Article

An Intercellular Heme-Trafficking Protein Delivers Maternal Heme to the Embryo during Development in C. elegans

Department of Animal & Avian Sciences and Department of Cell Biology & Molecular Genetics, University of Maryland, College Park, MD 20742, USA.
Cell (Impact Factor: 32.24). 05/2011; 145(5):720-31. DOI: 10.1016/j.cell.2011.04.025
Source: PubMed
ABSTRACT
Extracellular free heme can intercalate into membranes and promote damage to cellular macromolecules. Thus it is likely that specific intercellular pathways exist for the directed transport, trafficking, and delivery of heme to cellular destinations, although none have been found to date. Here we show that Caenorhabditis elegans HRG-3 is required for the delivery of maternal heme to developing embryos. HRG-3 binds heme and is exclusively secreted by maternal intestinal cells into the interstitial fluid for transport of heme to extraintestinal cells, including oocytes. 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 a role for HRG-3 as an intercellular heme-trafficking protein.

Full-text

Available from: Iqbal Hamza, Jan 21, 2014
An Intercellular Heme-Trafficking
Protein Delivers Maternal Heme to the
Embryo during Development in C. elegans
Caiyong Chen,
1
Tamika K. Samuel,
1,3
Jason Sinclair,
1,3
Harry A. Dailey,
2
and Iqbal Hamza
1,
*
1
Department of Animal & Avian Sciences and Department of Cell Biology & Molecular Genetics, University of Maryland, College Park,
MD 20742, USA
2
Biomedical and Health Sciences Institute, Department of Microbiology and the Department of Biochemistry and Molecular Biology,
University of Georgia, Athens, GA 30602, USA
3
These authors contributed equally to this work
*Correspondence: hamza@umd.edu
DOI 10.1016/j.cell.2011.04.025
SUMMARY
Extracellular free heme can intercalate into mem-
branes and promote damage to cellular macromole-
cules. Thus it is likely that specific intercellular
pathways exist for the directed transport, trafficking,
and delivery of heme to cellular destinations,
although none have been found to date. Here we
show that Caenorhabditis elegans HRG-3 is required
for the delivery of maternal heme to developing
embryos. HRG-3 binds heme and is exclusively
secreted by maternal intestinal cells into the intersti-
tial fluid for transport of heme to extraintestinal cells,
including oocytes. 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 zygoti c
hrg-3 expression. Our results establish a role for
HRG-3 as an intercellular heme-trafficking protein.
INTRODUCTION
Heme-containing proteins are found in nearly all phyla of organ-
isms (Hardison, 1996) and play essential roles in a wide range of
biological process (Faller et al., 2007; Kaasik and Lee, 2004;
Okano et al., 2010; Severance and Hamza, 2009). In mammalian
cells, heme is either imported from the extracellular milieu
through the plasma membrane (Uc et al., 2004; Worthington
et al., 2001) or synthesized within the mitochondria for export
to the cytoplasm for delivery to extramitochondrial compart-
ments for insertion into a repertoire of hemoproteins (Dailey,
2002; Severance and Hamza, 2009). Free heme is an amphi-
pathic planar macrocycle that can intercalate into membranes
where it may promote damage to cellular macromolecules (Balla
et al., 1991). Consequently, specific cellular pathways must exist
for the directed transport, trafficking, and delivery of heme to
numerous cellular destinations—but none have been found to
date (Severance and Hamza, 2009). Previously, we identified
the first bona fide metazoan heme importer HRG-1 (SLC48A1),
which we propose plays a critical role in regulating cellular
heme homeostasis in the roundworm Caenorhabditis elegans
and vertebrates (Rajagopal et al., 2008). Heme export is medi-
ated by a major facilitator superfamily protein, the feline leukemia
virus subgroup C cellular receptor (FLVCR), in red blood cells
and macrophages (Keel et al., 2008; Quigley et al., 2004). Hemo-
pexin, a serum heme-binding protein, may facilitate heme export
by physically interacting with FLVCR (Yang et al., 2010).
Together, these proteins constitute part of a larger, intricate
network to maintain organismal hem e homeostasis—a concept
heretofore poorly understood (Severance et al., 2010).
In an effort to identify additional components of the heme
transport pathways, we took advantage of C. elegans, a heme
auxotroph (Rao et al., 2005). In worms, nutritional heme is trans-
ported into the intestine by membrane-bound perm eases—
HRG-1 and HRG-4 (Rajagopal et al., 2008). However, it’s unclear
how tissues such as muscle, neurons, hypodermal cells, and
embryos acquire heme from the intestine. Here we identify
HRG-3, a heme-binding protein that functions to transport
heme from intestinal cells to extraintestinal tissues including
oocytes. Our results suggest that HRG-3 is an intercellular
heme carrier that is essential for early development in C. elegans.
RESULTS
Embryonic Heme Levels Are Affected by Maternal
Heme Availability
C. elegans wild-type N2 worms maintained axenically in
mCeHR-2 liquid medium are gravid adults in 3.5 days in the pres-
ence of optimal concentrations of heme (20 mM) (Rao et al.,
2005). However, their progeny are growth arrested at the fourth
larval stage (L4) in the absence of supplemented heme. To differ-
entiate the effects mediated by maternal heme from zygotic
heme, we cultured parental worms (P
0
) at 1.5, 20, and 750 mM
heme, all of which allow normal development and fertility, and
the ensuing progeny (F
1
) were maintained at either 0 or 20 mM
heme (Figure 1A and Figure S1 available online). Strikingly,
when grown at 0 mM heme, F
1
worms obtained from P
0
mothers
cultured at 1.5 mM heme were growth arrested at the first larval
720 Cell 145, 720–731, May 27, 2011 ª2011 Elsevier Inc.
Page 1
stage (L1), whereas F
1
worms derived from P
0
worms grown at
750 mM heme grew to young adults prior to becoming growth
arrested. Irrespective of the P
0
heme concentrations, F
1
progeny
developed normally when grown at 20 mM heme (Figure 1A and
Figure S1). These results suggest that larval development after
hatching is dependent upon maternal (P
0
) deposition of heme,
and that in the presence of heme, the F
1
progeny can overcome
maternally induced heme deficiency.
To corroborate these results, we used a transgenic heme
sensor strain in which the expression of intestinal GFP is
inversely correlated with heme levels in the worm (Sinclair
and Hamza, 2010). When worms were maintained at low
Figure 1. hrg-3 Is Induced by Heme De ficiency in C. elegans
(A) Parental worms were grown at the indicated heme concentrations for one generation, and the synchronized L1 (first stage larvae) progeny were inoculated into
axenic mCeHR-2 medium supplemented with 0 or 20 mM heme. Representative images of the progeny at day 5 are shown. L4, the fourth stage of larvae. Scale
bar, 20 mm.
(B) Environmental heme represses the expression of both maternal and embryonic GFP in the heme sensor strain IQ6011. I, maternal intestine; E, embryos. Scale
bar, 20 mm.
(C) GFP fluorescence quantification in the embryos derived from IQ6011 grown at 4 and 20 mM heme. Error bars represent SEM from three independent
experiments. *p < 0.001 compared with 4 mM heme.
(D) Northern blot analysis of hrg-3 (370 nucleotides) expression using total RNA isolated from worms grown at different heme concentrations. The blot was
reprobed with the internal control gpd-2.
(E) Quantification of hrg-3 mRNA by qRT-PCR. Relative fold changes were derived by normalizing the cycle threshold values to gpd-2 and then to samples
derived from 20 mM optimal heme using the DDCT method. The experiment was performed in triplicate, and the error bars indicate SEM.
(F) Comparison of HRG-3 proteins in C. elegans, C. briggsae, and C. remanei. Arrowhead, putative signal peptidase cleavage site and underline, hydrophobic
leader peptide.
See also Figure S1 and Table S2.
Cell 145, 720–731, May 27, 2011 ª2011 Elsevier Inc. 721
Page 2
concentrations of heme, strong GFP expression was observed
both in the maternal intestine and in the embryos. However,
embryonic GFP expression was severely attenuated, concomi-
tant with maternal GFP, when mothers were provided with
20 mM heme, further demonstrating that heme levels in the
embryos are linked to maternal heme status (Figures 1B and 1C).
