Stimulation of active uptake of nucleosides and amino acids by cyclic adenosine 3' :5'-monophosphate in the yeast Schizosaccharomyces pombe.

Abstract

In conditions of glucose starvation, the maximum velocity of the mediated transport of nonmetabolized and metabolized amino acids, uridine, adenosine, and sucrose across the plasma membrane is stimulated by a factor of two by the addition of 1 mM adenosine 3':5'-monophosphate to Schizosaccharomyces pombe 972h- wild strain, to the glucose-super-repressed and derepressed mutants COB5 and COB6, and to Saccharomyces cerevisiae strain IL 216-IA. The mediated uptake of 2-D-deoxyglucose and the apparently nonmediated uptake of guanosine are not stimulated by the cyclic nucleotide. N6,O2'-Dibutyryl adenosine 3':5'-monophosphate is also efficient, whereas theophylline, guanosine 3':5'-monophosphate, 5'-AMP, ATP, and adenosine are ineffective. The cellular ATP content of glycerol-grown S. pombe COB5 is about 10 nmol per mg of protein and is not decreased by further incubation in the starvation medium. The addition of 100 mM glucose markedly enhances transport without any increase of the cellular ATP content. The addition of antimycin A or Dio-9 decreases markedly both cellular ATP content and transport. The addition of 2.5 mM glucose to antimycin A-containing medium restores both transport is not necessarily of mitochondrial origin. The uptake of 2-D-deoxyglucose is unaffected by the respiratory inhibitors. Stimulation of uptake by cyclic adenosine 3':5'-monophosphate occurs only in glucose-deprived cells. The addition of 10 mM glucose elicits the disappearance of the stimulation and prevents the 30% decrease of the cellular adenosine 3':5'-monophosphate content produced by glucose starvation. Adenosine 3':5'-'monophosphate does not enhance the steady state ATP level but requires cellular ATP produced either by endogenous respiration or, in the absence of respiration blocked by antimycin A, by further addition of 2.5 mM glucose. Stimulation of active uptake by adenosine 3':5'-monophosphate does not require protein synthesis because the addition of cycloheximide or anisomycin does not prevent the stimulation of L-leucine uptake. In the absence of respiration, Dio-9, and ATPase inhibitor, suppresses instantaneously the cellular ejection of protons as well as the uptake of uridine and amino acids. It abolishes also the adenosine 3':5'-monophosphate-stimulated transport. In the presence of antimycin A, specific mitochondrial ATPase inhibitors such as venruricidin A do not inhibit metabolite uptakes and their stimulation by adenosine 3':5'-monophosphate. These results suggest that in these conditions, the target of Dio-9 is not the mitochondrial ATPase but a plasma membrane proton-translocating function generating an electrochemical gradient required for active transport. That adenosine 3':5'-monophosphate enhances the Dio-9-sensitive proton extrusion supports the view that the cyclic nucleotide might modulate the plasma membrane ATPase.