Chaudhuri B, Ingavale S, Bachhawat A K
Institute of Microbial Technology, Chandigarh, India.
Genetics. 1997 Jan;145(1):75-83. doi: 10.1093/genetics/145.1.75.
Mutants in the adenine biosynthetic pathway of yeasts (ade1 and ade2 of Saccharomyces cerevisiae, ade6 and ade7 of Schizosaccharomyces pombe) accumulate an intense red pigment in their vacuoles when grown under adenine-limiting conditions. The precise events that determine the formation of the pigment are however, still unknown. We have begun a genetic investigation into the nature and cause of pigmentation of ade6 mutants of S. pombe and have discovered that one of these pigmentation defective mutants, apd1 (adenine pigmentation defective), is a strict glutathione auxotroph. The gene apd1+ was found to encode the first enzyme in glutathione biosynthesis, gamma-glutamylcysteine synthetase, gcs1+. This gene when expressed in the mutant could confer both glutathione prototrophy and the characteristic red pigmentation, and disruption of the gene led to a loss in both phenotypes. Supplementation of glutathione in the medium, however, could only restore growth but not the pigmentation because the cells were unable to achieve sufficient intracellular levels of glutathione. Disruption of the second enzyme in glutathione biosynthesis, glutathione synthetase gsh2+, also led to glutathione auxotrophy, but only a partial defect in pigment formation. A reevaluation of the major amino acids previously reported to be present in the pigment indicated that the pigment is probably a glutathione conjugate. The ability of vanadate to inhibit pigment formation indicated that the conjugate was transported into the vacuole through a glutathione-conjugate pump. This was further confirmed using strains of S. cerevisiae bearing disruptions in the recently identified glutathione-conjugate pump, YCF1, where a significant reduction in pigment formation was observed. The pump of S. pombe is distinct from the previously identified vacuolar pump, hmt1p, for transporting cadystin peptides into vacuoles of S. pombe.
酵母腺嘌呤生物合成途径中的突变体（酿酒酵母的ade1和ade2，粟酒裂殖酵母的ade6和ade7）在腺嘌呤限制条件下生长时，其液泡中会积累强烈的红色色素。然而，决定色素形成的精确事件仍然未知。我们已经开始对粟酒裂殖酵母ade6突变体色素沉着的性质和原因进行遗传学研究，并发现其中一个色素沉着缺陷突变体apd1（腺嘌呤色素沉着缺陷）是严格的谷胱甘肽营养缺陷型。发现apd1+基因编码谷胱甘肽生物合成中的第一种酶，γ-谷氨酰半胱氨酸合成酶gcs1+。该基因在突变体中表达时可赋予谷胱甘肽原养型和特征性红色色素沉着，而该基因的破坏会导致两种表型丧失。然而，在培养基中补充谷胱甘肽只能恢复生长，而不能恢复色素沉着，因为细胞无法达到足够的细胞内谷胱甘肽水平。谷胱甘肽生物合成中的第二种酶谷胱甘肽合成酶gsh2+的破坏也导致谷胱甘肽营养缺陷型，但仅在色素形成方面存在部分缺陷。对先前报道的色素中存在的主要氨基酸的重新评估表明，该色素可能是谷胱甘肽缀合物。钒酸盐抑制色素形成的能力表明，该缀合物通过谷胱甘肽缀合物泵转运到液泡中。使用最近鉴定的谷胱甘肽缀合物泵YCF1存在破坏的酿酒酵母菌株进一步证实了这一点，在该菌株中观察到色素形成显著减少。粟酒裂殖酵母的泵与先前鉴定的用于将卡迪辛肽转运到粟酒裂殖酵母液泡中的液泡泵hmt1p不同。