Heme Deficiency Induces hrg-3 Expression in C. elegans
A previous transcriptome analysis identified several hundred
heme-responsive genes (hrgs) in worms grown in axenic
mCeHR-2 liquid culture supplemented with 4, 20, and 500 mM
heme ( Severance et al., 2010). To identify genes that may play
a role in heme delivery, we first sorted genes based on the
degree of upregulation under low heme conditions followed by
three additional criteria. They should encode proteins (1) with
a molecular weight of %30 kDa—a feature characteristic of met-
allochaperones (Kim et al., 2008), (2) with conserved amino acids
that bind heme (H, Y, or C), and (3) that lack multispan transmem-
brane domains. These criteria resulted in the identification of
F58E6.7, which was upregulated >70-fold by low heme in the
microarray. Northern blot analysis revealed the presence of
a single 370 nucleotide transcript (Figure 1D), and qRT-PCR
revealed that worms grown in 1.5 or 4 mM heme increased
the abundance of F58E6.7 mRNA by more than 900-fold and
400-fold, respectively, over what is found in worms grown in
20 mM heme (Figure 1E). Consistent with the northern blotting
results, 5
0
and 3
0
RACE confirmed the presence of an 377
nucleotide mRNA containing an 9 nucleotide 5
0
untranslated
region (UTR), three exons, and a 155 nucleotide 3
0
UTR (not
shown). The open reading frame (ORF) encodes a 70 amino
acid protein with a predicted molecular mass of 8.1 kDa
(Figure 1F). Within the amino terminus of F58E6.7 resides
a stretch of hydrophobic amino acids, which could serve as
either a transmembrane region or a signal peptide. BLAST
searches and gene prediction algorithms identified putative
homologs in other Caenorhabditis species (Figure 1F). These
homologs share >50% sequence identity at the amino acid level.
Consistent with genome nomenclature, we termed F58E6.7
as hrg-3.
Lack of hrg-3 in C. elegans Reveals Developmental
Phenotypes during Heme Deficiency
To determine the function of HRG-3 in C. elegans, we analyzed
worms containing a deletion in hrg-3. The tm2468 strain contains
a 218 bp deletion that encompasses part of the promoter, the
first two exons and the first intron, resulting in a null mutant
(Figures 2A and 2B). These mutant worms have no overt pheno-
types when fed the standard worm diet containing E. coli strain
OP50 (not shown). Because hrg-3 is highly upregulated in worms
grown at low heme conditions in mCeHR-2 liquid medium, and to
rigorously analyze the hrg-3 mutant phenotype, we sought to
recapitulate the heme deprivation conditions on agar plates
with E. coli as the food source. Because the OP50 E. coli strain
can synthesize heme endogenously, it was not possible to
deplete the bacteria of heme. The bacterial strain RP523 is
defective in hemB, which encodes 5-aminolevulinic acid dehy-
dratase (ALAD), the second enzyme in the heme biosynthesis
pathway (Li et al., 1988). Consequently, RP523 is dependent
upon exogenous heme for growth. By exposing worms to
RP523 grown with different concentrations of heme, one can
control heme levels in the worm via E. coli. Wild-type N2 worms
exhibited a 1 to 2 day growth delay when fed RP523 grown with
1 mM heme compared to those grown on OP50, a growth pheno-
type that was not present when wild-type worms were fed RP523
grown with 10 to 50 mM heme (not shown). hrg-3 mutant worms,
like wild-type worms, revealed the expected growth delay in the
parental (P
0
) generation when fed RP523 grown with 1 mM heme.
However, 40% of eggs laid by the mutant worms failed to hatch
(Figure 2C), and the F
1
embryos that did hatch were growth
arrested at the first larval stage (Figures 2D and 2E). The lethality
and growth retardation phenotypes were completely rescued
when hrg-3 mutants were fed RP523 that had been grown with
50 mM heme. Collectively, these results indicate that HRG-3 is
essential for both embryonic and postembryonic development
under heme-limiting conditions and that the absence of HRG-3
results in heme deficiency that is manifested specifically in the
F
1
generation.
HRG-3 Is Secreted by the Intestine into the Interstitial
Fluid
We determined the tissue and subcellular distribution of HRG-3.
Worms expressing Phrg-3::GFP transcriptional fusions had GFP
in the worm intestine, with the greatest levels in the anterior (int2
and int3) and mid-intestinal cells (int4–6). The anterior-most (int1)
and the posterior-most gut cells (int7–9) possessed low levels of
GFP (Figure 3A). GFP was only observed in worms that were
maintained in %6 mM heme in mCeHR2 medium—consistent
with the qRT-PCR results (not shown). Intestinal Phrg-3::GFP
expression was observed through all larval stages, and in both
hermaphrodites and males (Figure S2). Zygotic expression of
hrg-3 was first detected in late embryos at 300 min of develop-
ment (Figure S2).
To identify the subcellular distribution of HRG-3, we con-
structed transgenic worms that express the translational reporter
Phrg-3::HRG-3::YFP. Worms grown in 2 mM heme possessed
a weak HRG-3::YFP signal that was located in cytoplasmic
puncta within the worm intestine (Figure 3B, left panel).
Unexpectedly, the majority of HRG-3::YFP was present as
vesicular structures outside the intestine in coelomocytes—
macrophage-like scavenger cells located in the pseudocoelomic
cavity (Figure 3B, right panel).
To determine whether the HRG-3::YFP translational reporter
was inadvertently expressed in extraintestinal cells, we directed
the expression of hrg-3 from the vha-6 promoter, a well-charac-
terized intestinal promoter (Oka et al., 2001). Transgenic worms
expressing Pvha-6::HRG-3::mCherry revealed strong HRG-
3::mCherry localization in extraintestinal tissues including coelo-
mocytes, the pseudocoelom, gonadal sheath cells, and the
uterus (Figure 3C). Within intestinal cells, HRG-3::mCherry signal
was observed as distinct cytoplasmic vesicles that colocalized
with mannosidase::GFP (Mans-GFP), a protein that localizes to
the Golgi (Figure 3D). However, unlike HRG-3::mCherry, expres-
sion of Mans-GFP from the vha-6 promoter showed no extrain-
testinal localization (Figure 3D). Taken together, these results
strongly suggest that HRG-3 is secreted from the intestinal cells
into the pseudocoelom for uptake by extraintestinal tissues.
722 Cell 145, 720–731, May 27, 2011 ª2011 Elsevier Inc.
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HRG-3 Is Specifically Targeted to the Secretory Pathway
To determine whether HRG-3 is a membrane-anchored protein
within an exosome or a soluble secreted protein, we synthesized
truncated variants of HRG-3 that were tagged at the C terminus
with GFP. Expression of HRG-3-GFP in HEK293, a human
kidney cell line, resulted in perinuclear localization. To examine
the membrane orientation of HRG-3, we conducted fluores-
cence protease protection (FPP) assays (Lorenz et al., 2006). In
this procedure transfected cells are sequentially exposed to
digitonin to permeabilize the cells, followed by protease diges-
tion to cleave cytoplasmic-located proteins. For example, the
membrane protein prototype, CFP-CD3 d-YFP, which is targeted
to the endoplasmic reticulum (ER), contains a cytoplasmic YFP
domain that is susceptible to protease digestion compared to
the lumenal CFP domain, which remains intact (Figure 3E, upper
panels). We found that HRG-3-GFP in transfected cells was not
digested by the protease treatment, a result that was reproduc-
ible when the N-terminal 29 amino acids of HRG-3 (HRG-3N)
were expressed as a YFP fusion protein (Figure 3E). These
results indicate that the C terminus of HRG-3 is protected from
protease digestion and is not cytoplasmic.
To further identify the location of HRG-3 and the function of the
N-terminal region, we synthesized truncated forms of HRG-3.
Ectopic expression of these fusion proteins in HEK293 cells re-
vealed that full-length HRG-3 and HRG-3N colocalized with the
Golgi marker b 1,4-galactosyltransferase (GalT)-CFP, consistent
with the localization of HRG-3 in the C. elegans intestinal cells
(Figure 3F). However, deletion of the first 29 amino acids
(HRG-3DN) resulted in a cytoplasmic localization, indicating
that this N-terminal region is necessary for targeting HRG-3 to
the secretory pathway. The localization of HRG-3 was not due
to a large fluorescent protein tag because an HA epitope-tagged
HRG-3 also colocalized with the GalT marker (Figure 3F, right
panel), and coexpression of HRG-3-YFP and HRG-3-HA in the
same cell resulted in both proteins colocalizing with the GalT
marker (not shown).
Figure 2. hrg-3 Is Required for Early Development under Low Heme
(A) hrg-3 contains three exons. The deleted region in tm2468 allele is depicted by an underline. Open rectangles, exons; gray boxes, untranslated regions; +1,
transcription start site.
(B) RT-PCR was performed using the primer set shown as arrows in Figure 2A on total RNA extracted from the hrg-3 (tm2468) and N2 (wild-type control) worms
grown at low heme.
