Inhibition of an iron-responsive element/iron regulatory
protein-1 complex by ATP binding and hydrolysis
Zvezdana Popovic and Douglas M. Templeton
Laboratory Medicine and Pathobiology, University of Toronto, Canada
Mechanisms of post-transcriptional regulation of gene
expression include control of initiation of translation
and regulation of mRNA degradation. Among the
best-studied models for these processes is regulation of
proteins involved in iron homeostasis. These control
mechanisms involve functional iron-responsive ele-
ments (IREs) in the 5¢-UTRs or 3¢-UTRs of mRNAs
that interact with iron regulatory proteins (IRPs),
depending upon the amount of iron present in the cell.
Two IRPs have been identified: IRP-1, which contains
a 4Fe)4S iron–sulfur cluster [1], and IRP-2, which
does not [2,3]. IRP-1 has 30% amino acid identity to
mitochondrial aconitase [4], a 4Fe)4S enzyme involved
in the tricarboxylic acid cycle. IRP-1 is generally
believed to interconvert between an enzymatically inac-
tive IRE-binding state and a nonbinding form with
aconitase activity, the latter requiring an intact 4Fe-4S
cluster. Thus, the simple model for iron sensing by
IRP-1 involves direct association of iron with the iron–
sulfur center to form a complete 4Fe)4S cluster.
A linkage between cellular iron levels and energy
metabolism is suggested by the influence of agents that
Keywords
ATP binding; ATP hydrolysis; energy
metabolism; iron regulatory proteins; iron-
responsive element
Correspondence

[K
m
¼ 5.3 lm, V
max
¼ 3.4 nmolÆmin
)1
Æ(mg protein)
)1
] are consistent with
those of a number of other ATP-hydrolyzing proteins, including the RNA-
binding helicases. Although the iron-responsive element does not itself
hydrolyze ATP, its presence enhances iron regulatory protein-1’s ATPase
activity, and ATP hydrolysis results in loss of the complex in gel shift assays.
Abbreviations
AMP-PNP, adenosine 5¢-(b,c-imido)triphosphate; ATP-cS, adenosine 5¢-O-(3-thiotriphosphate); CCCP, carbonyl cyanide
m-chlorophenylhydrazone; EMSA, electrophoretic mobility shift assay; IRE, iron-responsive element; IRP, iron regulatory protein.
3108 FEBS Journal 274 (2007) 3108–3119 ª 2007 The Authors Journal compilation ª 2007 FEBS
affect ATP levels and regulate IRP-1. In addition to
iron, NO [5], H
2
O
2
[5] and oxidative stress in general
[6] influence the activity of IRP-1. All these agents
decrease the level of intracellular ATP, and concomit-
antly increase IRP-1 binding activity, suggesting integ-
rated regulatory mechanisms. In addition, IRP-1 may
be regulated by phosphorylation [7]. Generation of
NO results in an increase in IRP-1 activity, and a
decrease in cytosolic aconitase activity [8–12]. NO

ondrial function and limit ATP production [18], while
increasing IRP-1 binding activity. Hypoxia also decrea-
ses ATP as cells switch their primary means of energy
production from the tricarboxylic acid cycle to glyco-
lysis [19], and modulates cellular iron homeostasis in
human hepatoma and erythroleukemia cells by enhan-
cing IRP-1 binding capacity [20], although in rodent
cells it has been found to decrease total IRP binding
[21], perhaps due to a higher IRP-2 ⁄ IRP-1 ratio in the
rodent [20].
The present study was undertaken to examine the
possible interaction of ATP with the IRE–IRP system.
We have determined that ATP binds to IRP-1, is
hydrolyzed, and disrupts IRE–IRP-1 binding.
Results
Effect of uncoupling oxidative phosphorylation
on IRP binding activity
We treated HepG2 cells with iron (20 lgÆmL
)1
) for 3 h
in the presence or absence of carbonyl cyanide m-chlo-
rophenylhydrazone (CCCP), and RNA binding was
subsequently examined by electrophoretic mobility
shift assay (EMSA) with human ferritin H-chain IRE.
Iron treatment decreased IRP binding activity to 60%
of control values, but simultaneous treatment with iron
and 10 lm CCCP prevented the iron-dependent
inhibition of IRP binding activity (Fig. 1A). In
parallel, ATP was measured in samples subjected to
each treatment. In the absence of oxidative phosphory-

