SUPPLEMENTARY METHODS:
Cells: G1ER cells were provided by Dr. Stuart Orkin. Mouse erythroid leukemia
(MEL), human erythroid leukemia (K562), and osteosarcoma (Saos2) cells (ATCC,
Manassas, VA) were transfected or transduced with an Abcb6 expression vector or
with empty vector and cultured as described21. RNA was extracted with TriZol
reagent (Invitrogen) and standard northern analysis was performed. Abcb6 and
GAPDH were identified by using 32P-labeled gene-specific probes. The plasmids
used were pcDNA3; pcDNA3-Abcb6 containing a C-terminal V5 epitope or a Cterminal Flag epitope; and pcDNA3 HuPRP. The Walker domain mutant substituted
a glycine residue for the conserved lysine.
PCR, immunoblotting, and in situ hybridization: PCR primers are listed in
Supplementary Table 1. The amplification conditions were: 95C for 4 min; 25
cycles of 95C for 30 sec, 58C for 30 sec, and 68C for 30 sec; 72C for 5 min.
Immunoblot analysis of total cell or mitochondrial lysate was performed as previously
described21. For in situ hybridization, mouse embryo sections were fixed overnight at
4 ºC in 4% paraformaldehyde in PBS, incubated for a second night in 30% sucrose in
PBS at 4 ºC, mounted in OCT compound (Tissue Tek), and sectioned. Digoxigeninlabeled Abcb6 sense or antisense probes were used as previously described30.
Isolation and purification of mitochondrial membranes: Homogenized
mitochondria were centrifuged at 105,000  g for 60 min. The pellet was resuspended
in Buffer A (250 mM sucrose, 10 mM Tris-HCl, pH 7.4) and centrifuged at 11,500 
g for 15 min to separate the outer membrane (supernatant) and inner membrane and
matrix (pellet) fractions. The supernatant was centrifuged at 105,000  g for 60 min to
recover the outer membranes (pellet), which were resuspended in 2 ml Buffer A and
purified The pellet containing the inner membrane mitoplasts was resuspended in 22
ml 25.2% sucrose and purified by centrifugation through a discontinuous sucrose
gradient (25.2%, 37.7%, 51.7%, 61.5%). All sucrose solutions were buffered with 10
mM Tris-HCl, and all operations were conducted at 4C.
Submitochondrial localization of Abcb6: We used the procedure described by
Leighton and Schatz 31 with modifications. Briefly, cell pellets were washed with
PBS. The cells were resuspended in a hypotonic RSB buffer (10 M NaCl, 1.5 M
MgCl2, 10 M Tris-HCl [pH 7.5] containing complete proteinase inhibitor cocktail)
(Boehringer Mannheim) and allowed to swell (cell swelling was periodically
monitored by microscopy). The swollen cells were homogenized with a type B
Dounce homogenizer. MS buffer (210 M mannitol, 70 M sucrose, 5 M Tris-HCl
[pH 7.5], 1 M EDTA [pH 7.5]) was added immediately and the lysate was
centrifuged at 1000  g for 10 min at 4 ºC to remove nuclei and unbroken cells. The
supernatant was then centrifuged at 10,000  g for 30 min to isolate the mitochondrial
pellet. Isolated mitochondria were subjected to hypotonic shock by exposure to 20
mM KCl for 30 min on ice in the presence or absence of 1 mg/ml proteinase K. At
the end of incubation, proteinase K was inactivated by addition of cold trichloroacetic
acid. Mitoplasts were pelleted by brief centrifugation and analyzed by immunoblot,
using a chemiluminescence kit. Abcb6-Flag was detected with anti-Flag antibody
(Sigma). Other antibodies used were anti-cytochrome C (Molecular Probes; 1:500
dilution) and anti-cytochrome C oxidase subunit II (Molecular Probes; 1:500
dilution). Anti-VDAC (Sigma, l:500) anti-ANT (Santa Cruz, 1:500), anti-ATP
synthase (Molecular Probes, 1:500), and anti-Abcb6 antibodies were previously
described21.
Immunoprecipitation analysis: NIH3T3 cells were cultured in Dulbecco’s modified
Eagle’s medium (DMEM) containing 4500 mg/L glucose supplemented with 10%
FCS, 2 mM L-glutamine, and antibiotics (Gibco). Cells were transfected with
expression vectors for Abcb6-V5, Abcb6-Flag, or other combinations indicated in the
figure legends, by using Lipofectamine Plus. After 24 h, cells were washed with PBS
and harvested into 1 ml ice-cold PBS containing 1x protease inhibitor cocktail
(Complete, Roche). The cells were pelleted by centrifugation and resuspended in
Buffer A (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 10% glycerol, and 1 x protease
inhibitor cocktail [Roche]). After ultrasound disruption, the cells were solubilized in
NP-40 and centrifuged to remove debris. The supernatant was analyzed by
immunoblotting or immunoprecipitation after protein concentration was measured by
the Bradford method (Protein Assay, Bio-Rad Laboratories, Inc.). The lysate was
immunoprecipitated with either an anti-V5 (Invitrogen) or anti-Flag monoclonal
antibody (M2, Sigma). The immunoprecipitate was washed twice with Buffer A
containing 1% NP-40. The proteins were electrophoretically fractionated, transferred
to nitrocellulose membranes, and incubated with anti-V5 (MBL, Nagoya, Japan) or
anti-Flag (Sigma) polyclonal antibody. The blots were incubated with horseradish
peroxidase–conjugated anti-rabbit IgG F(ab’) 2 fragments (Amersham Pharmacia
Biotech) and an enhanced chemiluminescence kit (ECL, Amersham Pharmacia
Biotech) was used for detection.