(C) hrg-3 mutants and their wild-type broodmates were fed with heme-deficient strain RP523 grown at the indicated heme concentrations. Percentage of
unhatched embryos were scored following incubation at 20
C for 24 hr. Error bars indicate the SEM from three individua l experiments. *p < 0.001 compared with
wild-type broodmate controls under the same condition.
(D) Worm strains were grown on RP523 for two subsequent generations. F
1
worms were sorted by COPAS BioSort. Time of flight and extinction indicate the length
and the optical density of worms, respectively.
(E) Progeny grown on RP523 with 1 and 50 mM heme were imaged at day 6 and day 3 post-hatching, respectively. Representative images of gravid adults in
controls and the arrested larvae of hrg-3 mutants are shown. Scale bar, 20 mm.
Cell 145, 720–731, May 27, 2011 ª2011 Elsevier Inc. 723
Page 4
To examine whether the N terminus is cleaved or retained in
HRG-3, transfected HEK293 cell lysates were subjected to anal-
ysis by SDS-PAGE and immunoblotting. The full-length HRG-3
and the HRG-3DN proteins were found to be equivalent in size
(Figure 3G, lanes 1 and 2). Correspondingly, expression of
HRG-3N-GFP resulted in a protein that was indistinguishable
from GFP alone in size, suggesting that the N-terminal hydro-
phobic region is cleaved to produce the mature HRG-3-GFP
protein (Figure 3G, lanes 3 and 4). To verify this result, we
compared the molecular weight of HRG-3 that was generated
either by in vitro transcription and translation or by transfecting
HEK293 cells. Immunoblotting with anti-HA antibody revealed
that the in vitro generated protein had a larger molecular weight,
corresponding to the retention of the 25 amino acid leader
peptide (Figure 3G, lanes 5 and 6). These observations suggest
that the N-terminal portion of HRG-3 may be processed and
removed in the mature protein.
Specific Regulators of the Secretory Pathway Direct
HRG-3 Trafficking
To identify the membrane-trafficking components that regulate
HRG-3 secretion from the intestine, we used RNAi-mediated
depletion of 45 genes that encode regulators of endocytosis
and secretion (Table S1)(Balklava et al., 2007). We found
that depletion of 15 trafficking factors caused HRG-3::mCherry
to either accumulate within the maternal intestine (vha-1)or
Figure 3. HRG-3 Is Secreted from the Maternal Intestine
(A) IQ8031 harboring the transcriptional reporter Phrg-3::GFP was maintained at 2 mM heme in axenic mCeHR-2 medium. Arrow indicates GFP expression in the
intestine. Scale bars, 20 mm.
(B) Transgenic worms with the translational reporter Phrg-3::HRG-3::YFP were grown at 2 mM heme in axenic mCeHR-2 medium. YFP localization was examined
at day 4 by confocal microscopy. Arrowhead, Golgi apparatus in the intestine; Arrow, coelomocytes. Scale bars, 10 mm.
(C) HRG-3::mCherry was expressed using an intestine-specific vha-6 promoter and analyzed by confocal microscopy. HRG-3::mCherry was observed in
coelomocytes (C), pseudocoelom (P), gonads (G), and uterus (U). Scale bars, 20 mm.
(D) The HRG-3::mCherry transgenic strain was crossed into strain RT1315, which expresses the Golgi-localized mannosidase (Mans)-GFP fusion protein. Images
from these double transgenic worms were acquired by confocal microscopy. Arrowhead, Golgi apparatus in the intestine; Arrow and C, coelomocytes. Scale
bars, 5 mm.
(E) Fluorescence protease protection assays in transfected HEK293 cells treated with digitonin and proteinase K. Time-lapse images were acquired in live cells by
epifluorescence microscopy. Scale bars, 10 mm.
(F) Fluorescence (YFP) and immunofluorescence (HA) analyses of HRG-3 constructs in HEK293 cells. Galactosyltransferase (GalT)-CFP was used as a Golgi
marker. Scale bars, 10 mm.
(G) Immunoblots of HRG-3 constructs. Left panel shows western blots of HRG-3 proteins expressed in HEK293 cells and probed with GFP antibody. Right panel
shows the western blot of HRG- 3-HA expressed either in HEK293 cells (lane 5) or in an in vit ro transcription and translation system (IVT, lane 6) and probed with
a HA antibody. The size difference between HRG-3-GFP (lane 1) and HRG-3D N::GFP (lane 2, top band) is 0.4 kDa and between HRG-3N-GFP (lane 3) and GFP
(lane 4) is <0.1 kDa. Lane 2 contains HRG-3 fusion protein (asterisk) and a smaller degradation product.
See also related Figure S2.
724 Cell 145, 720–731, May 27, 2011 ª2011 Elsevier Inc.
Page 5
mislocalize in extraintestinal tissues (sec-23) or embryos
(sec-24.1)(Table 1 and Figure 4A). The majority of these candi-
date genes encoded for protein subunits that formed vesicle
coatomer and vacuolar ATPase complexes (Table S1).
To determine whether HRG-3 secretion was tissue
dependent, we ectopically expressed hrg-3::mCherry in the
hypodermis, specialized epithelial cells in C. elegans, using the
dpy-7 promoter (Rolls et al., 2002). Transgenic worms express-
ing Pdpy-7::HRG-3::mCherry revealed HRG-3::mCherry signal
within cytoplasmic puncta in the hypodermis and in extrahypo-
dermal cells including coelomocytes, the pseudocoelom, and
the uterus (Figure 4B and Figure S3). As observed for the regula-
tion of HRG-3 trafficking in the intestine, HRG-3 secretion from
the hypodermis was also regulated by general membrane-
trafficking components (Figure 4A versus Figure S3). Collec-
tively, these results indicate that HRG-3 trafficking is mediated
by general regulatory factors within the secretory pathway and
is cell-type independent.
To examine whether HRG-3 secretion was dependent on
organismal heme levels, we generated transgenic worms that
expressed hrg-3::YFP under the control of the inducible
hsp-16.2 promoter, which is strongly expressed in the intestine
and induced in response to heat shock. HRG-3::YFP accumu-
lated in the coelomocytes, which is indicative of secretion from
the intestine, within 60 min after induction and continued to
accumulate over time (Figures 4C and 4D). Importantly,
Phsp-16.2::HRG-3::YFP transgenic worms accumulated similar
amounts of HRG-3::YFP irrespective of heme concentrations in
the growth medium (Figure 4E). We were unable to examine
HRG-3::YFP secretion in worms grown at <1 mM heme because
these animals were severely growth retarded.
To directly demonstrate that maternal HRG-3 is deposited
within the embryo, we analyzed Pvha-6::HRG-3::mCherry
mosaic transgenic worms in which the transgene was
maintained as an extrachromosomal array with a transmission
efficiency of 60%. Thus, P
0
mothers that express HRG-
3::mCherry will lay F
1
progeny that either express or lack the
transgene (Figure 4F). Remarkably, 100% of F
1
embryos isolated
from transgenic mothers were positive for HRG-3::mCherry even
though 40% of these embryos did not express the transgene.
HRG-3::mCherry was visible at the time of gastrulation and
detectable up to the mid-larval stages (L2 and early L3). Impor-
tantly, 100% of the F
2
progeny, derived from nontransgenic
HRG-3::mCherry-positive F
1
mothers, lacked any detectable
HRG-3::mCherry signal and the transgene (Figure 4F). These
results confirm that maternal HRG-3 is transferred to all embryos
irrespective of the zygotic genotype.
Maternal HRG-3 Rescues the Growth Phenotypes
Our studies reveal that although hrg-3 is expressed in the intes-
tine, hrg-3 loss-of-function mutants show embryonic lethality
and growth retardation in the F
1
generation when grown under
heme-insufficient conditions. These phenotypes could be due
to HRG-3 deficiency either in P
0
mother, in the F
1
embryo, or
both. To answer this question, we created a Phrg-3::HRG-
3::ICS::GFP construct in which hrg-3 and gfp were under the
control of a single hrg-3 promoter but were separated by the
SL2 intercistronic sequence (ICS) from rla-1 (Figure 5A). In
C. elegans, the HRG-3::ICS::GFP transgene is transcribed as
a single polycistronic mRNA but yields two separate proteins:
HRG-3 and GFP. Thus, GFP fluorescence is indicative of trans-
gene expression (Figure 5A). Size and optical density analysis
of stably transformed worms using a COPAS Biosort provided
data that demonstrated that hrg-3 expression fully rescues the
severe growth phenotype in the F
1
progeny in hrg-3-deficient
worms in the presence of low heme (Figure 5B).
