Heme dynamics and trafficking factors revealed by genetically encoded fluorescent heme sensors
Heme dynamics and trafficking factors revealed by genetically encoded fluorescent heme sensors
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DOI：
10.1073/pnas.1523802113
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发表时间：
2016-07-05
影响因子：
11.1
通讯作者：
Reddi, Amit R.
中科院分区：
文献类型：
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作者：
Hanna, David A.;Harvey, Raven M.;Reddi, Amit R.
血红素是一种重要的辅因子和信号分子。血红素的蛋白质和血红素信号的收购最终依赖于动员不稳定血红素（LH）的能力。然而，LH池的性质，包括浓度，氧化态，分布，形态和动态，知之甚少。在此，我们阐明了LH的性质和动力学，使用遗传编码的比例荧光血红素传感器在单细胞真核生物酿酒酵母。我们发现LH的亚细胞分布是不均匀的;胞质溶胶将LH维持在类似于20-40 nM，而线粒体和细胞核将其维持在低于2.5 nM的浓度。此外，我们发现，信号分子一氧化氮可以启动快速动员血红素在细胞质和细胞核中的某些含巯基的因素。我们还发现，糖酵解酶甘油醛磷酸脱氢酶构成一个主要的细胞血红素缓冲，并负责维持血红素依赖性核转录因子血红素激活蛋白（Hap 1 p）的活性。总之，我们证明了血红素传感器可用于揭示血红素贩运和动力学的基本方面，并可用于多种生物，包括大肠杆菌，酵母和人类细胞系。
Heme is an essential cofactor and signalingmolecule. Heme acquisition by proteins and heme signaling are ultimately reliant on the ability to mobilize labile heme (LH). However, the properties of LH pools, including concentration, oxidation state, distribution, speciation, and dynamics, are poorly understood. Herein, we elucidate the nature and dynamics of LH using genetically encoded ratiometric fluorescent heme sensors in the unicellular eukaryote Saccharomyces cerevisiae. We find that the subcellular distribution of LH is heterogeneous; the cytosol maintains LH at similar to 20-40 nM, whereas the mitochondria and nucleus maintain it at concentrations below 2.5 nM. Further, we find that the signaling molecule nitric oxide can initiate the rapid mobilization of heme in the cytosol and nucleus from certain thiol-containing factors. We also find that the glycolytic enzyme glyceraldehyde phosphate dehydrogenase constitutes a major cellular heme buffer, and is responsible for maintaining the activity of the heme-dependent nuclear transcription factor heme activator protein (Hap1p). Altogether, we demonstrate that the heme sensors can be used to reveal fundamental aspects of heme trafficking and dynamics and can be used across multiple organisms, including Escherichia coli, yeast, and human cell lines.