title: Heme dynamics and trafficking factors revealed by genetically encoded fluorescent heme sensors.
abstract: 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 ∼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.
authors.affiliation: School of Chemistry and Biochemistry and Parker Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, GA 30332;
authors.forename: David A
authors.initials: DA
authors.lastname: Hanna
authors.affiliation: School of Chemistry and Biochemistry and Parker Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, GA 30332;
authors.forename: Raven M
authors.initials: RM
authors.lastname: Harvey
authors.affiliation: School of Chemistry and Biochemistry and Parker Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, GA 30332;
authors.forename: Osiris
authors.initials: O
authors.lastname: Martinez-Guzman
authors.affiliation: Department of Animal and Avian Sciences, University of Maryland, College Park, MD 20740; Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20740;
authors.forename: Xiaojing
authors.initials: X
authors.lastname: Yuan
authors.affiliation: School of Chemistry and Biochemistry and Parker Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, GA 30332;
authors.forename: Bindu
authors.initials: B
authors.lastname: Chandrasekharan
authors.affiliation: School of Chemistry and Biochemistry and Parker Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, GA 30332;
authors.forename: Gheevarghese
authors.initials: G
authors.lastname: Raju
authors.affiliation: Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC 29208.
authors.forename: F Wayne
authors.initials: FW
authors.lastname: Outten
authors.affiliation: Department of Animal and Avian Sciences, University of Maryland, College Park, MD 20740; Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20740;
authors.forename: Iqbal
authors.initials: I
authors.lastname: Hamza
authors.affiliation: School of Chemistry and Biochemistry and Parker Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, GA 30332; amit.reddi@chemistry.gatech.edu.
authors.forename: Amit R
authors.initials: AR
authors.lastname: Reddi
mesh_terms: D015374:Biosensing Techniques; D004926:Escherichia coli; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D057809:HEK293 Cells; D006418:Heme / Q000378:metabolism*; D006801:Humans; D009569:Nitric Oxide / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism
chemical_list: C000621829:Hap1p protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D009569:Nitric Oxide; D006418:Heme; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases
keywords: glyceraldehyde phosphate dehydrogenase; heme dynamics; heme sensors; heme trafficking; nitric oxide