To confirm that intestinal HRG-3 is crucial for heme delivery to
extraintestinal tissues, we used targeted gene rescue by ex-
pressing hrg-3 under the control of the intestine-specific vha-6
promoter. Unlike the hrg-3 promoter the vha-6 promoter is not
heme regulated. Furthermore, to distinguish between maternal
versus zygotic expressed HRG-3, we analyzed mosaic trans-
formants in which the transgene transmission efficiency was
40%. Thus, only P
0
mothers that express HRG-3::ICS::GFP
will lay progeny that either lack hrg-3 or express hrg-3 as an
extrachromosomal array. As expected, in the presence of low
heme, >30% embryos from hrg-3 loss-of-function mothers failed
to hatch and larvae that did hatch were growth arrested at the L1
stage. By contrast, <2% of embryos remained unhatched from
P
0
mothers expressing the hrg-3 transgene (Figure 5C). Impor-
tantly, a significant proportion of hatched embryos derived
from hrg-3-expressing mothers continued to grow past the L2
stage, even though these larvae did not express hrg-3 (Figure 5D
and Figure 5E, center and right panels). hrg-3-expressing
embryos derived from crosses between hrg-3 loss-of-function
mothers and HRG-3::ICS::GFP males were growth arrested.
Only 2 out of 65 F
1
progeny grew beyond the initial larval stages
(Figures 5F and 5G). These data strongly suggest that targeted
expression of hrg-3 from the maternal intestine is necessary
Table 1. Regulators of Membrane Trafficking that Affect
HRG-3-mCherry Expression and Localization
Gene ID
a
Name
Expression of HRG-3-mCherry
Intestine Embryo Extraintestinal
K02D10.5 snap-29 Increased Increased nc
T20G5.1 chc-1 Increased Increased nc
Y49A3A.2 vha-13 Increased nc nc
Y113G7A.3 sec-23 Increased Increased Lumenal
F12F6.6 sec-24.1 Increased Increased nc
R10E11.8 vha-1 Increased nc nc
Y25C1A.5 Increased na Lumenal
T01H3.1 vha-4 Increased nc nc
ZK970.4 vha-9 Increased nc nc
F59E10.3 Increased na Lumenal
R10E11.2 vha-2 Increased nc nc
Y55H10A.1 vha-19 Increased nc nc
ZK180.4 sar-1 Increased nc nc
F38E11.5 Increased na Lumenal
T14G10.5 Increased nc nc
na: these worms did not develop to the gravid stage.
nc: no change.
a
List of positive genes compiled from Table S1.
Cell 145, 720–731, May 27, 2011 ª2011 Elsevier Inc. 725
Page 6
and sufficient for embryonic development even when environ-
mental heme is limiting.
hrg-3 Mutant Embryos Have Reduced Heme Levels
Our results support a role for HRG-3 in heme delivery from the
maternal intestine to oocytes. Based on this evidence, we postu-
lated that when HRG-3 is available in limited quantities, greater
heme accumulation would be found in the maternal intestine
and a corresponding heme deficiency would exist in the devel-
oping embryos compared to wild-type worms. To estimate
heme levels in these tissues, we crossed the heme sensor strain
IQ6011 (Phrg-1::GFP) with hrg-3 null mutants (Rajagopal et al.,
2008; Severance et al., 2010; Sinclair and Hamza, 2010). The
resulting IQ8011 gravid worms had reduced GFP levels in the
intestine compared to IQ6011 worms (control) when grown in
medium containing 1.5 or 2 mM heme ( Figure 6A). Embryos
derived from these mothers showed reproducibly higher levels
of GFP, compared to wild-type controls (Figure 6B). As the
Figure 4. Regulators of the Secretory Pathway Direct HRG-3 Trafficking from the Maternal Intestine into the Embryos
(A) Depletion of candidate genes, selected from Table 1, alters HRG-3::mCherry expression in the intestine. RNAi against sec-23 and sec-24.1 resulted in
accumulation of HRG-3::mCherry in the intestinal lumen (arrows) and the embryos (arrowheads), respectively. Asterisks indicate the increased HRG-3-mCherry
level in the intestine due to vha-1 depletion. C, coelomocytes. Scale bars, 20 mm.
(B) HRG-3::mCherry was expressed using a hypodermis-specific dpy-7 promoter. Confocal images of the same cross-section but different focal planes are
shown to distinguish hypodermal cells from coelomocytes (C). Pdpy-7::HRG-3::mCherry construct is not expressed in the intestine (I). Arrowhead, Golgi
apparatus in the hypodermis; Arrows, coelomocytes. Scale bar, 10 mm.
(C) C. elegans expressing HRG-3::YFP under control of the hsp-16.2 promoter were heat shocked at 37
C for 30 min and then transferred to 20
C for 0, 30, 60, or
180 min. Representative confocal images of YFP and DIC are shown. Dotted circles in the first two images and arrows indicate the position of coelomocytes.
Scale bars, 20 mm.
(D) Quantification of HRG-3::YFP secretion from the intestine by measuring accumulation in coelomocytes at different time points after a 30 min induction by heat
shock. Asterisks indicate that this group is statistically different from any other groups (p < 0.001). Error bars indicate SEM from two independent experiments.
(E) Worms carrying Phsp-16.2::HRG-3::YFP grown on RP523 bacteria supplemented with 1, 4, or 20 mM heme for 72 hr were heat shocked at 37
C for 30 min.
Worms were transferred to 20
C for 60 min and YFP intensity in coelomocyte s was quantified and normalized to the coelomocyte volume. ns, not significant
(p > 0.05). Error bars indicate SEM from two independent experiments.
(F) Progeny derived from Pvha-6::HRG-3::mCherry mosaic transgenic worms with a transmission efficiency of 60% were analyzed by epifluorescence
microscopy for two successive generations. Representative images of transgenic (tg) and nontransgenic (non-tg) L1 larvae are shown. Numbers represent the
percentage of segregating progeny that contain the HR G-3::mCherry transgene. Although 100% of the embryos were positive for HRG-3::mCherry, in each
generation only 60% of the L1 progeny were transgenic. A hallmark of these transgenic worms was the accumulation of HRG-3::mCherry in coelomocytes
(arrows). Scale bars, 20 mm. Genotypes were confirmed in single worms by PCR amplification of the mCherry transgene using genomic DNA template (top panel).
See also related Figure S3.
726 Cell 145, 720–731, May 27, 2011 ª2011 Elsevier Inc.
Page 7
heme status is inversely correlated with the GFP expression in
heme sensor worms, these results suggest that deletion of
hrg-3 results in increased heme levels in the maternal intestine
and reduced heme levels in the embryos. The modest differ-
ences in embryonic GFP levels between wild-type and hrg-3
embryos could be due to incomplete penetrance of the embry-
onic lethal phenotype (40%; Figure 2C and Figure 5C) and
environmental modifiers such as nutrient heme (Figure 2C).
The consistently higher GFP (5 fold) content in the intestine
of the mother compared to the embryo could be attributed
to the endoreduplication of chromosomes and multi-nucleation
of the intestinal cells during worm development (Hedgecock
and White, 1985). Taken together, our results show that HRG-3
deficiency causes perturbation of heme homeostasis in the
maternal intestine and the embryo.
HRG-3 Is a Heme-Binding Protein
Although the genetic and cell biology data are compelling in
demonstrating that HRG-3 is involved in trafficking of heme
from the maternal intestine to eggs, the data do not discriminate
between direct or indirect functions of HRG-3 in heme homeo-
stasis. To determine whether HRG-3 directly interacts with
heme, we synthesized the mature secreted form of HRG-3 and
measured its ability in vitro to bind heme. Pure HRG-3 is readily
soluble in weak acidic solutions but becomes less soluble and
gradually precipitates at neutral pH. However, addition of ferric
Figure 5. Maternal Expression of hrg-3 Is Sufficient to Rescue the Early Embryonic Growth Phenotype
(A) hrg-3 and gfp were expressed using a single hrg-3 promoter but were separated by the intercistronic sequence (ICS) from rla-1. Introduction of this construct
into hrg-3 null mutant restored the growth of F
1
progeny at low heme. Progeny grown on RP523 with 1 mM heme were photographed at day 6 post-hatching.
Representative images of arrested or rescued F
1
worms are presented. Scale bar, 20 mm.
(B) Worm strains were grown on RP523 with 1 and 50 mM heme for two subsequent generations. The sizes of the F
1
worms were measured by COPAS BioSort.
