Active transport of basic amino acids driven by a proton motive force in vacuolar membrane vesicles of Saccharomyces cerevisiae.
The mechanism of transport of basic amino acids into vacuoles of cells of the yeast Saccharomyces cerevisiae was investigated in vitro. Right-side-out vacuolar membrane vesicles were prepared from purified vacuoles. Arginine was taken up effectively by the vesicles only in the presence of ATP, not in the presence of ADP or AMP-adenosyl-5'-yl imidodiphosphate. It was exchangeable and was released completely by a protonophore, 3,5-di-tert-butyl-4-hydroxybenzilidenemalononitrile (SF6847). The transport required Mg2+ ion but was inhibited by Cu2+, Ca2+, or Zn2+ ions. The transport activity was sensitive to the ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD), but not to oligomycin or sodium vanadate. SF6847 or nigericin blocked arginine uptake completely, but valinomycin had no effect. ATP-dependent formation of a delta pH across the membrane vesicles was shown by quenching of 9-aminoacridine fluorescence. These results indicate that DCCD-sensitive, Mg2+-ATPase of vacuolar membranes is essential as an energy-donating system for the active transport, and that an electrochemical potential difference of protons is a driving force of this basic amino acid transport. Arginine transport showed saturation kinetics with a Km value of 0.6 mM and the mechanism was well explained by an H+/arginine antiport.
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001120:Arginine / Q000378:metabolism*; D001597:Benzylidene Compounds / Q000494:pharmacology; D001693:Biological Transport, Active / Q000187:drug effects; D002413:Cations, Divalent; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D005614:Freeze Fracturing; D007700:Kinetics; D009570:Nitriles / Q000494:pharmacology; D009840:Oligomycins / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D014639:Vanadium / Q000494:pharmacology
D001597:Benzylidene Compounds; D002413:Cations, Divalent; D009570:Nitriles; D009840:Oligomycins; D014639:Vanadium; C001784:SF 6847; D004024:Dicyclohexylcarbodiimide; D001120:Arginine; D000251:Adenosine Triphosphatases