Heme allocation in eukaryotic cells relies on mitochondrial heme export through FLVCR1b to cytosolic GAPDH

Heme is an iron-containing cofactor essential for life. In eukaryotes heme is generated in the mitochondria and must leave this organelle to reach protein targets in other cell compartments. Mitochondrial heme binding by cytosolic GAPDH was recently found essential for heme distribution in eukaryotic cells. Here, we sought to uncover how mitochondrial heme reaches GAPDH. Experiments involving a human cell line and a novel GAPDH reporter construct whose heme binding in live cells can be followed by fluorescence revealed that the mitochondrial transmembrane protein FLVCR1b exclusively transfers mitochondrial heme to GAPDH through a direct protein-protein interaction that rises and falls as heme transfers. In the absence of FLVCR1b, neither GAPDH nor downstream hemeproteins received any mitochondrial heme. Cell expression of TANGO2 was also required, and we found it interacts with FLVCR1b to likely support its heme exporting function. Finally, we show that purified GAPDH interacts with FLVCR1b in isolated mitochondria and triggers heme transfer to GAPDH and its downstream delivery to two client proteins. Identifying FLVCR1b as the sole heme source for GAPDH completes the path by which heme is exported from mitochondria, transported, and delivered into protein targets within eukaryotic cells.


Introduction
Life evolved to widely utilize a special form of iron that is bound within the protoporphyrin IX ring (iron protoporphyrin IX; heme) 1,2 .Heme biosynthesis in eukaryotes is a complex process and its final steps occur inside the mitochondria 3 .However, how heme is then exported from mitochondria, transported, and inserted into numerous proteins that require heme for function but reside elsewhere in cells has been unclear.Because heme is chemically reactive and has promiscuous binding properties, its synthesis is tightly controlled and its intracellular transport has long been imagined to involve macromolecular carriers 2,4,5 .Of all the proteins or other macromolecules that have been proposed, the protein glyceraldehyde phosphate dehydrogenase (GAPDH), an enzyme in the glycolytic pathway that is ubiquitously expressed and known to perform alternative moonlighting functions [6][7][8] , has recently emerged as a premier intracellular heme chaperone 9 , based on findings that GAPDH binding of mitochondrially-generated heme is required for and coupled to intracellular heme delivery to numerous targets including hemoglobins a, b, and g 10 , myoglobin 10 , nitric oxide synthases [11][12][13] soluble guanylyl cyclase b-subunit (sGCb) 14 , cytochromes P450 15 , heme oxygenase 2 16 , indoleamine dioxygenase 1 (IDO1) and tryptophan dioxygenase (TDO) 17 .Insertion of the GAPDHsourced heme into recipient target proteins is the final downstream step in heme delivery, and is now understood to require the cell chaperone protein Hsp90, which is typically bound to the heme-free (apo-) forms of the recipient proteins and drives their heme insertions in an ATP-driven process 18 .Thus, GAPDH binding of mitochondrial heme enables its wide distribution in cells, most often capped by an Hsp90-assisted heme insertion into the protein target.However, one key question remains unanswered in the overall pathway, namely, how does mitochondrial heme reach GAPDH?Here, we address this question in experiments that utilize a cultured human cell line, isolated mouse mitochondria, and a newly described GAPDH reporter protein 19 whose heme binding in living cells can be followed in real-time based on the change in its fluorescence intensity.
Our results identify a transporter protein located in the outer mitochondrial membrane, Feline Leukemia Virus subgroup C Receptor 1b (FLVCR1b), as the sole conduit for mitochondrial heme export to GAPDH in the cells, operating through a mechanism that involves a direct FLVCR1b-GAPDH interaction for heme exchange.Overall, our findings elucidate the route of heme export from the mitochondria and show how this action is tightly coupled to heme distribution and insertion into final destination proteins inside eukaryotic cells.

