Mobilisation of vacuolar amino acids in leaf cells as affected by ATP and the level of cytosolic amino acids: ATP regulates but appears not to energize vacuolar amino-acid release

Abstract

The vacuoles of mesophyll cells are capable of storing amino acids which are mobilized on demand. An amino-acid carrier was characterized by studying the efflux of vacuolar amino acids from isolated mesophyll vacuoles using the silicon oil layer centrifugation technique. Efflux was slow in the absence of ATP. It was stimulated by a factor of more than 5 by the addition of ATP. A similar stimulation was brought about by adenylyl imidodiphosphate ( Ado PP [NH] P ), suggesting that efflux activation by ATP did not involve ATP hydrolysis. GTP, CTP, UTP and ADP were not effective, or were much less effective than ATP, in activating the amino-acid transport system. Neutral amino acids inhibited ATP-stimulated amino-acid efflux by an allosteric mechanism. External leucine, valine, phenylalanine and methionine were more effective in inhibiting efflux than alanine, arginine, lysine or glutamic acid when present outside the vacuole. Protein-modifying agents such as N -ethylmaleimide and p -chloromercuriphenylsulfonic acid activated amino-acid efflux from the vacuoles, whereas proteinase treatment led to an inhibition. Stimulation by ATP of amino-acid export from the vacuole and inhibition by specific amino acids may be part of a mechanism to stabilize cytoplasmic amino-acid levels during protein synthesis.