Department of Biological Sciences, Auburn University, Auburn, United States.
Department of Biochemistry, Microbiology and Immunology, University of Saskatchewan, Saskatoon, Canada.
Elife. 2021 Feb 16;10:e64690. doi: 10.7554/eLife.64690.
The mitochondrial carrier family protein SLC25A3 transports both copper and phosphate in mammals, yet in the transport of these substrates is partitioned across two paralogs: PIC2 and MIR1. To understand the ancestral state of copper and phosphate transport in mitochondria, we explored the evolutionary relationships of PIC2 and MIR1 orthologs across the eukaryotic tree of life. Phylogenetic analyses revealed that PIC2-like and MIR1-like orthologs are present in all major eukaryotic supergroups, indicating an ancient gene duplication created these paralogs. To link this phylogenetic signal to protein function, we used structural modeling and site-directed mutagenesis to identify residues involved in copper and phosphate transport. Based on these analyses, we generated an L175A variant of mouse SLC25A3 that retains the ability to transport copper but not phosphate. This work highlights the utility of using an evolutionary framework to uncover amino acids involved in substrate recognition by mitochondrial carrier family proteins.
线粒体载体家族蛋白 SLC25A3 在哺乳动物中既运输铜又运输磷酸盐，但在这些底物的运输中，它们被两个平行基因（PIC2 和 MIR1）分割。为了了解线粒体中铜和磷酸盐运输的原始状态，我们探索了 PIC2 和 MIR1 直系同源物在真核生物生命树中的进化关系。系统发育分析表明，PIC2 样和 MIR1 样直系同源物存在于所有主要的真核超群中，这表明一个古老的基因复制产生了这些平行基因。为了将这种系统发育信号与蛋白质功能联系起来，我们使用结构建模和定点诱变来鉴定参与铜和磷酸盐运输的残基。基于这些分析，我们生成了一种保留运输铜能力但不运输磷酸盐能力的小鼠 SLC25A3 L175A 变体。这项工作突出了使用进化框架来揭示线粒体载体家族蛋白参与底物识别的氨基酸的实用性。
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