Lin C M, Crawford B F, Kosman D J
Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York, Buffalo 14214.
J Gen Microbiol. 1993 Jul;139(7):1617-26. doi: 10.1099/00221287-139-7-1617.
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 64Cu by wild type and met3-containing (ATP sulphurylase-deficient) strains have been kinetically dissected. The Michaelis constant for uptake (4 microM) 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 64Cu also suggests the presence of this intermediate pool of bound copper. The Vmax for uptake includes a constant contribution from accumulation of 64Cu within the plasmalemma [0.1 nmol min-1 (mg protein)-1] plus that fraction of the 64Cu within the intermediate pool which diffuses away and is trapped on the cell wall as a metal sulphide. This latter contribution to Vmax can be two- to three-times greater than the intracellular uptake depending on the amount and type of sulphur supplementation provided in the 64Cu2+ 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 64Cu accumulation is strongly inhibited by Zn2+ and Ni2+. This inhibition is due to competition for cell-generated sulphide; in the presence of 65Zn2+, the decrease in 64Cu bound is quantitatively related to the amount of 65Zn which becomes cell-associated. In contrast, intracellular 64Cu uptake is not inhibited by these two metals (at 50 microM) 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.
在酿酒酵母中，铜与细胞的结合可能涉及铜与细胞壁的结合以及细胞内铜的积累。前一过程需要细胞通过硫酸盐还原同时产生硫化氢。已对野生型和含met3（缺乏ATP硫酸化酶）菌株摄取64Cu时这两个过程各自的贡献进行了动力学分析。摄取的米氏常数（4 microM）与正在发生的细胞结合类型无关，这表明（尽管不是必需的）两个过程都与一个共同的动力学中间体相关。64Cu与细胞结合的时间依赖性也表明存在这种结合铜的中间池。摄取的Vmax包括质膜内64Cu积累的恒定贡献[0.1 nmol min-1（mg蛋白质）-1]，加上中间池中扩散并作为金属硫化物被困在细胞壁上的那部分64Cu。对Vmax的后一种贡献可能比细胞内摄取大两到三倍，这取决于64Cu2+摄取缓冲液中提供的硫补充量和类型。两个过程都依赖能量，尽管依赖硫化物的周质积累对代谢抑制更为敏感。这可归因于硫酸盐还原为亚硫酸盐再还原为硫化物之前激活硫酸盐所需的ATP。周质64Cu积累受到Zn2+和Ni2+的强烈抑制。这种抑制是由于对细胞产生的硫化物的竞争；在存在65Zn2+的情况下，64Cu结合量的减少与细胞结合的65Zn量在数量上相关。相比之下，细胞内64Cu摄取不受这两种金属（50 microM）的抑制，表明铜转运途径具有金属特异性。这些观察结果提出了一个关于新到达的铜在细胞膜上的处理方式以及如何分配用于细胞内摄取的模型。