IRE complexation, we incubated HepG2 cell extracts
with different ATP concentrations prior to the addi-
tion of labeled IRE and subsequent EMSA (Fig. 3).
Binding activity was substantially decreased by 2.5 mm
ATP and was undetectable at 5 mm ATP. When puri-
fied recombinant IRP-1 was tested for IRE binding in
the presence of ATP, the results were similar (Fig. 3),
suggesting that interaction can occur among IRP-1,
IRE and ATP without involvement of cellular proteins
lacking IRP activity. Inclusion of 2% b-mercaptoetha-
nol in the reaction mixture had no effect with either
HepG2 extract, Ni
2+
–nitrolotriacetic acid-purified
recombinant protein, or affinity-purified recombinant
protein (Fig. 3B).
ATP is hydrolyzed to inhibit IRE–IRP binding
To determine the specificity of ATP’s effect on the
IRE–IRP interaction, complex formation was studied
Z. Popovic and D. M. Templeton ATP binding to IRP-1
FEBS Journal 274 (2007) 3108–3119 ª 2007 The Authors Journal compilation ª 2007 FEBS 3109
in the presence of other nucleotide triphosphates. CTP,
GTP, and UTP had no significant effect on IRP-1 bind-
ing in either HepG2 cell extracts (Fig. 4A) or with
recombinant protein (Fig. 4B). To determine whether
ATP hydrolysis is required to modulate IRP–IRE inter-
action, we analyzed IRE–IRP complex formation in the
presence of the ATP analogs adenosine 5¢-O -(3-thiotri-
phosphate) (ATP-cS) and adenosine 5¢-(b,c-imido)tri-
phosphate (AMP-PNP). The imidodiphosphate analog

and expressed relative to the values in untreated control cells. Values are means ± SD from three independent experiments expressed relat-
ive to control taken as 100% in each experiment, and statistical differences from control are indicated as in (A). The absolute concentration
in control cells was 2.27 ± 0.79 pmol ATPÆlg
)1
protein. (C) Level of IRP-1 in whole cell extracts following different treatments as in (A).
Equal amounts of protein (80 lg per lane) were subjected to western blot analysis with anti-IRP-1 serum. The position of IRP-1 just above
the 100 kDa molecular mass marker (compare Fig. 2) is indicated by the arrow.
ATP binding to IRP-1 Z. Popovic and D. M. Templeton
3110 FEBS Journal 274 (2007) 3108–3119 ª 2007 The Authors Journal compilation ª 2007 FEBS
establish whether IRE or IRP-1 has ATP hydrolytic
activity. ATPase activities were measured with a sensi-
tive assay that monitors the production of inorganic
[
32
P]phosphate from [
32
P]ATP[cP] by TLC. Recombin-
ant IRP-1 (200–800 ng; 250 nm to 1 lm) has ATPase
activity in the presence of 300 lm ATP (Fig. 5A,B)
that correlates with the amount of protein in the reac-
tion. A reaction with BSA as a protein control had
negligible activity. After boiling of IRP-1 for 5 min,
less than 3% activity remained. Size exclusion chroma-
tography of recombinant IRP-1 could not separate
IRE-binding activity from ATPase activity (data not
shown). Purified IRE RNA was devoid of ATPase
activity (Fig. 5C).
The kinetics of ATP hydrolysis were studied with
affinity-purified recombinant IRP-1. Release of inor-
ganic phosphate was linear with time (Fig. 6A) and

[
32
P]ATP[cP].
IRP-1 binds ATP
More direct proof that ATP binds directly to IRP-1
was sought in a photolabeling experiment. Recombin-
ant IRP-1 was incubated with 8-azido-[
32
P]ATP[aP]
for 2 min at 4 °C, and this was followed by UV-
induced covalent crosslinking of bound [a-
32
P]nucleo-
tide. The proteins were separated by SDS ⁄ PAGE, and
AB
C
Fig. 3. ATP inhibits IRP–IRE binding activity. IRE binding activity
was measured by EMSA using either cytosolic extract from HepG2
cells or recombinant human IRP-1 after addition of the indicated
concentration of ATP. A representative gel is shown in (A), and the
values in the histograms (C) are means ± SD from three independ-
ent experiments. Values marked *** differ from control ([ATP] ¼ 0)
at P < 0.001. (B) EMSA of HepG2 cell extracts, Ni
2+
–nitrilotriacetic
acid-purified ecombinant IRP-1 (Ni-IRP-1), and affinity-purified IRP-1
(A-IRP-1) without ATP, with 5 m
M ATP, or with 5 mM ATP plus 2%
b-mercaptoethanol (ME).
Fig. 2. Purification of IRP-1. Recombinant IRP-1 was purified from E. coli by Ni

