Active transport of basic amino acids driven by a proton motive force in vacuolar membrane vesicles of Saccharomyces cerevisiae.

Y Ohsumi,Y Anraku

doi:10.1016/s0021-9258(19)69736-x

Abstract

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.

Highlights

We are interested in the mechanism controlling the intracellular distribution of amino acidsin yeast cells, andto characterize the properties of transport systems for amino acids in vacuolar membrane we established a method for preparing pure vacuolar membrane vesicles from intact vacuoles
Arginine was taken up effectively by the vesicles only Chemicals-~-[U-’~C]Argini(n3e42 mCi/mmol) and other radioin the presence of ATP, not in the presenceof ADP or active compounds were purchased from Amersham
A r g iTnrianne sport of YVeaacsuMt oelamrbVraensiecles of intact vacuoles the resulting vesicles showed less than 104 of the arginine uptake activity of vesicles prepared by the standard procedure

Summary

Yoshinori Ohsumi and Yasuhiro Anrakus

From the Departmenotf Biology, Faculty of Science, University of Tokyo, Hongo, Tokyo 113, Japan isolatedvacuoles withoutany energy supply.Buthitherto attempts to show active transport by isolated vacuoles have failed [9]. We are interested in the mechanism controlling the intracellular distribution of amino acidsin yeast cells, andto characterize the properties of transport systems for amino acids in vacuolar membrane we established a method for preparing pure vacuolar membrane vesicles from intact vacuoles. This paperdescribes the mechanismof active transport of arginine by vacuolar membranes, which is well explained by H+/arginine antiport driven by a proton motive force

The mechanism of transport of basic amino ianctiods

AND DISCUSSION

ArgiTnriannesport of YVeaacsuMt oelamrbVraensiecles

Consistent with itseffect in inducing effluxof arginine from