Selective regulatory effects of purine and pyrimidine nucleotides on vacuolar transport of amino acids

The release of amino acids from their vacuolar store was studied in situ, i .e. in cells with selectively permeabilized plasma membrane and functionall y intact vacuoles. As we previously described [Roos et al., J. Biol. Chem. 272 (1997) 15849-15855], this transport process is regulated by extravacuol ar adenylates at their physiological concentrations. We now show, using our test object Penicillium cyclopium, that not only purine but also pyrimidin e nucleotides are involved in the control of efflux of vacuolar phenylalani ne. At 0.1 mM adenosine or guanosine phosphates inhibit, whereas cytidine o r uridine phosphates stimulate the rate of efflux. At 1 mM the same nucleot ides have no measurable impact on efflux but abolish the effects of other n ucleotides present at 0.1 mM. This argues for at least two interacting bind ing sites with different nucleotide affinities. The minimum structural requ irement for any of the observed effects is a non-cyclic ribonucleoside mono phosphate. In intact cells, cytosolic concentrations of ATP (representing p urine nucleotides) and CTP (representing pyrimidine nucleotides) are 1-2 mM and 0.05-0.2 mM, respectively. ATP is therefore assumed to dominate transp ort control and allow optimum efflux (and uptake) rates. Short-time starvat ion of carbon and nitrogen adjusts CTP and ATP at levels that cause declini ng efflux rates. During prolonged starvation both nucleotides fall below th eir transport-controlling concentrations and thus allow increasing rates of efflux from the still maintained vacuolar pool. Hence, efflux control unde r nutrient limitation includes an interplay of purine and pyrimidine nucleo tides which precisely regulates the release of vacuolar amino acids and ena bles flexible adjustment to either amino acid saving or cell survival. (C) 2000 Published by Elsevier Science B.V.