d
>30lm. To characterize the binding further, a
filter-binding assay was performed with up to 500 lm
[
32
P]ATP[aP] and gave a K
d
¼ 86 ± 17 lm (Fig. 7D).
ATPase activity is enhanced in the presence
of IRE
Binding to RNA and ATPase activity are characteris-
tics of RNA helicases, and RNA binding increases the
ATPase activity of these proteins [26]. To test whether
the ATPase activity of IRP-1 is also enhanced in the
presence of IRE, we measured hydrolysis of
[
32
P]ATP[cP] in the presence of 0–400 ng of IRE
(Fig. 8). Compared to the amount of ATP hydrolyzed
with 400 ng of IRP alone, addition of IRE increased
the release of inorganic [
32
P]phosphate by about 50%.
IRE alone at 400 ng had no hydrolytic activity
(Fig. 5C).
Discussion
We have demonstrated here that treatment of HepG2
cells with an uncoupler of oxidative phosphorylation
depleted them of ATP and prevented suppression of
Fig. 4. Requirement for ATP hydrolysis to inhibit IRP–IRE binding

3112 FEBS Journal 274 (2007) 3108–3119 ª 2007 The Authors Journal compilation ª 2007 FEBS
IRP activity by iron. A similar effect of CCCP-induced
ATP depletion was observed on the reconstitution of
cytosolic and mitochondrial aconitase activities after
removal of NO-generating agents [27]. That is, the
high IRE-binding activity of IRP-1 after NO exposure
remained elevated in CCCP-treated cells after cessation
of NO flux. Moreover, Bouton et al. [27] also observed
that IRP-1 cannot dissociate from IRE if mitochondria
are unable to produce ATP. They suggested that IRP-
1–IRE complex dissociation, an obligate step upstream
of 4Fe)4S cluster repair, utilizes an ATP-dependent
mechanism. Although this is a plausible explanation
for our results, we have not attempted to rule out
other possibilities. For instance, interference with mito-
chondrial function and dissipation of the mitochond-
rial transmembrane potential by CCCP could prevent
reconstitution by interfering with mitochondrial
4Fe)4S cluster synthesis, although Li et al. have dem-
onstrated that reconstitution of mammalian cytosolic
aconitase probably involves cytosolic forms of enzymes
of cluster synthesis [28].
Although iron treatment has a dramatic impact on
iron–sulfur proteins and the bioenergetic function of
mitochondria [29], 3 h of iron treatment of our HepG2
cultures did not significantly increase the ATP level
compared to that in control cells (Fig. 1). Oexle et al.
[30] found a significant increase in ATP after 24 h in
iron-treated, differentiating K562 cells as compared to
control or deferoxamine-treated cells, and aconitase

of other ATP-hydrolyzing proteins, e.g. Escherichia coli
DnaK (20 lm) [35], Hsc70 (1.4 lm) [36], and F1-
ATPase (15 lm) [37]. Furthermore, these proteins have
V
max
values in the range 1.1–3.5 nmolÆmin
)1
Æmg
)1
,
comparable to the value of 3.4 nmolÆmin
)1
Æmg
)1
measured here, supporting a physiologic relevance
of the observed ATP binding.
The requirement for more than 1 mm ATP to abol-
ish binding in EMSA (Fig. 3) is at first sight inconsis-
tent with a K
d
of 86 lm. The K
m
and K
d
values are in
reasonable agreement, and it may be that factors other
AB
CD
Fig. 6. Kinetics of ATP hydrolysis by affinity-
purified recombinant IRP-1. (A) Time

