Identification and Characterization of HRG-1 heme transporters in eukaryotes

Heme is a prosthetic group in proteins that perform diverse biological functions including respiration, gas sensing, xenobiotic detoxification, cell differentiation, circadian clock control and micro RNA processing. In most eukaryotes, heme is synthesized through a multi-step pathway with defined intermediates that are highly conserved through evolution. Despite our extensive knowledge about heme biosynthesis and degradation, the molecules and pathways involved in intracellular heme trafficking are unknown, primarily due to the inability to dissociate the tightly regulated processes of heme biosynthesis and degradation from intracellular trafficking events. Caenorhabditis elegans and related helminths are natural heme auxotrophs that rely solely on exogenous heme for normal development and reproduction. We performed a genome-wide microarray analysis and identified 288 genes that are regulated by heme at the transcriptional level in C. elegans. Here, we characterize two heme-responsive genes, hrg-1 and its paralog hrg-4, that are highly upregulated at low heme concentrations and demonstrate that HRG-1 and HRG-4 are heme transporters. Depletion of hrg-1 and hrg-4 in worms by RNAi results in the disruption of organismal heme homeostasis and abnormal response to heme analogs. HRG-4 traffics to the plasma membrane, and HRG-1 localizes to endo-lysosomal compartments. While hrg-4 appears to be specific to worms, hrg-1 has homologs in vertebrates. Knock-down of hrg-1 in zebrafish results in severe anemia and profound developmental defects, which are fully rescued by worm hrg-1. Human and worm HRG-1 proteins localize together. CeHRG-1, hHRG1 and CeHRG-4 all bind and transport heme. To further understand the in vivo functions of hrg-1 and hrg-4, we characterize the genetic deletions of these genes in C. elegans. Preliminary experiments suggest that the deletion mutants respond abnormally to heme analogs, although these results do not phenocopy the RNAi knock-down studies. We speculate that the deletion strains may have developed compensatory mechanisms in response to the genetic lesions in hrg-1 and hrg-4. Taken together, the studies described herein lay the foundation for identifying the molecular mechanisms for heme transport by the HRG-1 proteins in metazoans and delineating the heme trafficking pathways in C. elegans

A systematic genetic screen for genes involved in sensing inorganic phosphate availability in Saccharomyces cerevisiae

Saccharomyces cerevisiae responds to changes in extracellular inorganic phosphate (P_i) availability by regulating the activity of the phosphate-responsive (PHO) signaling pathway, enabling cells to maintain intracellular levels of the essential nutrient P_i. P_i-limitation induces upregulation of inositol heptakisphosphate (IP_7) synthesized by the inositol hexakisphosphate kinase Vip1, triggering inhibition of the Pho80/Pho85 cyclin-cyclin dependent kinase (CDK) complex by the CDK inhibitor Pho81, which upregulates the PHO regulon through the CDK target and transcription factor Pho4. To identify genes that are involved in signaling upstream of the Pho80/Pho85/Pho81 complex and how they interact with each other to regulate the PHO pathway, we performed genome-wide screens with the synthetic genetic array method. We identified more than 300 mutants with defects in signaling upstream of the Pho80/Pho85/Pho81 complex, including AAH1, which encodes an adenine deaminase that negatively regulates the PHO pathway in a Vip1-dependent manner. Furthermore, we showed that even in the absence of VIP1, the PHO pathway can be activated under prolonged periods of P_i starvation, suggesting complexity in the mechanisms by which the PHO pathway is regulated

Genome-Wide Analysis Reveals Novel Genes Essential for Heme Homeostasis in Caenorhabditiselegans

Heme is a cofactor in proteins that function in almost all sub-cellular compartments and in many diverse biological processes. Heme is produced by a conserved biosynthetic pathway that is highly regulated to prevent the accumulation of heme—a cytotoxic, hydrophobic tetrapyrrole. Caenorhabditis elegans and related parasitic nematodes do not synthesize heme, but instead require environmental heme to grow and develop. Heme homeostasis in these auxotrophs is, therefore, regulated in accordance with available dietary heme. We have capitalized on this auxotrophy in C. elegans to study gene expression changes associated with precisely controlled dietary heme concentrations. RNA was isolated from cultures containing 4, 20, or 500 mM heme; derived cDNA probes were hybridized to Affymetrix C. elegans expression arrays. We identified 288 heme-responsive genes (hrgs) that were differentially expressed under these conditions. Of these genes, 42% had putative homologs in humans, while genomes of medically relevant heme auxotrophs revealed homologs for 12% in both Trypanosoma and Leishmania and 24% in parasitic nematodes. Depletion of each of the 288 hrgs by RNA–mediated interference (RNAi) in a transgenic heme-sensor worm strain identified six genes that regulated heme homeostasis. In addition, seven membrane-spanning transporters involved in heme uptake were identified by RNAi knockdown studies using a toxic heme analog. Comparison of genes that were positive in both of the RNAi screens resulted in the identification of three genes in common that were vital for organismal heme homeostasis in C. elegans. Collectively, our results provide a catalog of genes that are essential for metazoan heme homeostasis and demonstrate the power of C. elegans as a genetic animal model to dissect the regulatory circuits which mediate heme trafficking in both vertebrate hosts and their parasites, which depend on environmental heme for survival

<i>VIP1</i> is required for constitutive activation of the PHO pathway in <i>ado1Δ</i> and <i>aah1Δ</i>.

