Department of Physiology, Pathophysiology & Toxicology and Center for Biomedical Education and Research (ZBAF), University of Witten/Herdecke, D-58453, Witten, Germany.
Department of Biochemistry, State University of New York (SUNY), Buffalo, New York, 14214, USA.
Sci Rep. 2018 Jan 9;8(1):211. doi: 10.1038/s41598-017-18584-4.
Much of iron and manganese metabolism occurs in mitochondria. Uptake of redox-active iron must be tightly controlled, but little is known about how metal ions enter mitochondria. Recently, we established that the divalent metal transporter 1 (DMT1) is present in the outer mitochondrial membrane (OMM). Therefore we asked if it mediates Fe and Mn influx. Mitochondria were isolated from HEK293 cells permanently transfected with inducible rat DMT1 isoform 1 A/+IRE (HEK293-rDMT1). Fe-induced quenching of the dye PhenGreen™SK (PGSK) occurred in two phases, one of which reflected OMM DMT1 with stronger Fe uptake after DMT1 overexpression. DMT1-specific quenching showed an apparent affinity of ~1.5 µM for Feand was blocked by the DMT1 inhibitor CISMBI. Fe influx reflected an imposed proton gradient, a response that was also observed in purified rat kidney cortex (rKC) mitochondria. Non-heme Fe accumulation assayed by ICPOES and stable Fe isotope incorporation by ICPMS were increased in HEK293-rDMT1 mitochondria. HEK293-rDMT1 mitochondria displayed higher Fe and Mn uptake relative to controls with Mn uptake blocked by the DMT1 inhibitor XEN602. Such transport was defective in rKC mitochondria with the Belgrade (G185R) mutation. Thus, these results support a role for DMT1 in mitochondrial Fe and Mn acquisition.
铁和锰的代谢大部分发生在线粒体中。必须严格控制氧化还原活性铁的摄取，但人们对金属离子如何进入线粒体知之甚少。最近，我们确定二价金属转运蛋白 1（DMT1）存在于线粒体外膜（OMM）中。因此，我们询问它是否介导 Fe 和 Mn 的流入。从用可诱导的大鼠 DMT1 同工型 1A/+IRE（HEK293-rDMT1）永久转染的 HEK293 细胞中分离出线粒体。Fe 诱导的 PhenGreen™SK（PGSK）染料猝灭发生在两个阶段，其中一个反映了 OMM DMT1，过表达 DMT1 后 Fe 摄取更强。DMT1 特异性猝灭对 Fe 的表观亲和力约为 1.5µM，并且被 DMT1 抑制剂 CISMBI 阻断。Fe 流入反映了强加的质子梯度，这种反应也在纯化的大鼠肾皮质（rKC）线粒体中观察到。用 ICPOES 测定非血红素 Fe 积累和用 ICPMS 测定稳定的 Fe 同位素掺入均增加了 HEK293-rDMT1 线粒体中的 Fe。与对照相比，HEK293-rDMT1 线粒体中的 Fe 和 Mn 摄取更高，Mn 摄取被 DMT1 抑制剂 XEN602 阻断。rKC 线粒体中的这种转运存在缺陷，带有 Belgrade（G185R）突变。因此，这些结果支持 DMT1 在线粒体 Fe 和 Mn 摄取中的作用。