max
¼ 3.4 ± 0.3
nmolÆmin
)1
Æmg
)1
.
Z. Popovic and D. M. Templeton ATP binding to IRP-1
FEBS Journal 274 (2007) 3108–3119 ª 2007 The Authors Journal compilation ª 2007 FEBS 3113
than binding and hydrolysis, e.g. a conformational
change induced by high ATP concentrations, are
necessary for disruption of RNA binding. However,
other factors in the EMSA protocol, including changes
in Mg
2+
and ATP concentrations during electropho-
retic separation, may influence binding, and we think
this the more likely explanation; direct comparison of
the ATP dependence of EMSA and binding of ATP to
purified protein under optimized conditions may not be
appropriate. Nor can the concentration of ATP neces-
sary to disrupt IRP–IRE binding on EMSA be readily
compared to effective concentrations in vivo, where
additional interactions may be involved.
Disruption of the 4Fe)4S cluster of IRP-1 with 2%
b-mercaptoethanol is widely used to uncover total
IRP-1 binding activity. However, in our experiments
addition of 2% b-mercaptoethanol did not reconstitute
IRP-1 binding inhibited by ATP, and nor was the pro-
tein degraded. Similarly, Gonzalez et al. [38] reported

2
O
2
signifi-
cantly. Pantopoulos & Hentze [41] conclude that
IRP-1 activation by H
2
O
2
in permeabilized cells
appears to require ATP and GTP, indicating an energy
dependence of the process and ⁄ or the involvement of a
phosphorylation–dephosphorylation cycle. We found
that ATP-cS causes only about 30% inhibition of
binding in whole cell extracts but 80% inhibition with
recombinant protein, and this difference may arise
from different characteristics of native HepG2 protein
and recombinant protein, such as the presence of a
His-tag on the latter. Although ATP-cS is often con-
sidered a nonhydrolyzable ATP analog, it undergoes
hydrolysis in some circumstances, and is a substrate
for RNA-dependent nucleotide hydrolysis by helicases
[23]. Because hydrolysis of a phosphorothioate group
is actually predicted to be faster than that of the
corresponding phosphate on the basis of chemical
considerations, it has been suggested that the rate-
determining step may be a conformational change that
takes place after substrate binding [23]. Native and
recombinant proteins may have different conforma-
A

P]ATP[aP] as indicated. The reaction mixture was
electrophoresed, silver stained, and autoradiographed. (D) Filter-
binding assay of 0.62 lg of IRP-1 incubated with 100 n
M
[
32
P]ATP[aP] and the indicated concentration of unlabeled ATP.
The reaction mixture was collected on nitrocellulose membranes
as described in Experimental procedures. The saturation plot
was fitted by nonlinear regression (r
2
¼ 0.98) to give values of
K
d
¼ 86 ± 17 lM and B
max
¼ 4.6 ± 0.3, typical of three such
experiments.
ATP binding to IRP-1 Z. Popovic and D. M. Templeton
3114 FEBS Journal 274 (2007) 3108–3119 ª 2007 The Authors Journal compilation ª 2007 FEBS
tional flexibilities. Alternatively, lower inhibition by
ATP-cS in cell extracts may be due to the presence of
additional cellular proteins from HepG2 cytosol that
are involved in IRP–IRE complex formation. Further-
more, ATP-cS, but not AMP-PNP, inhibited IRP–IRE
binding in extracts from fibroblasts treated with the
analogs in culture [41]. Therefore, the 30% inhibition
of binding in our cell extracts still suggests that ATP
should be hydrolyzed in order to interact with the
IRP-1–IRE complex. This is confirmed by the lack of

tion, and when the ATP level increases, the complex
dissociates. Increased ATP hydrolysis in the presence
of IRE is reminiscent of an important ATPase protein
family ) the helicases [48]. They unwind duplex RNAs
in concert with the hydrolysis of nucleoside. For
example, the RNA-binding helicase eIF4A undergoes
cyclic conformational changes upon ATP binding and
hydrolysis such that the eIF4A–ADP complex has a
greatly decreased affinity for ssRNA [26]. Our data do
not show whether ATP inhibits the binding of IRP-1
to IRE, or instead facilitates dissociation of the com-
plex. Enhancement of ATP hydrolysis by IRE in the
presence of IRP-1, but absence of hydrolysis in the
presence of IRE alone, suggests that ATP may interact
with the complex. However, the enhancement is not
dramatic, and whether ATP inhibits formation or
increases dissociation of the IRE–IRP-1 complex can-
not be definitively determined from the present data.
If formation of the IRP-1–IRE complex depends on
iron concentration, but its dissociation depends on
ATP concentration, then the expression of IRE-con-
taining genes would actually depend on both iron and
ATP levels. At low ATP levels, transferrin receptor
mRNA would be stable, and transferrin receptor on
the membrane would continue to take up iron. In par-
allel, ferritin mRNA (and other 5¢-IRE mRNAs)
would still have IRP-1 bound, and ferritin synthesis
would be blocked, so iron would not be stored in ferr-
itin but relocated to iron-binding proteins, many of
them located in mitochondria and involved in energy

