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 CUP 1R-containing strain and a strain carrying a deletion of the CUP1 locus (yeast copper metallothionein) exhibited quantitatively similar saturable energy-dependent 64Cu2+ uptake when cultures were pregrown in copper-free media (medium [Cu] approximately 15 nM). The kinetic constants for uptake by the wild type strain were Vmax = 0.21 nmol of copper/min/mg of protein and Km = 4.4 microM. 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 degrees C. When present at 50 microM, 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 approximately 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 microM 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 greater than 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.
Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York, Buffalo 14214.
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D001386:Azides / Q000494:pharmacology; D001693:Biological Transport, Active / Q000187:drug effects; D002352:Carrier Proteins; D002413:Cations, Divalent; D003300:Copper / Q000378:metabolism*; D003301:Copper Radioisotopes; D003513:Cycloheximide / Q000494:pharmacology; D004140:Dinitrophenols / Q000494:pharmacology; D007700:Kinetics; D008668:Metallothionein / Q000235:genetics / Q000502:physiology*; D009154:Mutation; D009532:Nickel / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013054:Spectrophotometry, Atomic; D015032:Zinc / Q000494:pharmacology
D001386:Azides; C500769:CUP1-1 protein, S cerevisiae; D002352:Carrier Proteins; D002413:Cations, Divalent; D003301:Copper Radioisotopes; D004140:Dinitrophenols; C007121:copper thionein; D003300:Copper; D009532:Nickel; D008668:Metallothionein; D003513:Cycloheximide; D015032:Zinc