Lin C M, Kosman D J
Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York, Buffalo 14214.
J Biol Chem. 1990 Jun 5;265(16):9194-200.
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.
利用(^{64}Cu^{2 +})和原子吸收分光光度法相结合的方法，对酿酒酵母中铜的摄取机制进行了研究。当培养物在无铜培养基（培养基中铜含量约为(15 nM)）中预培养时，含有野生型耐铜(CUP1^{R})的菌株和携带(CUP1)基因座缺失（酵母铜金属硫蛋白）的菌株表现出定量相似的可饱和能量依赖性(^{64}Cu^{2 +})摄取。野生型菌株摄取的动力学常数为(V_{max}=0.21) nmol铜/分钟/毫克蛋白质，(K_{m}=4.4) (\mu M)。原子吸收分光光度法证实，(^{64}Cu^{2 +})的这种积累代表净摄取。在葡萄糖饥饿的细胞中未观察到这种摄取，但在以甘油和乙醇为碳源生长的细胞中可以观察到。摄取受到(N_{3}^{-})和二硝基苯酚的抑制，在(4^{\circ}C)的培养物中几乎检测不到。当(Zn^{2 +})和(Ni^{2 +})浓度为(50) (\mu M)时，摄取受到(50%)的抑制，表明这种摄取过程对(Cu^{2 +})具有相对选择性。在含有冷(Cu^{2 +})的培养基中生长或预孵育的培养物中，(^{64}Cu^{2 +})的积累在定性和定量上都有所不同。这两种处理都会导致(^{64}Cu^{2 +})积累出现一个快速阶段（半衰期约为(1)分钟），这代表同位素交换，因为它不会导致细胞相关铜质量的增加；而且，它不依赖能量。(^{64}Cu^{2 +})与这个池的交换不受(Zn^{2 +})的抑制。用(Cu^{2 +})预处理也会导致净积累速率的变化；细胞在(5) (\mu M)培养基铜中孵育(3)小时，能量依赖性摄取速度增加了(1.6)倍。预先添加环己酰亚胺消除了这种(Cu^{2 +})依赖性增加，实际上，将(^{64}Cu^{2 +})摄取速度抑制了(85%)以上。在环己酰亚胺、(Cu^{2 +})处理的细胞中也不存在可交换池，这表明可交换(Cu^{2 +})源自最初通过能量依赖性过程摄取的铜。硫蛋白缺失突变体在对培养基铜和环己酰亚胺的反应上与野生型相似，表明铜金属硫蛋白在酵母中不直接参与(Cu^{2 +})的摄取（与保留不同）。
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