FEBS Journal 274 (2007) 3108–3119 ª 2007 The Authors Journal compilation ª 2007 FEBS 3115
were plated at a density of 4 · 10
6
cells per 60 mm dish,
allowed to attach and grow overnight, and then treated
with iron (20 lgÆmL
)1
) as ferric ammonium citrate, 10 lm
CCCP, or both. After 3 h, cells were harvested and protein
was extracted (see below).
Preparation of cytoplasmic extracts
Monolayers of HepG2 cells were scraped in NaCl ⁄ P
i
and
centrifuged at 11 000 g for 1 min with an Eppendorf 5415R
centrifuge. The pellet was resuspended in extraction buffer
(EB: 10 mm Hepes, pH 7.6, 3 mm MgCl
2
,40mm KCl,
1mm dithiothreitol, 0.2% Nonidet P-40). After sonication
for 5 s, the suspension was centrifuged for 1 min at
11 000 g with an Eppendorf 5415R centrifuge. The super-
natant was recentrifuged under the same conditions for use
in the IRE-binding assay [49]. The protein content of the
extracts was determined with a Bradford-based protein
determination kit (Bio-Rad, Mississauga, ON, Canada).
The protein extract was aliquoted and stored at ) 80 °C.
Preparation of RNA transcripts
Transcription was performed in vitro with 1 lgofBamH1
linearized plasmid pSPTfer [50], coding the human ferritin

lysate to a final concentration of 0.4 m, and the lysate was
tumbled with Ni
2
–nitrilotriacetic acid agarose beads for 1 h
at 4 °C. This mixture was poured into a column and
washed sequentially at 4 °C with buffer N plus 0.4 m KCl,
buffer N alone, and buffer N with 5 mm imidazole. IRP-1
was eluted with buffer N containing 50 mm imidazole.
Recombinant protein was further purified with the use of
streptavidin-conjugated paramagnetic spheres (Invitrogen
Canada Inc., Burlington, ON, Canada) and biotinylated
IRE RNA [52]. For biotinylation, 100 lL of a transcription
reaction mixture was prepared containing 5 lLof10mm
GTP, UTP and ATP, 2.5 lLof10mm CTP, and 2.5 lLof
10 mm biotin-14-CTP (Invitrogen Canada Inc.) according
to the manufacturer’s instruction. Biotin-14-CTP is a CTP
analog that contains biotin attached to the N4 position via
a 14-atom linker. Full-length transcripts were purified on
QuickSpin columns and used for affinity chromatography.
Streptavidin beads (100 lL) were washed two times with
200 lL of binding buffer (20 mm Hepes, pH 7.5, 150 mm
KCl, 1 mm phenylmethanesulfonyl fluoride), resuspended in
100 lL of binding buffer, and added to biotinylated IRE
(10 lg in 150 lL of binding buffer). Biotinylated IRE was
bound to streptavidin beads for 10 min at 4 °C, and
unbound IRE was washed with 100 lL of binding buffer.
Fifty micrograms of fresh IRP-1 was then added to mag-
netic beads in 100 mm KCl in 200 lL of binding buffer,
and tumbled for 90 min at 4 °C. Beads were washed three
times with 400 lL of washing buffer (20 mm Hepes,

nitrocellulose membranes, incubated with antibodies to
IRP-1 or IRP-2 (Alpha Diagnostic International, San Anto-
nio, TX) (1 : 5000 dilution), and detected using ECL west-
ern blot detection reagents (Amersham Bioscience, Baie
d’Urfe
´
, PQ, Canada).
ATP binding to IRP-1 Z. Popovic and D. M. Templeton
3116 FEBS Journal 274 (2007) 3108–3119 ª 2007 The Authors Journal compilation ª 2007 FEBS
8-Azido-[
32
P]nucleotide binding experiments
Recombinant human IRP-1 (0.2–4 lg) was combined with
8-azido-[
32
P]ATP[aP] (Affinity Labeling Technologies, Lex-
ington, KY; specific activity 10–15 CiÆmmol
)1
) at a final
concentration of 20 lm and assay buffer (25 mm Tris,
pH 7.6, 100 mm KCl, 5 mm MgCl
2
and 10% glycerol) in a
total volume of 20 lL according to the manufacturer’s pro-
tocol. After 2 min of incubation on ice, the samples were
crosslinked immediately with a 254 nm UV lamp for 2 min.
The samples were separated on either a 4–15% gradient or
8% SDS ⁄ PAGE gels (Bio-Rad), silver stained, dried, and
exposed to X-ray film.
Filter-binding assay