FLVCR1b supplies heme to GAPDH and enables downstream heme delivery
To explore how heme that is generated in mitochondria transfers to GAPDH in the cell, we tested the importance of FLVCR1b, a transmembrane protein exclusively localized in the outer mitochondrial membrane that has previously been implicated in mitochondrial heme export in erythrocytes during erythropoiesis 20 .We performed experiments in a human embryonic kidney cell line (HEK293T cells) transfected to express an HA-tagged human GAPDH reporter protein called TC-hGAPDH 19 , which after being labeled with FlAsH reagent 19,21 signals its heme binding in living cells or in solution by a fluorescence quenching of its FlAsH signal 19 .We determined how siRNA-targeted knockdown of cell FLVCR1b expression impacts heme loading onto TC-GAPDH in living cells in response to our stimulating their mitochondrial heme biosynthesis by providing the heme precursor molecules d-aminolevulinic acid (d-ALA) and ferric citrate (Fe) 22 , which we have previously shown causes the total heme level in the HEK293T cells, and the level of heme bound in the TC-hGAPDH, to increase by about 3-fold within 2 h 19 .The targeted siRNA treatment diminished cell FLVCR1b protein expression relative to a scrambled siRNA control by 70 +/-9% (n = 5) (Fig. 1A and S1A) and this did not impact the cell heme level from increasing in response to the d-ALA/Fe addition (Table S1), consistent with a previous report 20 .However, the FLVCR1b knockdown inhibited transfer of mitochondrial heme into TC-hGAPDH by more than 90%, as indicated by the TC-hGAPDH fluorescence signal in the FLVCR1b knockdown cells remaining stable with time after the d-ALA/Fe addition, compared to the large decrease in TC-hGAPDH fluorescence that occurred in similarly-treated cells given the scrambled siRNA (Fig. 1B).The FLVCR1b knockdown did not alter cell expression of TC-hGAPDH (Fig. S1B) nor did it inhibit TC-hGAPDH from obtaining heme when it was provided externally to the cell cultures (Fig. S2A and B).Thus, a targeted knockdown of FLVCR1b expression greatly restricted mitochondrial heme transfer to TC-hGAPDH in the living cells.
We next examined how the FLVCR1b knockdown would impact downstream delivery of mitochondrial heme to two protein targets that we expressed separately in the cells and that are known to have GAPDH-dependent heme deliveries (a TC-tagged version of soluble guanylyl cyclase b-subunit termed TC-sGCb, and indoleamine dioxygenase-1, IDO1) 14,17 .The FLVCR1b knockdown did not alter cell expression of the TC-sGCb and IDO1 target proteins (Fig. S1C and D) but it did completely block their heme deliveries, which otherwise took place following d-ALA/Fe addition (Fig. 1C and D).Thus, the mitochondrial heme exporter FLVCR1b is needed for GAPDH to obtain mitochondrial heme in the cells, and this in turn is required for delivery of the mitochondrial heme to two GAPDH-dependent protein targets, revealing that in the absence of a FLVCR1b-GAPDH heme transfer the cells had no alternative way to accomplish the heme deliveries.

FLVCR1b and GAPDH directly interact during mitochondrial heme export
We next investigated if mitochondrial heme export to GAPDH involved its interaction with FLVCR1b, whose expression is restricted to the outer mitochondrial membrane 20,23 .We utilized the Duolink proximity ligation assay (PLA) which detects the interaction of two proteins within a 0-40 nm distance, a typical range for protein partners that engage in direct interaction 24 .We compared the extent of FLVCR1b interaction with GAPDH before and after providing cells with d-ALA/Fe to stimulate their mitochondrial heme biosynthesis and to enable the consequent heme loading into GAPDH as shown above.Using the kinetic data from Fig. 1B as a guide, we assessed the level of GAPDH-FLVCR1b interaction at 0, 15, 30, 45, 60, and 120 min after adding d-ALA/Fe to the cells, which correspond to times before, during, and after mitochondrial heme loading into GAPDH takes place.The PLA data in Fig. 2A and B show there was a detectable level of GAPDH and FLVCR1b interaction present in the resting cells (2.5x greater than background signal).Following the d-ALA/Fe addition their interaction increased within 15 min and by 30 min had increased by 18-fold, and then gradually fell back to the original level by 60 min.Antibody pulldown experiments were done to independently assess the PLA results and they confirmed there is a temporal buildup in GAPDH-FLVCR1b association that peaked at 30 min after the d-ALA/Fe addition (Fig S3A and B).In separate PLA-based experiments, we found that knockdown of FLVCR1b expression in the cells prevented GAPDH interaction with the mitochondrial surface protein HADHA and the increase in their interaction that otherwise occurred in response to d-ALA/Fe addition in the control cells (Fig. 2C and D).This confirmed that the changes in GAPDH interaction specifically involved its interaction with mitochondrial FLVCR1b.
We further investigated the basis for the rise and fall in FLVCR1b-GAPDH interaction by expressing the H53A point mutant variant of HA-hGAPDH, which due to it having a 30-fold poorer heme binding affinity, 12 does not accumulate mitochondrially-generated heme in cells that are given d-ALA/Fe 12,19 .The PLA data in Fig. 2E and F show that there was a detectable level of H53A HA-GAPDH interaction with FLVCR1b present in the resting cells (about 2x above background), and upon d-ALA/Fe addition we observed a delayed and partly muted rise in their interaction relative to what took place in replica experiments using cells expressing wild-type HA-GAPDH (Fig. 2E and F).Immunoprecipitation experiments confirmed the PLA-based results (Fig S4A -C).This suggests that heme binding to GAPDH was coupled to the rise and fall in its interaction with FLVCR1b that occurred when cell mitochondrial heme biosynthesis is stimulated.

