Marchi Emmanuela, Lodi Tiziana, Donnini Claudia
Dipartimento di Genetica, Biologia dei Microrganismi, Antropologia, Evoluzione, University of Parma, Parma, Italy.
FEMS Yeast Res. 2007 Aug;7(5):715-21. doi: 10.1111/j.1567-1364.2007.00235.x. Epub 2007 Apr 12.
The original purpose of the experiments described in this article was to identify, in the biotechnologically important yeast Kluyveromyces lactis, gene(s) that are potentially involved in oxidative protein folding within the endoplasmic reticulum (ER), which often represents a bottleneck for heterologous protein production. Because treatment with the membrane-permeable reducing agent dithiothreitol inhibits disulfide bond formation and mimics the reducing effect that the normal transit of folding proteins has in the ER environment, the strategy was to search for genes that conferred higher levels of resistance to dithiothreitol when present in multiple copies. We identified a gene (KNQ1) encoding a drug efflux permease for several toxic compounds that in multiple copies conferred increased dithiothreitol resistance. However, the KNQ1 product is not involved in the excretion of dithiothreitol or in recombinant protein secretion. We generated a knq1 null mutant, and showed that both overexpression and deletion of the KNQ1 gene resulted in increased resistance to dithiothreitol. KNQ1 amplification and deletion resulted in enhanced transcription of iron transport genes, suggesting, for the membrane-associated protein Knq1p, a new, unexpected role in iron homeostasis on which dithiothreitol tolerance may depend.
本文所述实验的最初目的是，在具有重要生物技术意义的乳酸克鲁维酵母中，鉴定可能参与内质网（ER）内氧化蛋白折叠的基因，内质网内的氧化蛋白折叠通常是异源蛋白生产的一个瓶颈。由于用膜通透性还原剂二硫苏糖醇处理会抑制二硫键形成，并模拟折叠蛋白正常转运在ER环境中的还原作用，因此策略是寻找当以多拷贝存在时赋予对二硫苏糖醇更高抗性水平的基因。我们鉴定出一个基因（KNQ1），它编码一种对几种有毒化合物具有药物外排通透酶功能的蛋白，该基因以多拷贝存在时可增强对二硫苏糖醇的抗性。然而，KNQ1产物并不参与二硫苏糖醇的排泄或重组蛋白的分泌。我们构建了一个knq1基因缺失突变体，并表明KNQ1基因的过表达和缺失均导致对二硫苏糖醇的抗性增加。KNQ1基因的扩增和缺失导致铁转运基因的转录增强，这表明对于膜相关蛋白Knq1p而言，在铁稳态中具有一个新的、意想不到的作用，而二硫苏糖醇耐受性可能依赖于此。