Confocal microscopy. Confocal microscopy was performed as described32 with
minor changes. Briefly, mitochondrial membrane fractions were fixed to chamber
slides at 37C and immunostained with antibodies to Flag epitope (monoclonal,
Sigma), VDAC ( polyclonal, Sigma), ANT (polyclonal, Santa Cruz Biotechnology),
and ATP synthase (monoclonal, Molecular Probes). Secondary antibodies were Alexa
488 coupled to goat antimouse antibody and Alexa 594 coupled to goat antirabbit
antibody (Molecular Probes Inc.). The samples were examined in a Leica TCS NT SP
confocal laser scanning microscope with a 100X APO 1.4 NA oil immersion
objective.
Measurement of ATPase activity. Vanadate-sensitive ATPase activity was
measured in the crude mitochondrial fraction isolated from K562, K562-Abcb6-Flag,
K562-Abcb6-V5, and K562–Walker mutant Abcb6 (Abcb6MT)-V5 cells by
colorimetric assay as described previously32. Preparations of the isolated mitochondria
containing 30 g of protein were incubated with the indicated concentrations of
coproporphyrin III in methanol; the reaction was started by addition of 3.3 mM
MgATP. The vanadate-sensitive ATPase activity stimulated by coproporphyrinogen
III was measured as nanomoles of inorganic phosphate released per minute per
milligram of protein.
Generation of tetracycline-regulated Abcb6 expression vector: The human
Abcb6/pcDNA 3.1 topo vector carrying Flag-tagged Abcb6 was cut with HindIII and
XbaI, and the Abcb6-Flag–containing fragment was ligated into pcDNA4/TO vector
(Invitrogen). The resulting plasmid was used in site-directed mutagenesis to generate
a Walker A–mutant Abcb6-Flag. This clone was sequenced and verified to contain
only the Walker A mutation.
Transient induction of Abcb6-Flag and siRNA treatment of CHO cells: The
tetracycline-regulated (T-Rex) cell line T-Rex-CHO (Invitrogen) was transfected with
pcDNA4 encoding Abcb6-Flag or Walker A–mutant Abcb6-Flag by using
Lipofectamine 2000 reagent according to the manufacturer’s protocol. Abcb6-Flag
expression was induced with 1 g/ml tetracycline and Abcb6 expression was analyzed
by western blot with anti-Flag monoclonal antibody (Sigma). The siRNA
oligonucleotide and negative-control scrambled oligonucleotide were customsynthesized by Dharmacon (Lafeyette, CO). Both siRNA and control oligonucleotides
were used at a final concentration of 150 nM and were added to cells at the time of
transfection with pcDNA4-Abcb6-Flag expression vector.
siRNA 1: 5’ GCGCAUACUUUGUCACUGACA 3’
3’ UUCGCGUAUGAAACAGUGACUP 5’
siRNA 2: 5’ CCGAAUAGAUGGGCAGGACAU 3’
3’ UUGGCUUAUCUACCCGUCCUGP 5’
Scrambled oligo: 5’ UAGCGACUAAACACAUCAA 3’
Generation of Abcb6-targeted ES cells. An Abcb6 targeting vector was
constructed in the PKO vector by standard molecular procedures. Briefly, the 5’ arm
consisted of a 3.2-kb HindIII/XhoI fragment 5' of exon 1 that was ligated into the
pKONTKV1901 vector (Stratagene). The 3’ arm was a 5.4-kb EcoRI fragment that
contained exons 16 to 19. The fragments were verified by DNA sequence analysis.
The targeting vector was linearized by NotI, then electroporated into 129/SVJ-derived
ES cells. Genomic DNA from 798 ES clones that survived 2 weeks of G418 selection
was screened first by PCR analysis and subsequently by Southern blot analysis. Seven
clones had undergone correct homologous recombination.
