Adult humans have about 25 trillion red blood cells (RBCs) and each second we recycle about five million RBCs by erythrophagocytosis (EP) in macrophages of the reticuloendothelial system. Despite the central role for EP in mammalian iron metabolism, the molecules and pathways responsible for heme trafficking during EP remain unknown. Here, we show that the mammalian homolog of HRG1, a transmembrane heme permease in C. elegans, is essential for macrophage iron homeostasis and transports heme from the phagolysosome to the cytoplasm during EP. HRG1 is strongly expressed in macrophages of the reticuloendothelial system and specifically localizes to the phagolysosomal membranes during EP. Depletion of Hrg1 in mouse macrophages causes attenuation of heme transport from the phagolysosomal compartment. Importantly, missense polymorphisms in human HRG1 are defective in heme transport. Our results reveal HRG1 as the long-sought heme transporter for heme-iron recycling in macrophages and suggest that genetic variations in HRG1 could be modifiers of human iron metabolism.
Summary
Heme is a cytotoxic, hydrophobic tetrapyrrole that crosses multiple biological membranes for incorporation into proteins critical for numerous biological processes. Thus, a prima facie argument can be made that heme trafficking within the aqueous cellular milieu must be mediated by specific intra- and intercellular pathways. Embryonic development in Caenorhabditis elegans, a heme auxotroph, is inextricably dependent on maternal heme acquisition. Here we show that HRG-3 is required to deliver maternal heme to oocytes for zygotic development. HRG-3 binds heme and is exclusively secreted by the intestine during heme insufficiency into the interstitial fluid for transport of maternal heme to extra-intestinal cells. HRG-3 deficiency results either in death during embryogenesis or in developmental arrest immediately post hatching – phenotypes that are fully suppressed by maternal but not zygotic hrg-3 expression. Our results establish an unprecedented role for HRG-3 as an intercellular heme chaperone in zygotic development and maternal-embryonic nutrition in C. elegans.
Leishmania spp. are trypanosomatid parasites that replicate intracellularly in macrophages, causing serious human morbidity and mortality throughout the world. Trypanosomatid protozoa cannot synthesize heme, so must acquire this essential cofactor from their environment. Earlier studies identified LHR1 as a Leishmania amazonensis transmembrane protein that mediates heme uptake. Null mutants of LHR1 are not viable and single knockout strains have reduced virulence, but very little is known about the properties of LHR1 directly associated with heme transport. Here, we use functional assays in Saccharomyces cerevisiae to show that specific tyrosine residues within the first three predicted transmembrane domains of LHR1 are required for efficient heme uptake. These tyrosines are unique to LHR1, consistent with the low similarity between LHR1 and its corresponding homologs in C. elegans and human. Substitution of these tyrosines in LHR1 resulted in varying degrees of heme transport inhibition, phenotypes that closely mirrored the impaired ability of L. amazonensis to replicate as intracellular amastigotes in macrophages and generate cutaneous lesions in mice. Taken together, our results imply that the mechanism for heme transport by LHR1 is distinctive and may have adapted to secure heme, a limiting cofactor, inside the host. Since LHR1 is significantly divergent from the human heme transporter HRG1, our findings lay the groundwork for selective targeting of LHR1 by small molecule antagonists.
In this protocol, we present the required materials, and the procedure for making modified C. elegans Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.
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