Department of Chemistry, Texas A&M University, College Station, Texas, USA.
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, USA.
J Biol Chem. 2021 Jan-Jun;296:100141. doi: 10.1074/jbc.RA120.015907. Epub 2020 Dec 6.
Vacuoles are acidic organelles that store Fe polyphosphate, participate in iron homeostasis, and have been proposed to deliver iron to mitochondria for iron-sulfur cluster (ISC) and heme biosynthesis. Vma2Δ cells have dysfunctional V-ATPases, rendering their vacuoles nonacidic. These cells have mitochondria that are iron-dysregulated, suggesting disruption of a putative vacuole-to-mitochondria iron trafficking pathway. To investigate this potential pathway, we examined the iron content of a vma2Δ mutant derived from W303 cells using Mössbauer and EPR spectroscopies and liquid chromatography interfaced with inductively-coupled-plasma mass spectrometry. Relative to WT cells, vma2Δ cells contained WT concentrations of iron but nonheme Fe dominated the iron content of fermenting and respiring vma2Δ cells, indicating that the vacuolar Fe ions present in WT cells had been reduced. However, vma2Δ cells synthesized WT levels of ISCs/hemes and had normal aconitase activity. The iron content of vma2Δ mitochondria was similar to WT, all suggesting that iron delivery to mitochondria was not disrupted. Chromatograms of cytosolic flow-through solutions exhibited iron species with apparent masses of 600 and 800 Da for WT and vma2∆, respectively. Mutant cells contained high copper concentrations and high concentrations of a species assigned to metallothionein, indicating copper dysregulation. vma2Δ cells from previously studied strain BY4741 exhibited iron-associated properties more consistent with prior studies, suggesting subtle strain differences. Vacuoles with functional V-ATPases appear unnecessary in W303 cells for iron to enter mitochondria and be used in ISC/heme biosynthesis; thus, there appears to be no direct or dedicated vacuole-to-mitochondria iron trafficking pathway. The vma2Δ phenotype may arise from alterations in trafficking of iron directly from cytosol to mitochondria.
液泡是储存 Fe 多磷酸盐、参与铁稳态平衡的酸性细胞器，并且有人提出液泡将铁递送到线粒体用于铁-硫簇（ISC）和血红素生物合成。Vma2Δ 细胞的 V-ATP 酶功能失调，使它们的液泡呈非酸性。这些细胞的线粒体铁代谢失调，表明破坏了一种潜在的液泡到线粒体铁运输途径。为了研究这种潜在的途径，我们使用 Mössbauer 和 EPR 光谱以及与电感耦合等离子体质谱联用的液相色谱法，检查了源自 W303 细胞的 vma2Δ 突变体的铁含量。与 WT 细胞相比，vma2Δ 细胞含有 WT 浓度的铁，但非血红素 Fe 占据了发酵和呼吸 vma2Δ 细胞中铁含量的主导地位，表明 WT 细胞中存在的液泡铁离子已被还原。然而，vma2Δ 细胞合成了 WT 水平的 ISC/hemes，并且具有正常的 aconitase 活性。vma2Δ 线粒体的铁含量与 WT 相似，这表明向线粒体的铁输送没有受到干扰。细胞溶质流通过程的色谱图显示，WT 和 vma2∆ 的铁物种具有 600 和 800 Da 的表观质量。突变细胞含有高铜浓度和高浓度的一种被分配给金属硫蛋白的物质，表明铜代谢失调。先前研究的 BY4741 菌株的 vma2Δ 细胞表现出与先前研究更一致的铁相关特性，这表明存在微妙的菌株差异。在 W303 细胞中，具有功能 V-ATP 酶的液泡对于铁进入线粒体并用于 ISC/heme 生物合成似乎不是必需的；因此，似乎没有直接或专门的液泡到线粒体铁运输途径。vma2Δ 表型可能是由于铁从细胞质直接转运到线粒体的运输改变引起的。