Supplementary Table 1. PCR primers used in this study
Mouse Abcb6
Forward 5’ GCCGTGATATGAACACACAG 3’
Reverse 5’ GCCAAAAAGCACAAAGTCCC 3’
Mouse UDC
Forward 5’ AAGCCATCACCCTTACTCGAC 3’
Reverse 5’ GACTCAAAGAGCTGCAATGCC 3’
Mouse ALAS1
Forward 5’ CTCAAGGACCAACCTGTTCTC 3’
Reverse 5’ CTGGCTTCCAGTCATATTGTTC 3’
Mouse ALAS2
Forward 5’ CGTGTCTTGCAGGCCATAG 3’
Reverse 5’ GACTTCCTATACGAAGGTCAG 3’
Mouse Actin
Forward 5’ GTGACGAGGCCCAGAGCAAG 3’
Reverse 5’ AGGGGCCGGACTCATCGTAC 3’
Human Abcb6
Forward 5’ CTTCGTCCCCAGTCCTATAC 3’
Reverse 5’ CCTTCTCAGTCAGCAAGTTC 3’
Human Gapdh
Forward 5’ ACCACAGTCCATGCCATCAC 3’
Reverse 5’ TCCACCACCCTGTTGCTGTA 3’
Human TfR
Forward 5’ AGCACAGATATCCACACACC 3’
Reverse 5’ GATATTGAAACTCCACGCCC 3’
Human CPO
Forward 5’ TGTCTTTACCTCTAACTGCCC 3’
Reverse 5’ CATTCATCACTGACAGCATCC 3’
Human FC
Forward 5’ CACACAGTATCCACAGTACAG 3’
Reverse 5’ GCAGAAAACAGAATGACCACC 3’
Human FRXN
Forward 5’ GATGAGACCACCTATGAAAGAC 3’
Reverse 5’ TGGATGGAGAAGATAGCCAG 3’
Supplementary Figure 1.
Abcb6 is upregulated by both hemin and ALA. a) Northern blot shows Abcb6
mRNA upregulation by hemin. b) Northern blot shows Abcb6 mRNA upregulation
by ALA. c) Immunoblot shows time-dependent upregulation of Abcb6 protein by
ALA.
Supplementary Figure 2
Abcb6 localizes to the outer mitochondrial membrane. (a) K562 cells were treated
with hemin and mitochondria were isolated. The outer (OM) and inner (IM)
mitochondrial membranes were purified, and the total cell lysate (TL) and purified
OM and IM fractions were analyzed by immunoblot with probes for Abcb6, Abcb7
(an IM protein), VDAC (an OM protein), and ATP synthase (an IM marker). (b)
Mitochondria were isolated from cells transduced with Abcb6-Flag or empty vector.
The OM and IM fractions were probed for the IM marker ANT, the OM marker
VDAC, and Abcb6. (c-f) Purified mitochondrial IM and OM fractions were fixed on
glass slides, incubated with the indicated antibodies, and examined by confocal
microscopy. Abcb6 was detected by using anti-Flag antibody. Yellow color in overlay
indicates co-localization.
Supplementary Figure 3.
Abcb6 localizes to the outer mitochondrial membrane and binds hemin. a) A model
depicts the four possible submitochondrial positions of Abcb6 (outer or inner
membrane, with Flag-tagged C-terminus toward the cytoplasmic or intermembrane
side). At right, disruption of the outer membrane (OM) by osmotic shock (OS) forms
mitoplasts in which Flag-tag resists proteinase K (PK) digestion (scissors) only in the
orientation shown. b) Mitoplasts and mitochondria +/– OS were treated with PK
and/or SDS (as a control for complete lysis) and analyzed by immunoblot with
antibodies to Flag, cytochrome C (CytC, a soluble intermembrane protein), and
cytochrome oxidase subunit 2 (Cox 2, localized to the inner membrane [IM]). c)
Abcb6 is able to homodimerize. Transient expression of C-terminal Flag-tagged or V5
epitope–tagged Abcb6 was followed by immunoprecipitation (IP) and immunoblot
analysis.
Supplementary Figure 4
Overexpression of functional Abcb6 increases intracellular protoporphyrin IX (PPIX).
Intracellular PPIX was measured by FACS analysis as previously described 8. a) PPIX
fluorescence in Saos-2 cells transfected with Abcb6 (green), HuPRP (“mutant”, a
non–heme-binding Abcb6 splice variant; pink), or empty vector (red). b) PPIX
fluorescence in the MEL cells described8.
Supplementary Figure 5
Expression of selected enzymes important for heme biosynthesis is higher in K562
cells overexpressing Abcb6 than in vector-control cells. Fold change is expressed as
multiple of expression in vector-control cells. ALAD, aminolevulinate dehydratase;
ALAS, aminolevulinate synthase; CPO, coproporphyrinogen oxidase.
Supplementary References
31. Leighton, J. and Schatz, G. An ABC transporter in the mitochondrial inner
membrane is required for normal growth of yeast. EMBO J. 14, 188-195 (1995).
32. Ozvegy-Laczka C., et al. High-affinity interaction of tyrosine kinase inhibitors
with the ABCG2 multidrug transporter. Mol Pharmacol. 65:1485-95 (2004)