FLVCR1b in isolated mitochondria interacts with GAPDH to mediate heme export and delivery to target proteins
We next performed experiments with freshly isolated mitochondria from mouse brain and liver to expand on our cell culture findings.Upon incubating the isolated mitochondria for 30 min with mitochondria-free cell supernatant and d-ALA and then reisolating the mitochondria, we observed a 3-fold increase in their heme level (Fig. 3A & B), confirming earlier reports that in vitro heme biosynthesis takes place in this circumstance 25 .PLA experiments done with the re-isolated mitochondria showed that those that underwent the 30 min incubation with d-ALA and cytosol had a four-fold increase in FLVCR1b-GAPDH interaction (Fig. 3C & D), mirroring what took place in living cells upon d-ALA/Fe addition.
In follow-up experiments we incubated the isolated mitochondria for 30 min with cytosol and 14 C-d-ALA so they would generate and accumulate radiolabeled 14 C-heme, and then re-isolated these mitochondria for experiments.Upon placement in buffer solution at 37 C, the mitochondria released an inconsequential amount of 14 C-heme into the solution over a 60 min observation period, as indicated by the 14 C counts in the solution and mitochondria both remaining steady with time (Fig. 3E & F).However, the addition of purified GAPDH to these mitochondria resulted in an immediate and time-dependent export of the 14 C-heme out of the mitochondria and into the solution, whereas adding a comparable molar amount of glutathione S-transferase, which had previously been reported to stimulate mitochondrial heme release 26,27 caused much less 14 C-heme release (Fig. 3E & F).
We then performed an experiment in which purified FlAsH-labeled TC-hGAPDH was added to the heme-loaded and reisolated mitochondria to determine if the observed release of heme caused by the GAPDH addition was tied to its binding of the released heme.Addition of TC-hGAPDH stimulated the release of mitochondrial heme as before, which bound to it as indicated by the time-dependent decrease in its fluorescence intensity (Fig. 3G).Finally, we examined if the GAPDH addition to the reisolated mitochondria would enable the released heme to be delivered to the GAPDH client protein TC-apo-sGCb.As shown in Fig. 3H, the fluorescence of FlAsH-TC-apo-sGCb when present alone in the reaction solution remained steady over a 1 h time course.Addition of the reisolated mitochondria to this solution caused an immediate partial decrease in the FlAsH-TC-apo-sGCb fluorescence, which did not change with time and that we attribute to some build-up of heme that had become released from the mitochondrial preparation after re-isolation.Adding GAPDH to the reisolated mitochondria and FlAsH-TC-apo-sGCb solution immediately stimulated additional heme to transfer into the FlAsH-TC-apo-sGCb, as indicated by a time-dependent loss of its FlAsH fluorescence intensity (Fig. 3H).Thus, the main features of mitochondrial heme export to GAPDH and its subsequent delivery to a target protein that we had observed in living cells (i.e., a timedependent buildup of the FLVCR1b-GAPDH interaction coinciding with heme transfer to GAPDH and GAPDH heme transfer to the apo-sGCb target protein) could be recapitulated in a simple reconstitution system consisting only of isolated mitochondria, purified GAPDH, and the purified apo-sGCb target protein.In this system, GAPDH interacted with the mitochondrial FLVCR1b and had a strong stimulatory effect on the release of mitochondrial heme, which bound to the GAPDH and in turn allowed the heme to be delivered to the client protein.

