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

The cell association of copper in the yeast Saccharomyces cerevisiae c an involve both binding to the cell wall and the accumulation of coppe r within the cell. The former process requires the concurrent generati on 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 me t3-containing (ATP sulphurylase-deficient) strains have been kinetical ly dissected. The Michaelis constant for uptake (4 muM) is independent of the type of cell association which is occurring, suggesting, altho ugh not requiring, that both processes are associated with a common ki netic intermediate. The time dependence of the cell-association of Cu- 64 also suggests the presence of this intermediate pool of bound coppe r. The V(max) for uptake includes a constant contribution from accumul ation 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 dif fuses away and is trapped on the cell wall as a metal sulphide. This l atter contribution to V(max) can be two- to three-times greater than t he intracellular uptake depending on the amount and type of sulphur su pplementation provided in the Cu-64(2+) uptake buffer. Both processes are energy-dependent although the sulphide-dependent periplasmic accum ulation is somewhat more sensitive to metabolic inhibition. This can b e attributed to the ATP required for the activation of sulphate prior to its reduction to the level of sulphite and then sulphide. Periplasm ic Cu-64 accumulation is strongly inhibited by Zn2+ and Ni2+. This inh ibition is due to competition for cell-generated sulphide; in the pres ence of Zn-65(2+), the decrease in Cu-64 bound is quantitatively relat ed 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 i s handled at the cell membrane and is partitioned for intracellular up take.