DISTRIBUTION OF CU-64 IN SACCHAROMYCES-CEREVISIAE - KINETIC ANALYSES OF PARTITIONING

The cell association of copper in the yeast Saccharomyces cerevisiae can involve both binding to the cell wall and the accumulation of copper within the cell. The former process requires the concurrent generation of H2S by the cell via the reduction of sulphate. The contributions of each of these processes to the uptake of Cu-64 by wild type and met3-containing (ATP sulphurylase-deficient) strains have been kinetically dissected. The Michaelis constant for uptake (4 muM) is independent of the type of cell association which is occurring, suggesting, although not requiring, that both processes are associated with a common kinetic intermediate. The time dependence of the cell-association of Cu-64 also suggests the presence of this intermediate pool of bound copper. The V(max) for uptake includes a constant contribution from accumulation of Cu-64 within the plasmalemma [0.1 nmol min-1 (mg protein)-1] plus that fraction of the Cu-64 within the intermediate pool which diffuses away and is trapped on the cell wall as a metal sulphide. This latter contribution to V(max) can be two- to three-times greater than the intracellular uptake depending on the amount and type of sulphur supplementation provided in the Cu-64(2+) uptake buffer. Both processes are energy-dependent although the sulphide-dependent periplasmic accumulation is somewhat more sensitive to metabolic inhibition. This can be attributed to the ATP required for the activation of sulphate prior to its reduction to the level of sulphite and then sulphide. Periplasmic Cu-64 accumulation is strongly inhibited by Zn2+ and Ni2+. This inhibition is due to competition for cell-generated sulphide; in the presence of Zn-65(2+), the decrease in Cu-64 bound is quantitatively related to the amount of Zn-65 which becomes cell-associated. In contrast, intracellular Cu-64 uptake is not inhibited by these two metals (at 50 muM) showing that the copper translocation pathway is metal-specific. These observations suggest a model for the way newly arrived copper is handled at the cell membrane and is partitioned for intracellular uptake.