Time of flight and extinction indicate the length and the optical density of worms, respectively.
(C) The HRG-3::ICS::GFP construct (hrg-3ec) was expressed using an intestinal-specific vha-6 promoter. A transgenic strain with 40% transmission efficiency
was crossed to the hrg-3 mutant. GFP-expressing mothers gave rise to >98% live progeny at low heme. Error bars indicate the SEM from three individual
experiments. *p < 0.05 compared with hrg-3 mutant under the same condition.
(D) hrg-3 mutant worms with or without hrg-3ec construct were grown on RP523 with 1 mM heme for one generation. Their progeny were maintained under the
same condition for 5 days. Representative images of the arrested and rescued progeny are presented. ‘tg’ and ‘‘non-tg’ denote whether the hrg-3 null mutants
express or lack hrg-3ec construct, respectively. Scale bars, 20 mm.
(E) Progeny from Figure 5D were sorted based on size (extinction) and transgene (GFP) by COPAS BioSort.
(F) HRG-3::ICS::GFP males were crossed with hrg-3 mutant hermaphrodites that had been grown on RP523 supplemented with 1 mM heme for one generation.
The F
1
heterozygous progeny were maintained at the 1 mM heme for 5 days. Representative images of the arrested progeny are shown. Scale bars, 20 mm.
(G) F
1
progeny from (F) were sorted based on size (extinction) and transgene (GFP) by COPAS BioSort.
Cell 145, 720–731, May 27, 2011 ª2011 Elsevier Inc. 727
Page 8
protoheme to a solution of soluble HRG-3 at pH 7.0 resulted in
a distinct spectroscopic peak at 416 nm (Figure 6C), whereas
addition of ferrous protoheme resulted in a peak at 441 nm
(not shown). These peaks are shifted and distinct from those of
the free heme, indicating that heme is definitively binding
HRG-3. Although we were unable to obtain an accurate associ-
ation constant spectroscopically because the binding affinity of
HRG-3 for heme was weak, titration of both ferric and ferrous
heme revealed that, regardless of oxidation state, heme binds
to HRG-3 at a stoichiometry of 1:2 (heme:protein) (Figure 6D).
Notably, the soluble heme-bound HRG-3 slowly precipitates
over several hours as a bright red complex, indicating that the
precipitated protein remains bound to heme with significant
affinity.
DISCUSSION
As a heme auxotroph, embryonic and postembryonic develop-
ment in C. elegans is dependent on either maternal heme
deposition (Figure 1A, upper panel) or larval heme acquisition
(Figure 1A, lower panel). Our results uncover the crucial role of
HRG-3 in maintaining embryonic heme homeostasis and its
interdependence with maternal heme status (Figures 2C–2E
and Figures 5C–5E). C. elegans acquires environmental heme
through the coordinated functions of HRG-1 membrane-bound
heme permeases located in the intestine (Rajagopal et al.,
2008). Because a hermaphrodite worm has 959 somatic cells
of which 20 are polarized intestinal cells (McGhee, 2007), the
question remaining is how do extraintestinal cells acquire
heme? We postulate that this is partly accomplished through
HRG-3, which we have shown above is likely to be an intercel-
lular heme chaperone (Figure 6E). HRG-3 is transcriptionally
upregulated in response to heme insufficiency and secreted by
the maternal intestine into the pseudocoelom, the worm’s circu-
latory system, for mobilization of heme to extraintestinal tissues
including the gonads and uterus. In the absence of HRG-3, heme
accumulates in the intestine of gravid adults, whereas the
embryos are heme deficient resulting in embryonic lethality or
growth arrest immediately after the embryos hatch.
When and how does HRG-3 transfer heme to the embryo?
In C. elegans, oocyte fertilization results in a rapid assembly of
a trilamellate chitinous eggshell by the time pseudocleavage of
the one-cell embryo occurs (Johnston et al., 2006). The eggshell,
which surrounds the developing embryo until hatching, provides
a mechanical and osmotic barrier and ensures that early devel-
opmental events occur (Johnston et al., 2006). Given the imper-
vious nature of the eggshell matrix to environmental factors, we
speculate that heme deposition by HRG-3 must occur during
oocyte maturation and prior to fertilization. This maternal-to-
oocyte trafficking of heme exhibits striking similarity with the lipid
transport pathways by vitellogenins, the major yolk precursor
proteins. C. elegans contains six vitellogenins that are produced
by the maternal intestine to bind lipids and translocated to the
gonads via the pseudocoelom for yolk deposition in oocytes
(Blumenthal et al., 1984; Kimble and Sharrock, 1983; Spieth
and Blumenthal, 1985). However, unlike vitellogenins that are ex-
pressed only in the adult hermaphrodite (Blumenthal et al., 1984),
hrg-3 is expressed during all developmental stages in both
hermaphrodites and males (Figure S2). HRG-3 may, therefore,
play a more extensive role than vitellogenins by mobilizing
heme from intestinal cells to tissues other than embryos. Indeed,
Figure 6. Maternal-to-Embryonic Heme Transfer Is Perturbed in
hrg-3 Mutant Worms
(A) The IQ8011 (hrg-3; Phrg-1::GFP) strain was generated by crossing the
hrg-3 mutant into the heme sensor IQ6011 strain. Both strains were maintained
in axenic mCeHR-2 medium with the indicated heme concentr ations for
4 days. GFP levels were measured by fluorimetry in protein lysates prepared
from the gravid adults. Error bars indicate the SEM from four individual
experiments. *p < 0.01 compared with hrg-3 mutants under the same
conditions.
(B) IQ6011 and IQ8011 worms were maintained in axenic mCeHR-2 medium
with the indicated heme concentrations for 4 days. F
1
embryos obtained by
bleaching the gravid adults were homogenized and GFP levels measured by
fluorimetry. Error bars indicate the SEM from four individual experiments.
(C) Absorption spectra of HRG-3 peptide in the presence or absence of heme,
and free heme alone in buffer without added HRG-3. Heme-bound HRG-3
displays an absorbance peak at 416 nm, which is clearly shifted in both
wavelength and intensity from that of free heme.
(D) Absorption spectra of HRG-3 peptide in the presence of ferric or ferrous
heme. Increasing amounts of HRG-3 were added to 9 mM ferric heme or
ferrous heme (in the presence of dithionite), and the absorbance was moni-
tored at 415 nm or 441 nm, respectively.
(E) The proposed model of HRG-3 as a heme chaperone. Heme deficiency
induces the expression of HRG-1 and HRG-4, membrane-bound permeases
that import heme (red cross) into the intestine, and HRG-3, which is secreted
from the intestine into the circulatory system (pseudocoelom) for heme
delivery to extraintestinal cells.
728 Cell 145, 720–731, May 27, 2011 ª2011 Elsevier Inc.
Page 9
HRG-3-deficient F
1
larvae are growth arrested in the presence of
low heme, implying that, in addition to in utero development that
can be rescued by maternal HRG-3, sustained hrg-3 expression
in the larvae is essential for postembryonic development.
A similar pathway may also exist for other metals such as zinc,
which has been recently demonstrated to regulate meiotic matu-
ration of mammalian oocytes and early embryonic development,
implicating a role for zinc in the maternal legacy from egg to
embryo (Kim et al., 2010).
What are the cellular factors that regulate HRG-3 trafficking?
Of the 45 general regulators of membrane trafficking that were
recently identified from a genome-wide RNAi screen for modula-
tors of endocytosis and secretion of vitellogenin (Balklava
et al., 2007), RNAi depletion of 15 factors caused HRG-3 to
accumulate or mislocalize in both the intestine and hypodermis
(Table 1). Interestingly, these regulators broadly fall into two
categories—coatomer complex and vacuolar ATPase subunits.
HRG-3 trafficking and secretion may therefore be dependent
on assembly of vesicles and its acidification. Although maternal
HRG-3 persists from embryonic to larval stages, just like vitello-
genin (Chotard et al., 2010), HRG-3 is not part of the vitellogenin
complex because RNAi depletion of all six vitellogenins did not
alter HRG-3 secretion and trafficking (not shown). There are
several examples of maternal contributions to the embryo that
persist and function at later stages of development. For example,
maternal cyclin E, a cell-cycle checkpoint regulator, controls
G
1
/S progression and coordinates cell proliferation and differen-
tiation in C. elegans (Brodigan et al., 2003; Fay and Han, 2000).
Maternal cyclin E is sufficient to regulate G1 cell-cycle progres-
sion until the L3/L4 larval stages; cell-cycle defects only become
apparent when the maternal protein is exhausted in the F
1
progeny.
