RESUMEN
Heme is a cofactor and signaling molecule that is essential for much of aerobic life. All heme-dependent processes in eukaryotes require that heme is trafficked from its site of synthesis in the mitochondria to hemoproteins located throughout the cell. However, the mechanisms governing the mobilization of heme out of the mitochondria, and the spatio-temporal dynamics of these processes, are poorly understood. Here, using genetically encoded fluorescent heme sensors, we developed a live-cell assay to monitor heme distribution dynamics between the mitochondrial inner membrane, where heme is synthesized, and the mitochondrial matrix, cytosol and nucleus. Surprisingly, heme trafficking to the nucleus is â¼25% faster than to the cytosol or mitochondrial matrix, which have nearly identical heme trafficking dynamics, potentially supporting a role for heme as a mitochondrial-nuclear retrograde signal. Moreover, we discovered that the heme synthetic enzyme 5-aminolevulinic acid synthase (ALAS, also known as Hem1 in yeast), and GTPases in control of the mitochondrial dynamics machinery (Mgm1 and Dnm1) and ER contact sites (Gem1), regulate the flow of heme between the mitochondria and nucleus. Overall, our results indicate that there are parallel pathways for the distribution of bioavailable heme.This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Hemo , Saccharomycetales , GTP Fosfohidrolasas , Mitocondrias , Dinámicas MitocondrialesRESUMEN
Protoheme (hereafter referred to as heme) is an essential cellular cofactor and signaling molecule that is also potentially cytotoxic. To mitigate heme toxicity, heme synthesis and degradation are tightly coupled to heme utilization in order to limit the intracellular concentration of "free" heme. Such a model, however, would suggest that a readily accessible steady-state, bioavailable labile heme (LH) pool is not required for supporting heme-dependent processes. Using the yeast Saccharomyces cerevisiae as a model and fluorescent heme sensors, site-specific heme chelators, and molecular genetic approaches, we found here that 1) yeast cells preferentially use LH in heme-depleted conditions; 2) sequestration of cytosolic LH suppresses heme signaling; and 3) lead (Pb2+) stress contributes to a decrease in total heme, but an increase in LH, which correlates with increased heme signaling. We also observed that the proteasome is involved in the regulation of the LH pool and that loss of proteasomal activity sensitizes cells to Pb2+ effects on heme homeostasis. Overall, these findings suggest an important role for LH in supporting heme-dependent functions in yeast physiology.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Hemo/metabolismo , Plomo/farmacología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Estrés Fisiológico , Disponibilidad Biológica , Homeostasis , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/crecimiento & desarrollo , Transducción de SeñalRESUMEN
Cellular heme is thought to be distributed between a pool of sequestered heme that is tightly bound within hemeproteins and a labile heme pool required for signaling and transfer into proteins. A heme chaperone that can hold and allocate labile heme within cells has long been proposed but never been identified. Here, we show that the glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) fulfills this role by acting as an essential repository and allocator of bioavailable heme to downstream protein targets. We identified a conserved histidine in GAPDH that is needed for its robust heme binding both in vitro and in mammalian cells. Substitution of this histidine, and the consequent decreases in GAPDH heme binding, antagonized heme delivery to both cytosolic and nuclear hemeprotein targets, including inducible nitric-oxide synthase (iNOS) in murine macrophages and the nuclear transcription factor Hap1 in yeast, even though this GAPDH variant caused cellular levels of labile heme to rise dramatically. We conclude that by virtue of its heme-binding property, GAPDH binds and chaperones labile heme to create a heme pool that is bioavailable to downstream proteins. Our finding solves a fundamental question in cell biology and provides a new foundation for exploring heme homeostasis in health and disease.
Asunto(s)
Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Hemo/metabolismo , Chaperonas Moleculares/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cristalografía por Rayos X , Gliceraldehído-3-Fosfato Deshidrogenasas/química , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Hemo/química , Humanos , Ratones , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Datos de Secuencia Molecular , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Unión Proteica , Alineación de SecuenciaRESUMEN
Heme is an essential cofactor and signaling molecule. 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 â¼20-40 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.
Asunto(s)
Técnicas Biosensibles , Hemo/metabolismo , Escherichia coli , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Células HEK293 , Humanos , Óxido Nítrico/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Heme (iron protoporphyrin IX) is an essential protein prosthetic group and signaling molecule required for most life on Earth. All heme-dependent processes require the dynamic and rapid mobilization of heme from sites of synthesis or uptake to hemoproteins present in virtually every subcellular compartment. The cytotoxicity and hydrophobicity of heme necessitate that heme mobilization be carefully controlled to mitigate the deleterious effects of this essential toxin. Indeed, a number of disorders, including certain cancers, cardiovascular diseases, and aging and age-related neurodegenerative diseases, are tied to defects in heme homeostasis. However, the molecules and mechanisms that mediate heme transport and trafficking, and the dynamics of these processes, are poorly understood. This is in large part due to the lack of physical tools for probing cellular heme. Herein, we discuss the recent development of fluorescent probes that can monitor and image kinetically labile heme with respect to its mobilization and role in signaling. In particular, we will highlight how heme gazing with these tools can uncover new heme trafficking factors upon being integrated with genetic screens and illuminate the concentration, subcellular distribution, and dynamics of labile heme in various physiological contexts. Altogether, the monitoring of labile heme, along with recent biochemical and cell biological studies demonstrating the reversible regulation of certain cellular processes by heme, is challenging us to reconceptualize heme from being a static cofactor buried in protein active sites to a dynamic and mobile signaling molecule.
Asunto(s)
Grupo Citocromo b/química , Proteínas de Escherichia coli/química , Colorantes Fluorescentes/química , Hemo/química , Chaperonas Moleculares/química , Proteínas Recombinantes de Fusión/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Transporte Biológico , Línea Celular , Grupo Citocromo b/genética , Grupo Citocromo b/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/metabolismo , Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hemo/metabolismo , Humanos , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de SeñalRESUMEN
Heme b (iron protoporphyrin IX) is an essential but potentially cytotoxic cofactor, signaling molecule, and nutritional source of iron. Its importance in cell biology and metabolism is underscored by the fact that numerous diseases, including various cancers, neurodegenerative disorders, infectious diseases, anemias, and porphyrias, are associated with the dysregulation of heme synthesis, degradation, trafficking, and/or transport. Consequently, methods to measure, image, and quantify heme in cells are required to better understand the physiology and pathophysiology of heme. Herein, we describe fluorescence-based protocols to probe heme bioavailability and trafficking dynamics using genetically encoded fluorescent heme sensors in combination with various modalities, such as confocal microscopy, flow cytometry, and microplate readers. Additionally, we describe a protocol for measuring total heme and its precursor protoporphyrin IX using a fluorometric assay that exploits porphyrin fluorescence. Together, the methods described enable the monitoring of total and bioavailable heme to study heme homeostatic mechanisms in virtually any cell type and organism.