Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States of America.
PLoS One. 2011;6(6):e20896. doi: 10.1371/journal.pone.0020896. Epub 2011 Jun 28.
Mg homeostasis is critical to eukaryotic cells, but the contribution of Mg transporter activity to homeostasis is not fully understood. In yeast, Mg uptake is primarily mediated by the Alr1 transporter, which also allows low affinity uptake of other divalent cations such as Ni(2+), Mn(2+), Zn(2+) and Co(2+). Using Ni(2+) uptake to assay Alr1 activity, we observed approximately nine-fold more activity under Mg-deficient conditions. The mnr2 mutation, which is thought to block release of vacuolar Mg stores, was associated with increased Alr1 activity, suggesting Alr1 was regulated by intracellular Mg supply. Consistent with a previous report of the regulation of Alr1 expression by Mg supply, Mg deficiency and the mnr2 mutation both increased the accumulation of a carboxy-terminal epitope-tagged version of the Alr1 protein (Alr1-HA). However, Mg supply had little effect on ALR1 promoter activity or mRNA levels. In addition, while Mg deficiency caused a seven-fold increase in Alr1-HA accumulation, the N-terminally tagged and untagged Alr1 proteins increased less than two-fold. These observations argue that the Mg-dependent accumulation of the C-terminal epitope-tagged protein was primarily an artifact of its modification. Plasma membrane localization of YFP-tagged Alr1 was also unaffected by Mg supply, indicating that a change in Alr1 location did not explain the increased activity we observed. We conclude that variation in Alr1 protein accumulation or location does not make a substantial contribution to its regulation by Mg supply, suggesting Alr1 activity is directly regulated via as yet unknown mechanisms.
镁稳态对真核细胞至关重要，但镁转运体活性对稳态的贡献尚不完全清楚。在酵母中，镁的摄取主要由 Alr1 转运体介导，该转运体还允许其他二价阳离子（如 Ni(2+)、Mn(2+)、Zn(2+)和 Co(2+)）以低亲和力摄取。我们使用 Ni(2+)摄取来测定 Alr1 活性，发现镁缺乏条件下的活性约增加了九倍。mnr2 突变被认为阻止了液泡内镁库的释放，与 Alr1 活性增加有关，这表明 Alr1 受到细胞内镁供应的调节。与之前关于镁供应调节 Alr1 表达的报道一致，镁缺乏和 mnr2 突变都增加了羧基末端表位标记的 Alr1 蛋白（Alr1-HA）的积累。然而，镁供应对 ALR1 启动子活性或 mRNA 水平几乎没有影响。此外，虽然镁缺乏导致 Alr1-HA 积累增加了七倍，但 N 端标记和未标记的 Alr1 蛋白增加不到两倍。这些观察结果表明，C 端表位标记蛋白的镁依赖性积累主要是其修饰的一个人工制品。YFP 标记的 Alr1 的质膜定位也不受镁供应的影响，表明 Alr1 位置的变化不能解释我们观察到的活性增加。我们得出结论，Alr1 蛋白积累或位置的变化对其受镁供应的调节没有实质性贡献，这表明 Alr1 活性是通过未知的机制直接调节的。