Our biochemical studies demonstrate that the mature pro-
cessed HRG-3 protein binds both ferrous and ferric heme with
an apparent stoichiometry of two protein and one heme moiety.
The spectroscopic data are consistent with a five coordinate
high spin heme and reproducible by electron paramagnetic
resonance spectroscopy (A.N. Albetel, M.K. Johnson, H.A.D.,
and I.H., unpublished data). We propose that heme transfer by
HRG-3 to target sites may be dependent on affinity gradients,
as has been demonstrated for intracellular copper chaperones.
Copper transfer is thermodynamically favored from low- to
intermediate- to high-affinity sites driven by intracellular metallo-
chaperones; the hierarchy of copper binding among specific
chaperones is governed by fast metal transfer, specific
protein-protein recognition, and cellular compartmentalization
(Banci et al., 2010).
Although intercellular transport of iron by the transferrin-
transferrin receptor complex has been well documented, several
lines of evidence also support the existence of an intercellular
heme transport pathway in vertebrates. First, even though
knockout of the heme synthesis pathway in mice is embryonic
lethal, homozygous embryos survive at least until embryonic
day (E) 3.5, suggesting the existence of heme stores (Magness
et al., 2002; Okano et al., 2010). Second, zebrafish embryos
with loss-of-function mutations in heme synthesis genes can
survive from 10–25 days post-fertilization (Childs et al., 2000;
Dooley et al., 2008), plausibly because these embryos may
contain either maternal-derived mRNA for heme synthesis
enzymes or direct deposition of maternal heme. Third, human
patients with acute attacks of porphyrias, genetic diseases due
to defects in heme synthesis enzymes, are administered heme
intravenously as an effective therapeutic treatment, which
results in reduction of heme synthesis intermediates and
a concomitant increase in liver heme-dependent enzyme
activities for cytochrome P450 and tryptophan 2,3-dioxygenase,
indicating that infused heme in the blood stream is utilized in toto
by peripheral tissues (Puy et al., 2010; Bonkovsky et al., 1991).
Lastly, cell culture studies with human colon-derived Caco-2
cells and mouse macrophages reveal that a portion of heme,
derived from dietary sources or senescent red blood cells, is
released into the blood stream as an intact metalloporphyrin
(Knutson et al., 2005; Uc et al., 2004). A potential candidate for
an intercellular heme delivery protein would be hemopexin,
which scavenges heme with an apparent dissociation constant
(K
D
) 10
15
M and clears it from the circulatory system (Hrkal
et al., 1974). The heme-hemopexin complex binds to the LRP/
CD91 receptor and is endocytosed, and the majority of the he-
mopexin is degraded in the endo-lysosome of hepatocytes,
macrophages, and syncytiotrophoblasts (Hvidberg et al., 2005;
Tolosano et al., 2010). Surprisingly, hemopexin null mice are
viable and fertile and present no evidence of tissue damage
due to oxidative stress from abnormal heme and/or iron deposi-
tion under normal conditions; heme overload and hemolytic
damage, however, cause tissue damage in these mice (Tolosano
et al., 1999, 2010 ). We speculate that a functional homolog of
HRG-3 may also exist in vertebrates as an alternate pathway
to facilitate the targeted delivery and redistribution of heme
between tissues and specific cell types and maintain systemic
heme homeostasis.
Even though heme uptake and transport pathways are clearly
conserved across metazoans (Severance and Hamza, 2009),
heme auxotrophic organisms, such as C. elegans and parasitic
helminthes, are crucially dependent on these pathways for utili-
zation of environmental heme for growth and reproduction (Rao
et al., 2005). Helminths affect more than a quarter of the world’s
population (Chan et al., 1994; Hotez et al., 2008) and cause tens
of billions of dollars of loss in animal and plant production annu-
ally (Fuller et al., 2008; Jasmer et al., 2003). Moreover, anthel-
minthics are becoming less effective in humans and livestock
because of rampant drug resistance (Fuller et al., 2008; Jasmer
et al., 2003). We propose that an excellent anthelminthic target
would be the HRG-3-mediated pathway for transporting heme
to developing oocytes, especially in parasites such as hook-
worms, which infect more than a billion people worldwide and
feed on host red blood cell hemoglobin (Held et al., 2006;
Wu et al., 2009).
EXPERIMENTAL PROCEDURES
Worm Growth Assays on RP523
The heme-deficient E. coli strain RP523 was maintained in liquid LB medium
supplemented with 1 mM heme at 37
C(Li et al., 1988). To prevent unequal
growth of the RP523, overnight cultures were diluted into fresh medium with
different concentrations of heme, grown for 5.5 hr, and heat inactivated at
65
C for 2–5 min. A 0.2 optical density (OD) 600 of bacteria was seeded on
each 35 mm nematode growth medium (NGM) agar plate. Synchronized L1
Cell 145, 720–731, May 27, 2011 ª2011 Elsevier Inc. 729
Page 10
larvae of hrg-3 (tm2468) allele and its wild-type broodmates were place d onto
RP523 plates and incubated at 20
C for 3–5 days. Five gravid hermaphrodites
from each plate were allowed to lay eggs for 12–16 hr on a new RP523 seeded
plate. The embryos that did not hatch after 24–32 hr were considered dead.
The growth of F
1
larvae was scored when the wild-type worms reached young
adult stage. Those larvae that did not progress past L2 stage were considered
growth arrested. DIC images were acquired on the F
1
worms when the wild-
type broodmates reached gravid stage.
Worm Analysis with COPAS BioSort
Nematodes from plates containing RP523 were frozen when worms reached
gravid stage. Worms from each sample (100) were analyzed for length (time
of flight) and optical density (extinction) by using a COPAS BioSort (Union
Biometrica, Holliston, MA, USA). Gating parameters of time of flight 30–800
and extinction 15–800 were set by using synchronized L1s and mixed
worm populations. Raw data outside this range were filtered to exclude
particulates and bubbles. For rescue experiments, hrg-3 mutants expressing
Phrg-3::HRG-3::ICS::GFP (integrated) or Pvha-6::HRG-3::ICS::GFP (extra-
chromosomal array) were generated by genetic crosses. For zygotic rescue
experiment, hrg-3 mutants were grown on RP523 with 1 mM heme for one
generation, followed by crossing with male worms expressing Phrg-
3::HRG-3::ICS::GFP. The progeny were maintained at low heme for 5 days.
Both worm size and GFP intensity were analyzed using COPAS BioSort for
maternal and zygotic rescue experiments. The settings for GFP measure-
ments in the zygotic rescue experiments were gain = 3.5 and PMT voltage =
750 but for all other experiments the settings were gain = 3.0 and PMT
voltage = 600.
Maternal Transfer of HRG-3::mCherry
C. elegans transmitting the transgene Pvha6::HRG-3::mCherry with 60%
efficiency to its progeny were grown on NGM plates seeded with RP523
supplemented with 4 mM heme. Embryos from adult gravid worms either
with or without the transgene were released from the uterus using a needle.
These embryos were analyzed for mCherry expression using a Leica DMIRE2
inverted microscope and Simple PCI software. In parallel, transgenic P
0
gravid
worms were individually picked onto new plates and allowed to produce F
1
progeny and analyzed for mCherry expression by epifluorescence micros-
copy. These F
1
worms were separated and grown till they lay F
2
progeny,
which were analyzed for the mCherry transgene.
Fluorescence Protease Protection Assay
The procedure for fluorescence protease protection (FPP) assay was modified
from the protocol of Lorenz et al. (2006). HRG-3-GFP and control plasmid
pCFP-CD3d-YFP were transfected into HEK293 cells grown on Lab-Tek
chambered coverglass (Nunc). After 24 hr, the cells were washed with KHM
buffer (110 mM potassium acetate, 2 mM MgCl
2
, and 20 mM HEPES,
pH 7.3), and the cell chambers were moved to a DMIRE2 epifluorescence
microscope (Leica) connected to a Retiga 1300 cooled Mono 12-bit camera.
Time-lapse images were acquired before and after digitonin treatment
(30 mM digitonin/2 min) and following proteinase K (50 mg/ml) digestion.
Statistical Analysis
All data are presented as mean ± standard error of the mean (SEM). Statistical
significance was tested using one-way ANOVA followed by the Tukey-Kramer
Multiple Comparisons Test in GraphPad INSTAT version 3.01 (GraphPad,
San Diego, CA, USA). A p value of < 0.05 was considered as statistically
significant.
Additional material is available in the Extended Experime ntal Procedures.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Extended Experimental Procedures, three
figures, and two tables and can be found with this article online at doi:10.1016/
j.cell.2011.04.025.
