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.
Heme is an essential prosthetic group in proteins that reside in virtually every subcellular compartment performing diverse biological functions. Irrespective of whether heme is synthesized in the mitochondria or imported from the environment, this hydrophobic and potentially toxic metalloporphyrin has to be trafficked across membrane barriers, a concept heretofore poorly understood. Here we show, using subcellular-targeted, genetically encoded hemoprotein peroxidase reporters, that both extracellular and endogenous heme contribute to cellular labile heme and that extracellular heme can be transported and used in toto by hemoproteins in all six subcellular compartments examined. The reporters are robust, show large signal-to-background ratio, and provide sufficient range to detect changes in intracellular labile heme. Restoration of reporter activity by heme is organelle-specific, with the Golgi and endoplasmic reticulum being important sites for both exogenous and endogenous heme trafficking. Expression of peroxidase reporters in Caenorhabditiselegans shows that environmental heme influences labile heme in a tissue-dependent manner; reporter activity in the intestine shows a linear increase compared with muscle or hypodermis, with the lowest heme threshold in neurons. Our results demonstrate that the trafficking pathways for exogenous and endogenous heme are distinct, with intrinsic preference for specific subcellular compartments. We anticipate our results will serve as a heuristic paradigm for more sophisticated studies on heme trafficking in cellular and whole-animal models.
Heme is an essential cofactor and signaling molecule required for virtually all aerobic life. However, excess heme is cytotoxic. Therefore, heme must be safely transported and trafficked from the site of synthesis in the mitochondria or uptake at the cell surface, to hemoproteins in most subcellular compartments. While heme synthesis and degradation are relatively well characterized, little is known about how heme is trafficked and transported throughout the cell. Herein, we review eukaryotic heme transport, trafficking, and mobilization, with a focus on factors that regulate bioavailable heme. We also highlight the role of gasotransmitters and small molecules in heme mobilization and bioavailability, and heme trafficking at the host-pathogen interface.
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.
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 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 (Pb) 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 Pb effects on heme homeostasis. Overall, these findings suggest an important role for LH in supporting heme-dependent functions in yeast physiology.
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