) [36]. IRP-1 was added to 10 nm [
32
P]ATP
[cP] in 25 mm Tris ⁄ HCl (pH 7.6), 100 mm KCl, 5 mm
MgCl
2
, 10% glycerol and varying concentration of ATP in
a final volume of 20 lL. After incubation for 60 min at
room temperature, 2 lL was spotted on polyethyleneimine
cellulose TLC plates (Sigma, St Louis, MO, USA). IRE
(100–800 ng) was analyzed under the same conditions. The
spotted samples were resolved using 0.5 m lithium chloride
in 0.5 m formic acid and visualized by autoradiography.
Spots containing the released [
32
P]phosphate were cut and
measured by scintillation counting.
ATP measurement
To extract ATP, cells were harvested in cold 0.6 m HClO
4
and centrifuged at 9000 g for 1 min with an Eppendorf
5415R centrifuge. The pellet was saved for protein deter-
mination by Lowry’s method [54,55]. The supernatant was
neutralized with 5 m KOH and 0.4 m imidazole, and centri-
fuged again. Aliquots of ATP-containing extract were saved
at ) 80 °C. ATP was measured with a commercial lucifer-
in ⁄ luciferase (ENLITEN) kit (Promega) according to the
manufacturer’s protocol. Briefly, 100 l L of a 1 : 500 diluted
sample was added to a microplate, and 50 lL of ENLITEN
rL ⁄ L reagent was added immediately before measurement

regulation. J Biol Chem 269, 30904–30910.
3 Guo B, Yu Y & Leibold EA (1994) Iron regulates cyto-
plasmic levels of a novel iron-responsive element-bind-
ing protein without aconitase activity. J Biol Chem 269,
24252–24260.
4 Rouault TA, Stout CD, Kaptain S, Harford JB &
Klausner RD (1991) Structural relationship between an
iron-regulated RNA-binding protein (IRE-BP) and aco-
nitase: functional implications. Cell 64, 881–883.
5 Martins EL, Robalinho RL & Meneghini R (1995) Oxi-
dative stress induces activation of a cytosolic protein
Z. Popovic and D. M. Templeton ATP binding to IRP-1
FEBS Journal 274 (2007) 3108–3119 ª 2007 The Authors Journal compilation ª 2007 FEBS 3117
responsible for control of iron uptake. Arch Biochem
Biophys 316, 128–134.
6 Pantopoulos K & Hentze MW (1995) Rapid responses
to oxidative stress mediated by iron regulatory protein.
EMBO J 14, 2917–2924.
7 Schalinske KL & Eisenstein RS (1996) Phosphorylation
and activation of both iron regulatory proteins 1 and 2
in HL-60 cells. J Biol Chem 271, 7168–7176.
8 Pantopoulos K & Hentze MW (1995) Nitric oxide sig-
naling to iron-regulatory protein: direct control of ferri-
tin mRNA translation and transferrin receptor mRNA
stability in transfected fibroblasts. Proc Natl Acad Sci
USA 92, 1267–1271.
9 Pantopoulos K, Weiss G & Hentze MW (1996) Nitric
oxide and oxidative stress (H2O2) control mammalian
iron metabolism by different pathways. Mol Cell Biol
16, 3781–3788.