ACKNOWLEDGMENTS
We thank A. Golden and M. Krause for critical discussions and reading of the
manuscript; B. Grant, D. Hall, and J. McGhee for insights into intestinal regu-
lation in C. elegans; J. Lippincott-Schwartz for the FPP assay; B. Grant for
RNAi clones and C. elegans strain RT1315; J. Hanover for use of the COPAS
BioSort; T. Blumenthal for the SL2 ICS sequence; and National Bioresource
Project and S. Mitani for the hrg-3 strain. This work was supported by funding
from the National Institutes of Health R01DK74797 (I.H.). C.C., T.K.S., J.S., and
I.H. designed studies and interpreted data. H.A.D. conducted the biochemical
heme-binding studies. C.C. and I.H. wrote the manuscript. All authors dis-
cussed the results and commented on the manuscript.
Received: October 27, 2010
Revised: March 18, 2011
Accepted: April 27, 2011
Published: May 26, 2011
REFERENCES
Balklava, Z., Pant, S., Fares, H., and Grant, B.D. (2007). Genome-wide analysis
identifies a general requirement for polarity proteins in endocytic traffic. Nat.
Cell Biol. 9, 1066–1073.
Balla, G., Vercellotti, G.M., Muller-Eberhard, U., Eaton, J., and Jacob, H.S.
(1991). Exposure of endothelial cells to free heme potentiates damage medi-
ated by granulocytes and toxic oxygen species. Lab. Invest. 64, 648–655.
Banci, L., Bertini, I., Ciofi-Baffoni, S., Kozyreva, T., Zovo, K., and Palumaa, P.
(2010). Affinity gradients drive copper to cellular destinations. Nature 465,
645–648.
Blumenthal, T., Squire, M., Kirtland, S., Cane, J., Donegan, M., Spieth, J., and
Sharrock, W. (1984). Cloning of a yolk protein gene family from Caenorhabditis
elegans. J. Mol. Biol. 174, 1–18.
Bonkovsky, H.L., Healey, J.F., Lourie, A.N., and Gerron, G.G. (1991). Intrave-
nous heme-albumin in acute intermittent porphyria: evidence for repletion of
hepatic hemoproteins and regulatory heme pools. Am. J. Gastroenterol. 86,
1050–1056.
Brodigan, T.M., Liu, J., Park, M., Kipreos, E.T., and Krause, M. (2003). Cyclin E
expression during development in Caenorhabditis elegans. Dev. Biol. 254,
102–115.
Chan, M.S., Medley, G.F., Jamison, D., and Bundy, D.A. (1994). The evaluation
of potential global morbidity attributable to intestinal nematode infections.
Parasitology 109 , 373–387.
Childs, S., Weinstein, B.M., Mohideen, M.A., Donohue, S., Bonkovsky, H., and
Fishman, M.C. (2000). Zebrafish dracula encodes ferrochelatase and its
mutation provides a model for erythropoietic protoporphyria. Curr. Biol. 10,
1001–1004.
Chotard, L., Skorobogata, O., Sylvain, M.A., Shrivastava, S., and Rocheleau,
C.E. (2010). TBC-2 is required for embryonic yolk protein storage and larval
survival during L1 diapause in Caenorhabditis elegans. PLoS ONE 5, e15662.
Dailey, H.A. (2002). Terminal steps of haem biosynthesis. Biochem. Soc.
Trans. 30, 590–595.
Dooley, K.A., Fraenkel, P.G., Langer, N.B., Schmid, B., Davidson, A.J., Weber,
G., Chiang, K., Foott, H., Dwyer, C., Wingert, R.A., et al; Tu
¨
bingen 2000 Screen
Consortium. (2008). montalcino, A zebrafish model for variegate porphyria.
Exp. Hematol. 36, 1132–1142.
Faller, M., Matsunaga, M., Yin, S., Loo, J.A., and Guo, F. (2007). Heme is
involved in microRNA processing. Nat. Struct. Mol. Biol. 14, 23–29.
Fay, D.S., and Han, M. (2000). Mutations in cye-1, a Caenorhabditis elegans
cyclin E homolog, reveal coordination between cell-cycle control and vulval
development. Development 127, 4049–4060.
Fuller, V.L., Lilley, C.J., and Urwin, P.E. (2008). Nematode resistance. New
Phytol. 180, 27–44.
Hardison, R.C. (1996). A brief history of hemoglobins: plant, animal, protist,
and bacteria. Proc. Natl. Acad. Sci. USA 93,
5675–5679.
730 Cell 145, 720–731, May 27, 2011 ª2011 Elsevier Inc.
Page 11
Hedgecock, E.M., and White, J.G. (1985). Polyploid tissues in the nematode
Caenorhabditis elegans. Dev. Biol. 107, 128–133.
Held, M.R., Bungiro, R.D., Harrison, L.M., Hamza, I., and Cappello, M. (2006).
Dietary ir on content mediates hookworm pathogenesis in vivo. Infect. Immun.
74, 289–295.
Hotez, P.J., Brindley, P.J., Bethony, J.M., King, C.H., Pearce, E.J., and Jacob-
son, J. (2008). Helminth infections: the great neglected tropical diseases.
J. Clin. Invest. 118, 1311–1321.
Hrkal, Z., Vodra
´
zka, Z., and Kalousek, I. (1974). Transfer of heme from ferrihe-
moglobin and ferrihemoglobin isolated chains to hemopexin. Eur. J. Biochem.
43, 73–78.
Hvidberg, V., Maniecki, M.B., Jacobsen, C., Højrup, P., Møller, H.J., and
Moestrup, S.K. (2005). Identification of the receptor scavenging hemopexin-
heme complexes. Blood 106, 2572–2579.
Jasmer, D.P., Goverse, A., and Smant, G. (2003). Parasitic nematode interac-
tions with mammals and plants. Annu. Rev. Phytopathol. 41, 245–270.
Johnston, W.L., Krizus, A., and Dennis, J.W. (2006). The eggshell is required for
meiotic fidelity, polar-body extrusion and polarization of the C. elegans
embryo. BMC Biol. 4, 35.
Kaasik, K., and Lee, C.C. (2004). Reciprocal regulation of haem biosynthes is
and the circadian clock in mammals. Nature 430, 467–471.
Keel, S.B., Doty, R.T., Yang, Z., Quigley, J.G., Chen, J., Knoblaugh, S., Kings-
ley, P.D., De Domenico, I., Vaughn, M.B., Kaplan, J., et al. (2008). A heme
export protein is required for red blood cell differentiation and iron homeo-
stasis. Science 319, 825–828.
Kim, A.M., Vogt, S., O’Halloran, T.V., and Woodruff, T.K. (2010). Zinc
availability regulates exit from meiosis in maturing mammalian oocytes.
Nat. Chem. Biol. 6, 674–681.
Kim, B.E., Nevitt, T., and Thiele, D.J. (2008). Mechanisms for copper acquisi-
tion, distribution and regulation. Nat. Chem. Biol. 4, 176–185.
Kimble, J., and Sharrock, W.J. (1983). Tissue-specific synthesis of yolk
proteins in Caenorhabditis elegans. Dev. Biol. 96, 189–196.
Knutson, M.D., Oukka, M., Koss, L.M., Aydemir, F., and Wessling-Resnick, M.
(2005). Iron release from macrophages after erythrophagocytosis is up-
regulated by ferroportin 1 overexpression and down-regulated by hepcidin.
Proc. Natl. Acad. Sci. USA 102, 1324–1328.
Li, J.M., Umanoff, H., Proenca, R., Rus sell, C.S., and Cosloy, S.D. (1988).
Cloning of the Escherichia coli K-12 hemB gene. J. Bacteriol. 170, 1021–1025.
Lorenz, H., Hailey, D.W., and Lippincott-Schwartz, J. (2006). Fluorescence
protease protection of GFP chimeras to reveal protein topology and subcel-
lular localization. Nat. Methods 3, 205–210.
Magness, S.T., Maeda, N., and Brenner, D.A. (2002). An exon 10 deletion in the
mouse ferrochelatase gene has a dominant-negative effect and causes mild
protoporphyria. Blood 100, 1470–1477.
McGhee, J.D. (2007). The C. elegans intestine. WormBook, 1–36.
Oka, T., Toyomura, T., Honjo, K., Wada, Y., and Futai, M. (2001). Four subunit
a isoforms of Caenorhabditis elegans vacuolar H+-ATPase. Cell-specific
expression during development. J. Biol. Chem. 276, 33079–33085.
