Copper uptake in wild type and copper metallothionein-deficient Saccharomyces cerevisiae: Kinetics and mechanism

The mechanism of copper uptake in Saccharomyces cerevisiae has been investigated using a combination of ⁶⁴Cu²⁺ and atomic absorption spectrophotometry. A wild type copper-resistant CUP1^R-containing strain and a strain carrying a deletion of the CUP1 locus (yeast copper metallothionein) exhibited quantitatively similar saturable energy-dependent ⁶⁴Cu²⁺ up-take when cultures were pregrown in copper-free media (medium [Cu] ≈ 15 nM). The kinetic constants for uptake by the wild type strain were V_max = 0.21 nmol of copper/min/mg of protein and K_m = 4.4 μM. This accumulation of ⁶⁴Cu²⁺ represented net uptake as confirmed by atomic absorption spectrophotometry. This uptake was not seen in glucose-starved cells, but was supported in glycerol- and ethanol-grown ones. Uptake was inhibited by both N₃^- and dinitrophenol and was barely detectable in cultures at 4 °C. When present at 50 μM, Zn²⁺ and Ni²⁺ inhibited by 50% indicating that this uptake process was relatively selective for Cu²⁺. ⁶⁴Cu²⁺ accumulation was qualitatively and quantitatively different in cultures either grown in or preincubated with cold Cu²⁺. Either treatment resulted in the appearance of a fast phase (t_1/2 ≈ 1 min) of ⁶⁴Cu²⁺ accumulation which represented isotopic exchange since it did not lead to an increase in the mass of cell-associated copper; also, it was not energy-dependent. Exchange of ⁶⁴Cu²⁺ into this pool was not inhibited by Zn²⁺. Pretreatment with Cu²⁺ caused a change in the rate of net accumulation as well; a 3-h incubation of cells in 5 μM medium Cu²⁺ caused a 1.6-fold increase in the velocity of energy-dependent uptake. Prior addition of cycloheximide abolished this Cu²⁺-dependent increase and, in fact, inhibited the ⁶⁴Cu²⁺ uptake velocity by > 85%. The exchangeable pool was also absent in cycloheximide, Cu²⁺-treated cells suggesting that exchangeable Cu²⁺ derived from the copper taken up initially by the energy-dependent process. The thionein deletion mutant was similar to wild type in response to medium Cu²⁺ and cycloheximide indicating that copper metallothionein is not directly involved in Cu²⁺ uptake (as distinct from retention) in yeast.