Assessing roles for the Progesterone Receptor Membrane Component 2 (PGRMC2) and the Transport and Golgi Organization 2 (TANGO2) proteins in FLVCR1b heme export to GAPDH and its heme allocation
Because two other proteins (PGRMC2 and TANGO2) have recently been implicated in intracellular heme trafficking [28][29][30][31] we tested for their involvement in our system.A siRNA knockdown of PGRMC2 expression in HEK293T cells reduced PGRMC2 expression by an average of 75% (Fig. S5A and B) but did not impact cell expression of other relevant proteins (Fig. S5C-E) nor did it inhibit mitochondrial heme transfer to TC-hGAPDH or inhibit the delivery of mitochondrial heme to downstream target proteins sGCb or IDO1 in cells that were given d-ALA/Fe (Fig. S6A-C).This indicated PGRMC2 was not involved and so it was not studied further.In contrast, a knockdown of TANGO2 expression in the HEK293T cells (Fig. S7A and B) strongly inhibited mitochondrial heme transfer to TC-hGAPDH following d-ALA/Fe addition (Fig. 4A) and also blocked the downstream heme deliveries to sGCb and to IDO1 (Fig. 4B and C), without impacting the expression levels of these proteins (Fig. S7C-E) or the cell heme level (Table S2).Thus, TANGO2 appeared to be involved in mitochondrial heme transfer to GAPDH, and we further investigated its role.
Protein interaction studies using the PLA showed that knockdown of TANGO2 expression did not greatly alter the level of GAPDH interaction with FLVCR1b in resting cells, but it did prevent their interaction from increasing after d-ALA/Fe was added (Fig. 4D & E).
Further interrogation showed that TANGO2 interacted directly with FLVCR1b in the resting cells (5x above background) (Fig. S8A and B), and their level of interaction remained constant in cells that were given d-ALA/Fe to increase mitochondrial heme production (Fig. S8A-C) and also did not change in cells that underwent a knockdown of GAPDH expression (Fig. S9A-C).In comparison, PLA results showed TANGO2 engaged in only a weak interaction with GAPDH (2.5x greater than background) both prior to or following d-ALA/Fe addition to the cells (Fig. S10).Together, these results are consistent with TANGO2 primarily interacting with the FLVCR1b exporter to enable the mitochondrial heme transfer to GAPDH.
Because TANGO2 is thought to be localized to both cell cytosol and mitochondria 32,33 , we further dissected its role by utilizing isolated mitochondria to examine the importance of cytosolic versus mitochondrial TANGO2 in enabling the mitochondrial heme transfer to TC-hGAPDH.We incubated isolated mitochondria with d-ALA and with mitochondria-free cytosols that were prepared either from normal or from TANGO2 knockdown HEK293T cells, and then reisolated the mitochondria after the incubation and compared their extents of FLVCR1b-GAPDH interaction by PLA and their abilities to transfer heme to added TC-hGAPDH.The two sets of reisolated mitochondria behaved identically regarding their having an increased level of FLVCR1b-GAPDH interaction (Fig. 4F and   S11) and in their ability to transfer heme to TC-hGAPDH (Fig. 4G).These results discount a role for cytosolic TANGO2 and instead implicate mitochondrial TANGO2 in enabling the FLVCR1b heme transfer to GAPDH.

