Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York 13210, USA.
J Biol Chem. 2013 Apr 19;288(16):11366-77. doi: 10.1074/jbc.M112.419259. Epub 2013 Mar 1.
Vacuolar H(+)-ATPases (V-ATPases) acidify intracellular organelles and help to regulate overall cellular pH. Yeast vma mutants lack V-ATPase activity and allow exploration of connections between cellular pH, iron, and redox homeostasis common to all eukaryotes. A previous microarray study in a vma mutant demonstrated up-regulation of multiple iron uptake genes under control of Aft1p (the iron regulon) and only one antioxidant gene, the peroxiredoxin TSA2 (Milgrom, E., Diab, H., Middleton, F., and Kane, P. M. (2007) Loss of vacuolar proton-translocating ATPase activity in yeast results in chronic oxidative stress. J. Biol. Chem. 282, 7125-7136). Fluorescent biosensors placing GFP under transcriptional control of either an Aft1-dependent promoter (P(FIT2)-GFP) or the TSA2 promoter (P(TSA2)-GFP) were constructed to monitor transcriptional signaling. Both biosensors were up-regulated in the vma2Δ mutant, and acute V-ATPase inhibition with concanamycin A induced coordinate up-regulation from both promoters. PTSA2-GFP induction was Yap1p-dependent, indicating an oxidative stress signal. Total cell iron measurements indicate that the vma2Δ mutant is iron-replete, despite up-regulation of the iron regulon. Acetic acid up-regulated P(FIT2)-GFP expression in wild-type cells, suggesting that loss of pH control contributes to an iron deficiency signal in the mutant. Iron supplementation significantly decreased P(FIT2)-GFP expression and, surprisingly, restored P(TSA2)-GFP to wild-type levels. A tsa2Δ mutation induced both nuclear localization of Aft1p and P(FIT2)-GFP expression. The data suggest a novel function for Tsa2p as a negative regulator of Aft1p-driven transcription, which is induced in V-ATPase mutants to limit transcription of the iron regulon. This represents a new mechanism bridging the antioxidant and iron-regulatory pathways that is intimately linked to pH homeostasis.
液泡 H(+)-ATP 酶（V-ATPases）使细胞内细胞器酸化，并有助于调节细胞整体 pH 值。酵母 vma 突变体缺乏 V-ATPase 活性，可用于探索所有真核生物共有的细胞 pH 值、铁和氧化还原稳态之间的联系。之前在 vma 突变体中的微阵列研究表明，在 aft1p（铁调节基因）控制下，多个铁摄取基因上调，而只有一个抗氧化基因，即过氧化物酶 TSA2（Milgrom，E.，Diab，H.，Middleton，F.和 Kane，P. M.（2007 年）酵母中液泡质子转运 ATP 酶活性的丧失导致慢性氧化应激。J. Biol. Chem. 282，7125-7136）。构建了将 GFP 置于 aft1 依赖性启动子（P（FIT2）-GFP）或 TSA2 启动子（P（TSA2）-GFP）转录控制下的荧光生物传感器，以监测转录信号。这两种生物传感器在 vma2Δ 突变体中均上调，用康纳霉素 A 急性抑制 V-ATPase 诱导两个启动子的协同上调。TSA2-GFP 诱导依赖 Yap1p，表明存在氧化应激信号。总细胞铁测量表明，尽管铁调节基因上调，vma2Δ 突变体仍是铁充足的。乙酸在野生型细胞中上调 P（FIT2）-GFP 表达，表明 pH 控制丧失导致突变体中的铁缺乏信号。铁补充剂显著降低了 P（FIT2）-GFP 的表达，令人惊讶的是，将 P（TSA2）-GFP 恢复到野生型水平。tsa2Δ 突变诱导 aft1p 的核定位和 P（FIT2）-GFP 表达。数据表明，Tsa2p 作为 aft1p 驱动转录的负调节剂具有新功能，该功能在 V-ATPase 突变体中被诱导，以限制铁调节基因的转录。这代表了一种将抗氧化和铁调节途径联系起来的新机制，与 pH 稳态密切相关。
