Maeta Kazuhiro, Izawa Shingo, Okazaki Shoko, Kuge Shusuke, Inoue Yoshiharu
Laboratory of Molecular Microbiology, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
Mol Cell Biol. 2004 Oct;24(19):8753-64. doi: 10.1128/MCB.24.19.8753-8764.2004.
Methylglyoxal (MG) is synthesized during glycolysis, although it inhibits cell growth in all types of organisms. Hence, it has long been asked why such a toxic metabolite is synthesized in vivo. Glyoxalase I is a major enzyme detoxifying MG. Here we show that the Yap1 transcription factor, which is critical for the oxidative-stress response in Saccharomyces cerevisiae, is constitutively concentrated in the nucleus and activates the expression of its target genes in a glyoxalase I-deficient mutant. Yap1 contains six cysteine residues in two cysteine-rich domains (CRDs), i.e., three cysteine residues clustering near the N terminus (n-CRD) and the remaining three cysteine residues near the C terminus (c-CRD). We reveal that any of the three cysteine residues in the c-CRD is sufficient for MG to allow Yap1 to translocate into the nucleus and to activate the expression of its target gene. A Yap1 mutant possessing only one cysteine residue in the c-CRD but no cysteine in the n-CRD and deletion of the basic leucine zipper domain can concentrate in the nucleus with MG treatment. However, substitution of all the cysteine residues in Yap1 abolishes the ability of this transcription factor to concentrate in the nucleus following MG treatment. The redox status of Yap1 is substantially unchanged, and protein(s) interaction with Yap1 through disulfide bond is hardly detected in cells treated with MG. Collectively, neither intermolecular nor intramolecular disulfide bond formation seems to be involved in Yap1 activation by MG. Moreover, we show that nucleocytoplasmic localization of Yap1 closely correlates with growth phase and intracellular MG level. We propose a novel regulatory pathway underlying Yap1 activation by a natural metabolite in the cell.
甲基乙二醛（MG）在糖酵解过程中合成，尽管它会抑制所有类型生物体中的细胞生长。因此，长期以来人们一直想问，为什么体内会合成这种有毒的代谢物。乙二醛酶I是一种主要的解毒MG的酶。在这里我们表明，对酿酒酵母中的氧化应激反应至关重要的Yap1转录因子，在乙二醛酶I缺陷型突变体中持续集中在细胞核中，并激活其靶基因的表达。Yap1在两个富含半胱氨酸的结构域（CRD）中包含六个半胱氨酸残基，即靠近N端聚集的三个半胱氨酸残基（n-CRD）和靠近C端的其余三个半胱氨酸残基（c-CRD）。我们发现，c-CRD中的三个半胱氨酸残基中的任何一个都足以使MG促使Yap1转运到细胞核并激活其靶基因的表达。在c-CRD中仅具有一个半胱氨酸残基但在n-CRD中没有半胱氨酸且缺失碱性亮氨酸拉链结构域的Yap1突变体，经MG处理后可集中在细胞核中。然而，Yap1中所有半胱氨酸残基的替换消除了该转录因子在MG处理后集中在细胞核中的能力。Yap1的氧化还原状态基本不变，在用MG处理的细胞中几乎检测不到通过二硫键与Yap1相互作用的蛋白质。总体而言，分子间和分子内二硫键的形成似乎都不参与MG对Yap1的激活。此外，我们表明Yap1的核质定位与生长阶段和细胞内MG水平密切相关。我们提出了一种细胞中天然代谢物激活Yap1的新调控途径。