Discussion
How heme generated inside the mitochondria is released, distributed, and inserted into proteins that reside outside this organelle but require heme for function has been a longstanding unanswered question in cell biology 1,4,5,34 .Our finding that the outer mitochondrial membrane transporter FLVCR1b exclusively provides heme to GAPDH finally completes an outline for the entire process.The unveiling of the key steps and players involved in eukaryotic heme allocation indicate the process is surprisingly simple and specific, and this information can now be leveraged to investigate the molecular details and regulation of this essential process.The results to date support the mechanism outlined in Fig. 5: Heme made in the mitochondria is provided to GAPDH in the cytosol solely through the FLVCR1b exporter.The heme transfer involves a direct protein-protein interaction between FLVCR1b and GAPDH, with their level of interaction being dynamic and changing according to mitochondrial heme availability and the extent of heme loading into GAPDH.After GAPDH obtains heme it dissociates from the mitochondria, which enables it to transport heme and make it available to numerous client proteins that are located in the cytosol or in other cell compartments.The target heme proteins are present in cells in their heme-free states and are typically are in complex with the cell chaperone Hsp90, which drives their heme insertions in an ATP-dependent manner, possibly with assistance from co-chaperone proteins 13,35 .Hsp90 then dissociates from the heme-replete mature proteins, allowing their biological function 18 .
Regarding TANGO2, it had recently been proposed to act as a heme chaperone in cells or to aid in heme transfer from heme-enriched compartments, perhaps by acting independent of other heme-transporting proteins 30,31 .Our current findings clarify its role by showing TANGO2 interacts with FLVCR1b and not with GAPDH and enables FLVCR1b export of mitochondrial heme to GAPDH.This role is consistent with knockout of TANGO2 causing an increase in the cell mitochondrial heme level 31 , with TANGO2 displaying a poor affinity toward binding heme 31 , and with a recent study that cast doubt on its functioning in intracellular heme transport 36 .
Given that there are other membrane transporters that may enable mitochondrial heme or porphyrin transport 37,38 , and that membrane to membrane heme transfer has been invoked as a means for heme distribution in cells 34,39 , it is surprising that mitochondrial heme provision to GAPDH and its downstream targets solely relies on the FLVCR1b transporter.This heralds a much broader role for FLVCR1b in enabling intracellular heme distribution, which prior to our study had only been shown to be required for the hemoglobinization that occurs during erythropoiesis 20 .Indeed, the FLVCR1b-GAPDH heme transfer pathway that we describe can now explain why FLVCR1b is needed for hemoglobinization, because cellular heme delivery to apo-hemoglobin actually depends on GAPDH obtaining heme 10 .In our current study, we also found that knockdown of FLVCR1b expression prevented delivery of mitochondrial heme to two other GAPDHdependent client proteins (sGCb and IDO1).This is consistent with FLVCR1b being the sole provider of mitochondrial heme to GAPDH, and with GAPDH being the sole heme source for these two downstream heme deliveries.Our findings can also explain why TANGO2 knockdown was found to prevent mitochondrial heme from reaching apomyoglobin 31 , because myoglobin acquisition of heme also depends on GAPDH obtaining heme in cells 10 .Thus, without a functional FLVCR1b heme exporter to provide mitochondrial heme to GAPDH, it is unlikely that any GAPDH client protein will receive heme, thus severely compromising general heme allocation in cells.Indeed, one can envision GAPDH acting like a fountain that supplies mitochondrial heme to many proteins in the cell, and FLVCR1b acting as the only pipe that can supply the fountain with mitochondrial heme.How FLVCR1b transfers mitochondrial heme to GAPDH should now be considered.
FLVCR1b belongs to the major facilitator superfamily of transport proteins, which typically consist of 12 transmembrane-spanning helices connected by hydrophilic linkers that are exposed on either side of the membrane 40,41 .These proteins fold into two domains consisting of transmembrane helices 1-6 and 7-12, forming an extensive interface between the domains that functions by a rocker mechanism to create alternating open channels on either side of the membrane that facilitate small ligand passage 40 .FLVCR1b is the mitochondrially-targeted version of the larger cell membrane FLVCR1a transporter and is missing the first six transmembrane helices 20 .Although the structures of FLVCR1a and b remain to be elucidated, FLVCR1b is expected to form a dimer in the membrane to enable the rocking mechanism for heme ligand passage 42 .Within this context, it is fascinating that GAPDH stimulated heme export from the isolated mitochondria upon its addition.This behavior is reminiscent of how the addition of the heme-binding protein hemopexin increased heme flux out of cell membrane vesicles that contained the FLVCR1a exporter 43 .Our findings indicate that GAPDH makes direct contact with FLVCR1b in cells and in isolated mitochondria, and imply their interaction mediates FLVCR1b heme export.Moreover, the GAPDH-FLVCR1b interaction increased as mitochondrial heme biosynthesis increased and then decreased in conjunction with heme loading into the GAPDH.This behavior may reflect structural changes in FLVCR1b and GAPDH.For example, an increase in mitochondrial heme availability might at first favor a conformational subpopulation of FLVCR1b that better interacts with GAPDH, followed by a subsequent change in GAPDH conformation upon its heme binding that diminishes their mutual affinity.Indeed, the affinity of GAPDH toward its client proteins is sensitive to their heme binding status 18 .The basis for these changes in FLVCR1b-GAPDH interaction and how they may drive the heme transfer warrant further study.

Conclusions
Our findings reveal that heme provision in eukaryotic cells relies on a surprisingly simple pathway that involves just four principal proteins: two of them (TANGO2 and FLVCR1b) enable mitochondrial heme export to one cytosolic protein (GAPDH) that then broadly provides it for distribution inside cells, and a fourth protein (Hsp90) that enables heme to become inserted into multiple protein clients.Together, this achieves a milestone in our understanding of heme biology and provides a foundation to investigate the cellular mechanisms that control heme allocation in eukaryotes and whether dysregulation occurs and underpins disease.

Reagents
All chemicals were purchased from Sigma unless otherwise noted.

Transfection of siRNA and gene silencing
Expression levels of select proteins in HEK293T cells were reduced using siRNA directed against human mRNAs of GAPDH (Dharmacon, #D-001830-02-20), FLVCR1b (Dharmacon, #CTM840033), PGRMC2 (Santa Cruz, #sc-88944), or TANGO2 (Sigma, #EHU064871).siRNA's were used at a final concentration of 100 nM in the cell cultures along with Lipofectamine 2000.The siRNA-treated cells were cultured for 48 h before they received transfections with protein expression plasmids as described above.

