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):1605-15. doi: 10.1099/00221287-139-7-1605.
The metabolism of copper in the yeast Saccharomyces cerevisiae has been studied with respect to the distribution and stability to exchange of newly arrived 64Cu. Cells pre-incubated with 10 microM-Cu2+ accumulated 64Cu into two pools distinguishable by cellular locale and lability to exchange with extracellular cold copper. One pool was non-exchangeable and was localized to protoplasts. Size-exclusion chromatography of a soluble cell (protoplast) extract showed that this 64Cu was associated with up to four species. Two were identified as copper metallothionein and Cu,Zn superoxide dismutase based on comparisons of chromatograms derived from strains in which the genes for these two proteins had been deleted. A third species was identified as copper-glutathione based on chromatographic and biochemical assays. A second pool was exchangeable and was localized to the cell wall. In contrast to its rapid copper-stimulated exchange (t1/2 approximately 1 min), this pool exhibited only slow efflux (10% 64Cu loss per 60 min). Zn2+ did not stimulate the loss of 64Cu from this pool indicating that it was selective for copper. This pool was released into the supernatant upon protoplast formation and was found in the cell wall debris obtained when cells were mechanically disrupted. This 64Cu eluted in the void volume (peak Pv) of the column used to size-fractionate copper-binding species. The metal in Pv was exchangeable in vivo and in vitro. However, the corresponding chromatographic fraction obtained from copper-naive cells when labelled in vitro could bind less than 20% of the 64Cu bound to it in vivo indicating that the deposition of copper in this pool was primarily cell-dependent. In fact, this deposition was shown to be dependent on the cellular reduction of medium sulphate or sulphite to the level of sulphide, or on the addition of sulphide to the 64Cu uptake buffer. 64Cu in the non-exchangeable protoplast pool was not mobilized by cellular sulphide generation, indicating that cellular sulphide generation did not causally lead to the partitioning of 64Cu to the cell wall pool. The data indicate that the appearance of copper sulphide(s) on the cell wall in S. cerevisiae is gratuitous and does not represent a sulphide-based mechanism of copper resistance in this yeast.
关于酿酒酵母中铜的代谢，已针对新引入的64Cu的分布和交换稳定性进行了研究。用10微摩尔/升的Cu2+预孵育的细胞将64Cu积累到两个池中，这两个池可根据细胞位置和与细胞外冷铜交换的不稳定性来区分。一个池是不可交换的，定位于原生质体。对可溶性细胞（原生质体）提取物进行尺寸排阻色谱分析表明，这种64Cu与多达四种物质相关。基于对来自这两种蛋白质基因已被删除的菌株的色谱图的比较，其中两种被鉴定为铜金属硫蛋白和铜锌超氧化物歧化酶。基于色谱和生化分析，第三种物质被鉴定为铜 - 谷胱甘肽。第二个池是可交换的，定位于细胞壁。与其快速的铜刺激交换（半衰期约为1分钟）相反，这个池仅表现出缓慢的流出（每60分钟64Cu损失10%）。Zn2+不会刺激这个池中64Cu的损失，表明它对铜具有选择性。这个池在原生质体形成时释放到上清液中，并在细胞机械破碎时获得的细胞壁碎片中发现。这种64Cu在用于对铜结合物质进行尺寸分级的柱的空体积（峰Pv）中洗脱。峰Pv中的金属在体内和体外都是可交换的。然而，当在体外标记时，从未接触过铜的细胞中获得的相应色谱级分结合的64Cu不到其在体内结合量的20%，这表明该池中铜的沉积主要依赖于细胞。事实上，这种沉积被证明依赖于细胞将培养基中的硫酸盐或亚硫酸盐还原为硫化物的水平，或者依赖于向64Cu摄取缓冲液中添加硫化物。不可交换的原生质体池中的64Cu不会因细胞产生硫化物而被调动，这表明细胞产生硫化物不会因果导致64Cu分配到细胞壁池中。数据表明，酿酒酵母细胞壁上硫化铜（们）的出现是无端的，并不代表该酵母中基于硫化物的铜抗性机制。