<p>The <i>PHO84</i> reporter levels in all the strains in Fig 6 were averaged over 3 measurements and normalized to the <i>PHO84</i> reporter level in the wild type in 1 mM P_i conditions. Error bars represent the standard deviation of the normalized <i>PHO84</i> reporter levels in the mutants.</p

The PHO pathway in <i>vip1Δ</i> mutant is inducible in 50 uM P_i, but its induction kinetics are slower than the wild type.

<p>(A) The <i>PHO84</i> reporter levels of the wild type over time in 50 uM P_i. (B) The <i>PHO84</i> reporter levels of <i>vip1Δ</i> over time in 50 uM P_i. Time 0 data in (A) and (B) were obtained in 10 mM P_i before cells were inoculated into 50 μM P_i.</p

Adenine nucleotide levels in the wild type in no P_i and in <i>adk1Δ</i>, <i>aah1Δ</i> and <i>ado1Δ</i> in high P_i.

<p>(A) [ATP], [ADP], and [AMP] in wild type (WT) cells over time grown in no P_i medium. All the adenine nucleotide concentrations at each time point were normalized to those in 10 mM P_i. Note that the PHO pathway in no P_i is activated within 15 minutes. Adenine nucleotide levels at each time point were measured three times and the error bars in (A) are standard errors. (B) [ATP], [ADP], and [AMP] in WT, <i>adk1Δ</i>, <i>aah1Δ</i>, and <i>ado1Δ</i> in 10 mM P_i. Adenine nucleotide levels in the three mutants were measured two times and the error bars in (B) are standard errors.</p

Identification of mutants with altered <i>PHO84</i> expression in low and high P_i conditions.

<p>(A) Generation of single mutants harboring the <i>PHO84</i> reporter with the SGA method. The <i>PHO84</i> reporter consists of <i>PHO84</i> promoter-driven Venus and <i>TEF2</i> promoter-driven mCherry. Each single mutant in the library denoted by <i>xxx</i>Δ is kanamycin (G418)-resistant. (B) The distributions of the <i>PHO84</i> reporter levels in single cells in the <i>pho81Δ</i> and <i>pho80Δ</i> strains. Log₂ intensity ratio of Venus to mCherry (log₂(YFP/RFP)) was used to quantify the <i>PHO84</i> expression level. (C, D) The <i>PHO84</i> reporter levels of single mutants in the library measured in 50 μM P_i and 1 mM P_i conditions. The <i>PHO84</i> reporter level of each mutant was normalized to that of the wild type value in each P_i concentration (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176085#sec002" target="_blank">Materials and methods</a>). Red dashed lines in (C) and (D) indicate the <i>PHO84</i> reporter levels with p-values less than 0.001 estimating the maximum range of the <i>PHO84</i> reporter levels that the wild type exhibits in each P_i concentration. The mutants in black are previously identified mutants and the one in red is identified in this study.</p

Identification of genes acting upstream of the Pho80/Pho85/Pho81 kinase complex.

<p>(A) A schematic diagram depicting the expected outcome of epistasis analysis depending on whether or not a mutant is defective in the signaling process upstream of Pho80/Pho85. Genes in red act upstream of Pho80/Pho85 and those in blue do not act upstream of Pho80/Pho85. (B) The <i>PHO84</i> reporter levels of double mutants carrying the less induced hits and <i>pho80DΔ</i> in 50 μM P_i conditions. All 380 less induced hits were used to generate the double mutants. The <i>PHO84</i> reporter levels of double mutants in (B) were normalized to that of <i>pho80DΔ</i>. A red dashed line in (B) indicates the maximum <i>PHO84</i> reporter level of double mutants generated by one of the known downstream genes (<i>pho80DΔ gcn5Δ</i>). (C) The <i>PHO84</i> reporter levels of double mutants carrying the less repressed hits and <i>pho81Δ</i> in 1 mM P_i conditions. All 243 less repressed hits were used to generate the double mutants. The <i>PHO84</i> reporter levels of double mutants in (C) were normalized to that of <i>pho81Δ</i>. In (B) and (C), mutants in blue and red are defective in signaling process downstream and upstream of Pho80/Pho85, respectively. (D) A schematic diagram depicting adenine nucleotide metabolism. A gene in red is identified in this study and those in bold black are previously identified.</p

Transcriptional regulation of the PHO regulon in high and low P_i conditions.

<p>Transcriptional regulation of the PHO regulon in high and low P_i conditions.</p