Cell supernatant preparation
HEK293T cells were harvested in using 50 mM Tris-HCl pH 7.4 buffer with 0.1% Triton X-100, 5 mM Na-molybdate and EDTA-free protease inhibitor cocktail (Roche).After three freeze-thaw cycles the lysate was centrifuged at 10,000 x g for 15 min at 4 o C and the supernatant was collected.Bradford (Bio-Rad # 500-0006) or DC method (Bio-Rad #5000111) were used to measure the protein concentration.

Image analysis of Western blots
Image J quantification software (Image J; http://rsb.info.nih.gov/ij/) was used to quantify band intensities on western blots.Densitometric analysis (Image J; http://rsb.info.nih.gov/ij/) was used to measure relative protein amounts.The relative abundances of the protein of interest were determined by dividing its band intensity in each sample by the band intensity of a relevant protein control (i.e., FLVCR1b, actin) that was present and analyzed in the same sample.

Measurement of total heme
Total heme in cell supernatants was measured by an established fluorometric method 45 .
Briefly, 20 µl of cell supernatant was mixed with 980 µl of 2 M oxalic acid and boiled for 1h.After cooling to room temperature and centrifugation, the total porphyrin was measured by its fluorescence emission at 662 nm (excitation 400 nm) relative to standard curves generated with freshly-prepared heme solutions.

Isolation of cell cytoplasmic and mitochondrial fractions
To prepare mitochondria-free cytosol, HEK293T cells with and without TANGO2 silencing were lysed in cytosol extraction buffer (10 mM Tris-HCl, 0.34 mM Sucrose, 3 mM CaCl2, 2 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 5 mM Na-molybdate and EDTA-free protease inhibitor cocktail (Roche) on ice, then centrifuged at 720 x g for 5 min.The supernatant recovered was centrifuged at 10,000 x g for 5 min, and then was centrifuged 1 h in a Beckman Coulter ultracentrifuge (Optima L-100 XP) at 100,000 x g and then aliquoted and stored for use.Mitochondria were isolated from HEK293T cells at 4 o C following a previous procedure 20 .Cells were lysed in mitochondrial extraction buffer (0.25 M sucrose, 10 mM HEPES pH 7.4, 5 mM Na-molybdate and EDTA-free protease inhibitor cocktail (Roche).The cell suspension was passed through a 1 mL syringe and 27 gauge needle 10 times.The homogenate was centrifuged twice at 600 x g for 5 min and then the supernatant was centrifuged at 10,000 x g for 10 min to pellet crude mitochondria.The crude mitochondrial pellet was diluted with mitochondrial extraction buffer to a 1 mg/ml protein concentration.For IP studies the mitochondrial suspension was then solubilized with 1% Triton X-100 and protease inhibitor on ice for 30 min, centrifuged at 12,000 x g for 10 min at 4 o C, and the supernatant used following protein quantification (Bio-Rad #5000111).

Immunoprecipitation of mitochondrial samples
Solubilized mitochondrial supernatant samples (0.5 mg protein) were mixed with anti-FLVCR antibody (Novus Biologicals, #NB100-1481) and Protein G agarose beads (Cytiva # 17061801) to pull down the antibody-protein complex.The beads were washed three times with 1 mL lysis buffer (50 mM Tris-HCl pH 7.4 buffer with 0.1% Triton X-100, 5 mM Na-molybdate and EDTA-free protease inhibitor cocktail) by centrifugation (1000 x g for 5 min at 4 o C).The beads were then boiled in Laemmli buffer, resolved onto 10% SDS-PAGE and Western transferred to PVDF membrane (Bio-Rad # 1620177) to probe for proteins of interest, using the antibodies listed above.
Live cell heme binding kinetics using FIAsH-labeled TC-hGAPDH HEK293T cells in black walled 96-well plates (Greiner Bio-One, # 655090) that had been grown in heme-deficient conditions, transfected to express TC-hGAPDH, and had or had not undergone FLVCR1b, PGRMC2, or TANGO2 silencing, were labelled with FlAsH using the method described previously 19 .Briefly, the cell monolayers were washed once with phenol red-free Dulbecco's modified Eagle's medium (DMEM) containing 1 g/L glucose and then given FlAsH-EDT2 (5 μM) in Opti-MEM for 30 min at 37 °C.Afterward, the cell monolayers were washed twice with Phenol red free DMEM containing 10% heme depleted serum.Kinetic analyses were then performed in a FlexStation 3 plate reader (Molecular Devices) at 37 °C with excitation at 508 nm and emission at 528 nm.The fluorescence emission of FlAsH-labelled TC-hGAPDH in the cells was followed versus time with or without δ-ALA (1 mM) plus ferric citrate (100 µM) or hemin chloride alone (5 µM) being added to the cells at time = 0.

