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.

Unraveling the mystery of the ring: Tracking heme dynamics in living cells

“…Because a population of cells is combined into a bulk measurement, any information about the heterogeneity of heme levels across cells, as well as temporal information about changes in heme concentration, is lost. Finally, in these experiments, there is no way to differentiate between labile and bound heme (6). To gain greater insight into the details of heme biology, it is necessary to detect heme in live cells in a noninvasive manner.…”

“…cytochrome b 562 domain, the red emission signal of the sensor becomes an internal standard that can be used to normalize the altered emission of GFP upon heme binding (6).…”

“…Two commonly used methods for validating sensor efficacy are to express analogous sensors of varying affinities to ensure the measured concentration of analyte is not an artifact of sensor affinity and to measure sensor saturation as a function of sensor expression level (11)(12)(13). Hanna et al (6) were able to use the high-affinity and low-affinity HS1 sensors in both WT yeast and a yeast strain in which heme levels were manipulated by perturbing the heme biosynthetic pathway to verify sensor functionality. Moreover, because yeast genetics are well studied, HS1 could be put under the control of different promoters that give rise to high, medium, and low levels of expression.…”

“…Although there have long been tools to study heme structure and synthesis in vitro, as well as tools to study the total heme content in populations of cells, there are few tools to study the dynamics of labile heme in individual, live cells (5). In recent work in PNAS, Hanna et al (6) develop a new genetically encoded fluorescent sensor for heme, HS1, and uncover the dynamic regulation of resting labile heme, document the mobilization of labile heme in response to NO, and identify glyceraldehyde phosphate dehydrogenase (GAPDH) as a heme buffer in yeast.…”

Label-Free Imaging of Heme Dynamics in Living Organisms by Transient Absorption Microscopy

“…22–25 Typically, these sensors recognize only labile heme. 22,23,25 Moreover, the FRET sensors could perturb normal heme homeostasis by sequestering heme and distorting labile heme. 22,23,25 Recently, a horseradish peroxidase (HRP)-based sensor was engineered based on the enhancement of HRP activity upon heme-binding.…”