Okano, S., Zhou, L., Kusaka, T., Shibata, K., Shimizu, K., Gao, X., Kikuchi, Y.,
Togashi, Y., Hosoya, T., Takahashi, S., et al. (2010). Indispensable function for
embryogenesis, expression and regulation of the nonspecific form of the
5-aminolevulinate synthase gene in mouse. Genes Cells 15, 77–89.
Puy, H., Gouya, L., and Deybach, J.C. (2010). Porphyrias. Lancet 375,
924–937.
Quigley, J.G., Yang, Z., Worthington, M.T., Phillips, J.D., Sabo, K.M., Sabath,
D.E., Berg, C.L., Sassa, S., Wood, B.L., and Abkowitz, J.L. (2004). Identifica-
tion of a human heme exporter that is essential for erythropoiesis. Cell 118,
757–766.
Rajagopal, A., Rao, A.U., Amigo, J., Tian, M., Upadhyay, S.K., Hall, C., Uhm,
S., Mathew, M.K., Fleming, M.D., Paw, B.H., et al. (2008). Haem homeostasis
is regulated by the conserved and concerted functions of HRG-1 proteins.
Nature 453, 1127–1131.
Rao, A.U., Carta, L.K., Lesuisse, E., and Hamza, I. (2005). Lack of heme
synthesis in a free-living eukaryote. Proc. Natl. Acad. Sci. USA 102, 4270–
4275.
Rolls, M.M., Hall, D.H., Victor, M., Stelzer, E.H., and Rapoport, T.A. (2002). Tar-
geting of rough endoplasmic reticulum membrane proteins and ribosomes in
invertebrate neurons. Mol. Biol. Cell 13, 1778–1791.
Severance, S., and Hamza, I. (2009). Trafficking of heme and porphyrins in
metazoa. Chem. Rev.
109,
4596–4616.
Severance, S., Rajagopal, A., Rao, A.U., Cerqueira, G.C., Mitreva, M.,
El-Sayed, N.M., Krause, M., and Hamza, I. (2010). Genome-wide analysis
reveals novel genes essential for heme homeostasis in Caenorhabditis
elegans. PLoS Genet. 6, e1001044.
Sinclair, J., and Hamza, I. (2010). A novel heme-responsive element mediates
transcriptional regulation in Caenorhabditis elegans. J. Biol. Chem. 285,
39536–39543.
Spieth, J., and Blumenthal, T. (1985). The Caenorhabditis elegans vitellogenin
gene family includes a gene encoding a distantly related protein. Mol. Cell.
Biol. 5, 2495–2501.
Tolosano, E., Hirsch, E., Patrucco, E., Camaschella, C., Navone, R., Silengo,
L., and Altruda, F. (1999). Defective recovery and severe renal damage after
acute hemolysis in hemopexin-deficient mice. Blood 94, 3906–3914.
Tolosano, E., Fagoonee, S., Morello, N., Vinchi, F., and Fiorito, V. (2010). Heme
scavenging and the other facets of hemopexin. Antioxid. Redox Signal. 12,
305–320.
Uc, A., Stokes, J.B., and Britigan, B.E. (2004). Heme transport exhibits polarity
in Caco-2 cells: evidence for an active and membrane protein-mediated
process. Am. J. Physiol. Gastrointest. Liver Physiol. 287, G1150–G1157.
Worthington, M.T., Cohn, S.M., Miller, S.K., Luo, R.Q., and Berg, C.L. (2001).
Characterization of a hum an plasma membrane heme transporter in intestinal
and hepatocyte cell lines. Am. J. Physiol. Gastrointest. Liver Physiol. 280,
G1172–G1177.
Wu, B., Novelli, J., Foster, J., Vaisvila, R., Conway, L., Ingram, J., Ganatra, M.,
Rao, A.U., Hamza, I., and Slatko, B. (2009). The heme biosynthetic pathway of
the obligate Wolbachia endosymbiont of Brugia malayi as a pot ential anti-
filarial drug target. PLoS Negl. Trop. Dis. 3, e475.
Yang, Z., Philips, J.D., Doty, R.T., Giraudi, P., Ostrow, J.D., Tiribelli, C., Smith,
A., and Abkowitz, J.L. (2010). Kinetics and specificity of feline leukemia virus
subgroup C receptor (FLVCR) export function and its dependence on hemo-
pexin. J. Biol. Chem. 285, 28874–28882.
Cell 145, 720–731, May 27, 2011 ª2011 Elsevier Inc. 731
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  • Source
    • "Heme carrier protein 1 (HCP1) was reported to be a heme importer in mammalian intestine [69]. However, HCP1 is actually a high-affinity, proton coupled folate transporter [82]. It remains to be tested whether the low affinity heme transport activity of this folate transporter has physiological relevance. "
    [Show abstract] [Hide abstract] ABSTRACT: Heme is an iron-containing tetrapyrrole that plays a critical role in regulating a variety of biological processes including oxygen and electron transport, gas sensing, signal transduction, biological clock, and microRNA processing. Most metazoan cells synthesize heme via a conserved pathway comprised of eight enzyme-catalyzed reactions. Heme can also be acquired from food or extracellular environment. Cellular heme homeostasis is maintained through the coordinated regulation of synthesis, transport, and degradation. This review presents the current knowledge of the synthesis and transport of heme in metazoans and highlights recent advances in the regulation of these pathways.
    Full-text · Article · Jun 2015 · Science China. Life sciences
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    • "It is likely that homeostatic adaptation is controlled at the systemic level by bidirectional signaling between the intestine and extraintestinal tissues 484 Biometals (2015) 28:481–489 critically low heme conditions to ensure that oocytes developing within the germline receive the heme required for embryogenesis and larval development (Chen et al. 2011). This is accomplished by upregulation of HRG-3, an *8 kDa protein that is expressed in the intestine and binds to heme in a 2:1 (protein:heme) stoichiometric ratio (Fig. 2) (Chen et al. 2011). Although hrg-3 expression is upregulated more than 900 fold during heme deficiency, it is barely detectable when worms are grown in the presence of 6 lM heme. "
    [Show abstract] [Hide abstract] ABSTRACT: Heme is an essential cofactor for proteins involved in diverse biological processes such as oxygen transport, electron transport, and microRNA processing. Free heme is hydrophobic and cytotoxic, implying that specific trafficking pathways must exist for the delivery of heme to target hemoproteins which reside in various subcellular locales. Although heme biosynthesis and catabolism have been well characterized, the pathways for trafficking heme within and between cells remain poorly understood. Caenorhabditis elegans serves as a unique animal model for uncovering these pathways because, unlike vertebrates, the worm lacks enzymes to synthesize heme and therefore is crucially dependent on dietary heme for sustenance. Using C. elegans as a genetic animal model, several novel heme trafficking molecules have been identified. Importantly, these proteins have corresponding homologs in vertebrates underscoring the power of using C. elegans, a bloodless worm, in elucidating pathways in heme homeostasis and hematology in humans. Since iron deficiency and anemia are often exacerbated by parasites such as helminths and protozoa which also rely on host heme for survival, C. elegans will be an ideal model to identify anti-parasitic drugs that target heme transport pathways unique to the parasite.
    Full-text · Article · Feb 2015 · BioMetals
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    • "Mature HRG-3 is secreted into the worm's circulation and taken up by extracellular tissues and developing oocytes. When hrg-3 null worms are grown under heme limiting conditions, they show embryonic lethality and delayed growth, indicating a role for hrg-3 in the distribution of heme from the intestine during early embryonic and larval development (Chen et al., 2011). "
    [Show abstract] [Hide abstract] ABSTRACT: Heme is an iron-containing porphyrin ring that serves as a prosthetic group in proteins that function in diverse metabolic pathways. Heme is also a major source of bioavailable iron in the human diet. While the synthesis of heme has been well-characterized, the pathways for heme trafficking remain poorly understood. It is likely that heme transport across membranes is highly regulated, as free heme is toxic to cells. This review outlines the requirement for heme delivery to various subcellular compartments as well as possible mechanisms for the mobilization of heme to these compartments. We also discuss how these trafficking pathways might function during physiological events involving inter- and intra-cellular mobilization of heme, including erythropoiesis, erythrophagocytosis, heme absorption in the gut, as well as heme transport pathways supporting embryonic development. Lastly, we aim to question the current dogma that heme, in toto, is not mobilized from one cell or tissue to another, outlining the evidence for these pathways and drawing parallels to other well-accepted paradigms for copper, iron, and cholesterol homeostasis.
    Full-text · Article · Jun 2014 · Frontiers in Pharmacology
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