Heme transfer kinetics into TC-sGCβ (1-619) using FIAsH-EDT2
Heme transfer into FIAsH-labelled TC-sGCβ (1-619) expressed in HEK293T cells that had been grown in heme-deficient conditions and treated with corresponding siRNAs was monitored using methods reported previously 44 and as described above for TC-hGAPDH.
Briefly, after FlAsH labelling and cell monolayer washing the cell fluorescence was monitored starting with or without addition of δ-ALA (1mM) and ferric citrate (100 µM) at time = 0. Kinetic analyses were then performed in a FlexStation 3 (Molecular Devices) plate reader at 37 °C with excitation at 508 nm and emission at 528 nm.

Determination of 14 C-labelled heme in IDO1
HEK293T cells were grown in DMEM and silenced for FLVCR1b, PGRMC2, or TANGO2 expression for 48 h before being transfected to express FLAG-IDO1.Transfected cells were then given 14 C-δ-ALA (14 µM) and 10 µM ferric citrate and further cultured for 48 h.The cells were lysed using 50 mM Tris-HCl pH 7.4 buffer with 0.1% Triton X-100, 5 mM Na-molybdate, and EDTA-free protease inhibitor cocktail (Roche), and following centrifugation immunoprecipitations were done using supernatant (I mg protein) and anti-FLAG antibody (Sigma #F1804; dilution 1:1000) and Protein G agarose beads (60 µL, Cytiva # 17061801) were used to pull down the antibody-protein complex.The beads were washed and isolated as described above and tubes containing the beads were inserted into scintillation vials which received 4 ml of scintillation fluid (Liquiscint, National Diagnostics # LS-121), and 14 C counts were recorded with a scintillation counter as described 12 .

IDO1 activity assay
IDO1 activity was determined by colorimetric measure of the accumulated L-Kynurenine product in the cell culture medium.Cells were given L-Trp (2 mM) in phenol red free DMEM containing 10% FBS with or without δ-ALA (1 mM) and ferric citrate (100 µM).
After 5 h of incubation the culture medium was de-proteinized by adding an equal volume of 3% trichloroacetic acid (Sigma # T6399) and incubation at 50 o C for 30 min.After centrifugation at 9000 x g for 10 min the supernatants were mixed with an equal volume of p-dimethyl-aminobenzaldehyde (Sigma # 109762; 20 mg/ml) in glacial acetic acid at room temperature and incubated 3 min to allow for chromophore formation.Absorbance at 492 nm was measured in a plate reader (Molecular Devices) and similarly-processed standards containing L-Kyn (Sigma #K8625) were used to calculate the sample L-Kyn concentrations.

Mitochondrial preparation from mouse brain and liver
Mitochondria were isolated from brain or liver of 3-4 weeks old mice (18-22 g).The procedures were approved by Institutional Animal Care and Use Committee (IACUC) of the Cleveland Clinic.Mice were sacrificed by CO2 asphyxiation and cervical dislocation.
The brain and liver were removed immediately and immersed in ice cold mitochondrial isolation buffer (MIB, 0.25 M sucrose, 0.5mM EDTA, 10 mM Tris-HCl, pH 7.4), and mitochondria were isolated by a discontinuous Percoll gradient method at 4°C as described previously 46 .Briefly, Percoll (Sigma Aldrich #P1644) was diluted with MIB to obtain final concentrations of 12%, 26%, and 40% (v/v).Dounce homogenizers with glass pestles were used to homogenize each brain or liver sample in 12% Percoll.A gradient was prepared by pouring 26% Percoll on 40% Percoll.The homogenate in 12% Percoll was layered onto the gradient and was centrifuged at 30,000 x g in a Beckman Coulter ultracentrifuge (Optima L-100 XP) for 5 min.The second fraction was collected after removing the top layer and diluted 1:4 in MIB followed by centrifugation at 15,000 x g for 10 min.The pellet was resuspended in 1 ml of MIB and centrifuged at 15,000 x g for 5 min.The final mitochondrial pellet was resuspended in 100 µl of MIB, its protein concentration measured, and it was then used immediately for studies.
At the end of the incubations, mitochondria were pelleted by centrifugation (10,000 x g), washed twice, and then resuspended in incubation buffer (80 mM KC1, 50 mM MOPS, 5 mM K-phosphate, 1 mM EGTA, pH 7.4).Their heme content was quantified using the heme chromogen assay 45 .Briefly, 90 µl of mitochondrial suspension was mixed with 160 µl of heme chromogen reagent (40:60 pyridine:H2O, 200 mM NaOH), the heme iron was reduced by adding a few grains of sodium dithionite, the absorbance was measured in a 96 well plate at 556 nm, and was quantified by standard curve based on freshly made heme samples.Heme made in the mitochondria transfers to GAPDH solely through the mitochondrial FLVCR1b exporter.FLVCR1b functions in association with TANGO2 (T2), and its heme export involves a direct protein-protein interaction between FLVCR1b and GAPDH that is dynamic and influenced by the mitochondrial heme level and the extent of heme loaded onto the GAPDH.After GAPDH obtains heme, it dissociates from the mitochondria, and this makes heme broadly available to client proteins located in the cytosol or in other cell compartments.Client proteins are typically in complex with cell chaperone Hsp90, which is needed to drive their heme insertions in an ATPdependent process.Hsp90 then dissociates, enabling heme protein biologic functions.