Sci 1012, 1–13.
18 Heck DE, Kagan VE, Shvedova AA & Laskin JD
(2005) An epigrammatic (abridged) recounting of the
myriad tales of astonishing deeds and dire conse-
quences pertaining to nitric oxide and reactive oxygen
species in mitochondria with an ancillary missive con-
cerning the origins of apoptosis. Toxicology 208,
259–271.
19 Harris A (2002) Hypoxia ) a key regulatory factor in
tumour growth. Nat Rev Cancer 2, 38–47.
20 Toth I, Yuan LP, Rogers JT, Boyce H & Bridges KR
(1999) Hypoxia alters iron-regulatory protein-1 binding
capacity and modulates cellular iron homeostasis in
human hepatoma and erythroleukemia cells. J Biol
Chem 274, 4467–4473.
21 Hanson ES, Foot LM & Leibold EA (1999) Hypoxia
post-translationally activates iron-regulatory protein 2.
J Biol Chem 274, 5047–5052.
22 Popovic Z & Templeton DM (2005) A Northwestern
blotting approach for studying iron regulatory element-
binding proteins. Mol Cell Biochem 268 , 67–74.
23 Peck ML & Herschlag D (2003) Adenosine 5¢-O-(3-thio)
triphosphate (ATPcS) is a substrate for the nucleotide
hydrolysis and RNA unwinding activities of eukary-
otic translation initiation factor eIF4A. RNA 9, 1180–
1187.
24 Erecinska M & Wilson DF (1982) Regulation of cellular
energy metabolism. J Membrane Biol 70, 1–14.
25 Eisenstein RS, Tuazon PT, Schalinske KL, Anderson
SA & Traugh JA (1993) Iron-responsive element-bind-

Biochemistry, 4th edn. W.H. Freeman, New York, NY.
33 Meyron-Holtz EG, Ghosh MC, Iwai K, LaVaute T,
Brazzolotto X, Berger UV, Land W, Ollivierre-Wilson
H, Grinberg A, Love P et al. (2004) Genetic ablations
of iron regulatory proteins 1 and 2 reveal why iron
ATP binding to IRP-1 Z. Popovic and D. M. Templeton
3118 FEBS Journal 274 (2007) 3108–3119 ª 2007 The Authors Journal compilation ª 2007 FEBS
regulatory protein 2 dominates iron homeostasis.
EMBO J 23, 386–395.
34 Mo
¨
rikofer-Zwez S & Walter P (1989) Binding of ADP
to rat liver cytosolic proteins and its influence on the
ratio of free ATP ⁄ free ADP. Biochem J 259, 117–124.
35 Liberek K, Marszalek J, Ang D, Georgopoulos C &
Zylicz M (1991) Escherichia coli DnaJ and GrpE heat
shock proteins jointly stimulate ATPase activity of
DnaK. Proc Natl Acad Sci USA 88, 2874–2878.
36 Sadis S & Hightower LE (1992) Unfolded proteins sti-
mulate molecular chaperone Hsc70 ATPase by acceler-
ating ADP ⁄ ATP exchange. Biochemistry 31 , 9406–9412.
37 Yasuda R, Noji H, Yoshida M, KinositaK Jr & Itoh H
(2001) Resolution of distinct rotational substeps by sub-
millisecond kinetic analysis of F1-ATPase. Nature 410,
898–904.
38 Gonzalez D, Drapier JC & Bouton C (2004) Endogen-
ous nitration of iron regulatory protein-1 (IRP-1) in
nitric oxide-producing murine macrophages: further
insight into the mechanism of nitration in vivo and its
impact on IRP-1 functions. J Biol Chem 279, 43345–

Interaction of nucleotides with membrane-associated
cystic fibrosis transmembrane conductance regulator.
J Biol Chem 268, 15336–15339.
47 Pavelka MS Jr, Hayes SF & Silver RP (1994) Charac-
terization of KpsT, the ATP-binding component of the
ABC-transporter involved with the export of capsular
polysialic acid in Escherichia coli K1. J Biol Chem 269,
20149–20158.
48 Caruthers JM & McKay DB (2002) Helicase
structure and mechanism. Curr Opin Struct Biol 12,
123–133.
49 Parkes JG, Liu Y, Sirna JB & Templeton DM (2000)
Changes in gene expression with iron loading and chela-
tion in cardiac myocytes and non-myocytic fibroblasts.
J Mol Cell Cardiol 32, 233–246.
50 Mu
¨
llner EW, Neupert B & Ku
¨
hn LC (1989) A specific
mRNA binding factor regulates the iron-dependent sta-
bility of cytoplasmic transferrin receptor mRNA. Cell
58, 373–382.
51 Gray NK, Quick S, Goossen B, Constable A, Hirling
HK, Ku
¨
hn LC & Hentze MW (1993) Recombinant
iron-regulatory factor functions as an iron-responsive-
element-binding protein, a translational repressor and
an aconitase. A functional assay for translation repres-