Department of Biological Sciences, Indian Institute of Science Education and Research, Mohali, S.A.S. Nagar 140 306, India.
J Biosci. 2020;45.
The adenine biosynthetic mutants ade1 and ade2 of Saccharomyces cerevisiae accumulate a characteristic red pigment in their vacuoles under adenine limiting conditions. This red pigmentation phenotype, widely used in a variety of genetic screens and assays, is the end product of a glutathione-mediated detoxification pathway, where the glutathione conjugates are transported into the vacuole. The glutathione conjugation step, however, has still remained unsolved. We show here, following a detailed analysis of all the members of the thioredoxinfold superfamily, the involvement of the monothiol glutaredoxin GRX4 as essential for pigmentation. GRX4 plays multiple roles in the cell, and we show that the role in ade pigmentation does not derive from its regulatory role of the iron transcription factor, Aft1p, but a newly identified GST activity of the protein that we could demonstrate using purified Grx4p. Further, we demonstrate that the GRX domain of GRX4 and its active site cysteine C171 is critical for this activity. The findings thus solve a decades old enigma on a critical step in the formation of this red pigmentation.
酿酒酵母的腺嘌呤生物合成突变体 ade1 和 ade2 在腺嘌呤限制条件下其液泡中积累一种特征性的红色色素。这种广泛用于各种遗传筛选和测定的红色着色表型是谷胱甘肽介导的解毒途径的终产物，其中谷胱甘肽缀合物被转运到液泡中。然而，谷胱甘肽缀合步骤仍然未解决。我们在这里展示，在对硫氧还蛋白折叠超家族的所有成员进行详细分析后，单硫醇谷胱甘肽还原酶 GRX4 的参与对于着色是必不可少的。GRX4 在细胞中发挥多种作用，我们表明，在 ade 色素沉着中的作用不是来自其对铁转录因子 Aft1p 的调节作用，而是来自该蛋白新鉴定的 GST 活性，我们可以使用纯化的 Grx4p 证明这一点。此外，我们证明 GRX4 的 GRX 结构域及其活性位点半胱氨酸 C171 对于该活性至关重要。这些发现解决了一个关于这种红色着色形成的关键步骤的几十年未解之谜。