Fig. 1 :
Fig. 1: FLVCR1b mediates mitochondrial heme transfer to GAPDH and enables its downstream heme delivery in cells.A Ta r g e t e d siRNA-based knockdown of cell FLVCR1b expression in HEK293T cells.Mean +/-SD of 5 independent trials.B Knockdown of cell FLVCR1b expression inhibits mitochondrial heme transfer to FlAsH-labeled TC-hGAPDH in living cells whose mitochondrial heme biosynthesis was stimulated by d-ALA/Fe addition at time = 0. Representative of three independent trials, mean +/-SD of triplicates.C, D FLVCR1b knockdown inhibits the delivery of mitochondrial heme to two GAPDH client proteins in the living cells after the d-ALA/Fe addition, as judged by C it blocking the decrease in fluorescence intensity for FlAsH-labeled TC-sGCb and D it blocking the increase in the enzymatic activity and 14 C heme content of IDO1.C, Representative of three independent trials, mean +/-SD of triplicates.A, Significance: **** p < 0.001 based on two-tailed student t-test.t= 17.45, DF= 8 D, Mean +/-SD of 3 independent trials.Significance: *** p < 0.001 and **** p < 0.0001 vs. the compared group based on a one-way ANOVA test.F= 109.1,DF= 6 for 14 C counts and F= 681.9, DF= 6 for kynurenine.

Fig. 4 :
Fig. 4: TANGO2 enables FLVCR1b heme export to GAPDH and consequent heme delivery to target proteins.A Ta r g e t e d s i R N A -based knockdown of cell TANGO2 expression inhibited mitochondrial heme transfer to FlAsH-labeled TC-hGAPDH in live HEK293T cells after their mitochondrial heme biosynthesis was stimulated by d-ALA/Fe addition at time = 0. B, C TANGO2 knockdown inhibited the delivery of mitochondrial heme to two GAPDH client proteins in the living cells after the d-ALA/Fe addition, as judged by B it blocking the decrease in fluorescence intensity for FlAsHlabeled TC-sGCb and by C it blocking the gain in the enzymatic activity and 14 C heme content of IDO1.D Representative fluorescence microscope images of cells stained with DAPI for nuclei (blue), using HADHA antibody for mitochondria (green), and the GAPDH-FLVCR1b interaction by PLA (red) in cells processed after the indicated times of d-ALA/Fe exposure.BG, background.E Quantification of the PLA results to show the relative levels of GAPDH-FLVCR1b interaction.BG is the background PLA signal level.F PLA data comparing the relative interaction level of FLVCR1b and GAPDH on mitochondria that had been reisolated after having been incubated for the indicated times with d-ALA plus supernatants prepared either from control or TANGO2 knockdown HEK293T cells.BG is the background PLA signal level.G Heme transfer from the reisolated mitochondria to FlAsH-labeled TC-hGAPDH after mitochondria were added at time = 0. A, B, G Representative of three independent trials, values are the mean +/-SD of triplicates.C, E, F Three independent trials, mean +/-SD.Significance: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. the compared group based on a one-way ANOVA test.ns, not significant.C, F= 1989, DF= 6 for 14 C counts and F= 782.9, DF= 6 for kynurenine.E, F= 22.65, DF= 8 for control siRNA, F= 47.62, DF= 8 for TANGO2 siRNA.F, F= 43.90, DF= 8.