inhibitors and especially by its exclusive preference for the (S)-forms of reticuline and scoulerine while the (R)-enantiomers which do not occur in this plant species were strictly discriminated. The carrier has a high affinity for (S)-reticuline with a K m=0.3 μM. The rate of alkaloid uptake was 6 pmol·h(-1)·μl(-1) vacuole, and 0.03 mg alkaloid·mg(-1) vacuolar protein were taken up. Transport was stimulated five-to seven-fold by ATP and was inhibited by the ATPase inhibitors N,N'-dicyclohexylcarbodiimide and 4-4'-diisothiocyanatostilbene-2,2' disulfonic acid, as well as by the protonophore carbonyl cyanide m-chlorophenylhydrazone. A number of alkaloids did not compete with labelled (S)-reticuline for uptake into vacuoles. The uptake system is absolutely specific for alkaloids indigenous to the plant from which the vacuoles were isolated. Slight modifications of the topography of an alkaloid molecule even with full retention of its electrical charge results in its exclusion. Alkaloid efflux was also shown to be mediated by a highly specific energy-dependent carrier. These results contradict the previously proposed ion-trap mechanism for alkaloid accumulation in vacuoles. A highly specific carrier-mediated and energy-dependent proton antiport system for alkaloid uptake and release is postulated.