REVIEWS
TIBS 24 – FEBRUARY 1999
Non-mitochondrial ATP
transport
Herbert H. Winkler and
H. Ekkehard Neuhaus
Exchange of organelle ATP with cytosolic ADP through the ADP/ATP carrier is
a well-characterized feature of mitochondrial metabolism. Obligate intracellular bacteria, such as Rickettsia prowazekii, and higher-plant plastids possess another type of adenylate transporter, which exchanges bacterial or
plastidic ADP for ATP from the eukaryotic (host cell) cytoplasm. The bacterial
and plastidic transporters are similar but do not share significant sequence
similarities with the mitochondrial carrier. Recent molecular and biochemical
studies are providing deeper insight into the functional and evolutionary
relationships between the bacterial and the plant transport proteins.
THE CONCENTRATION OF ATP within
cells is orders of magnitude higher than
we would predict from the equilibrium
constant for ATP hydrolysis. The synthesis of ATP at such a high concentration
and the use of this ATP as the common
energy currency are a large part of cellular metabolism. The size and charge of
ATP prevents its crossing biological membranes without a carrier protein. Because
ATP is essentially absent from the extracellular environment, and the precious
ATP pool within the cytoplasm would be
put at risk if an ATP carrier was present
in the cell membrane, plasma-membrane
ATP transporters have not evolved. Thus,
conventional wisdom supports the generalization that the cell membrane is impermeant to ATP. However, the internal
membranes of the cell, organelle membranes, are not included in this generalization, and the cell membranes of strange
creatures that inhabit the cytoplasm of
other cells are also an exception. These
membranes, in contrast to the typical
cell membrane, separate an external
compartment where ATP is an available
substrate from an internal compartment
where ATP would be beneficial.
of plastids – can exchange ATP for ADP.
Mitochondria must transport the ATP
they synthesize from their matrix to the
cytosol (see Fig. 1). The mitochondrial
ADP/ATP transport system was discovered in the 1960s as a result of investigations stemming from the poisoning of
children who ate certain Sicilian thistles
containing atractyloside, which inhibits
this transport system1–3. This inhibitor,
as well as another inhibitor, bongkrekic
acid, continues to play an important
role in the elucidation of the properties
of the mitochondrial ADP/ATP transport
system4. The mitochondrial transporter
is a dimer. The monomer contains three
(a)
(b)
100-residue repeating units, and each repeating unit contains two transmembrane
helices and a connecting hydrophilic
loop.
In 1969, Heldt5 found that spinach-leaf
chloroplasts possess a carrier protein
for ATP/ADP transport. Subsequently,
ATP import was also observed in isolated chromoplasts, leucoplasts and
amyloplasts, which confirmed the general presence of ATP/ADP transporters
in plastids6. (All types of plastids are derived from undifferentiated proplastids.
Chloroplasts harbor the photosynthetic
machinery; amyloplasts store starch;
chromoplasts contain pigments, and
leucoplasts are located in roots.) Studies
of isolated plastids and proteoliposomes
demonstrated that these ATP/ADP
transporters are specific for ATP and
ADP, and exhibit Km values for both substrates in the range 15–40 mM7,8. However,
in contrast to mitochondrial adenylate
transporters, all non-mitochondrial ATP/
ADP transporters appear to be insensitive
to atractyloside or bongkrekic acid.
In eukaryotic cells, microsomal vesicles
include the Golgi complex and the endoplasmic reticulum (ER). Several reactions
in these organelles depend on energy in
the form of ATP. For example, protein
phosphorylation in the Golgi and ER, and
protein import and folding in ER, require
ATP at the luminal side. Vesicles from
both types of organelle possess transport
proteins that allow uptake of ATP from
the cytosol9,10, but the characteristics of
ATP transport have been determined in
detail only for enriched ER vesicles.
(c)
Atractyloside
Bongkrekic acid
ADP
ATP
ADP
ATP
ADP
ATP
F1-ATPase
+ Proton
H
extrusion
+
H
+
H
Organelle adenylate transport
Membrane-bound organelles – mitochondria, chloroplasts and other types
Thylakoid
Rickettsia
H. H. Winkler is at the Dept of Microbiology
and Immunology, University of South Alabama
College of Medicine, Mobile, AL 36688, USA;
and H. E. Neuhaus is at the Dept of Plant
Physiology, University of Osnabrück,
Osnabrück, D-49069, Germany.
Email: herbertw@sungcg.usouthal.edu
64
Mitochondrion
Chloroplast
Figure 1
Examples of adenylate-transport systems. (a) Rickettsia. (b) Mitochondrion. (c) Chloroplast.
The preferential directions of transport are indicated, as are the directions of proton extrusion and the proton-driven ATPase. The outer membranes are shown as blue lines and the
inner membranes as red lines.
0968 – 0004/99/$ – See front matter © 1999, Elsevier Science. All rights reserved.
PII: S0968-0004(98)01334-6
REVIEWS
TIBS 24 – FEBRUARY 1999
Uptake of ATP into enriched ER vesicles from rat liver and bakers’ yeast is
protein mediated and exhibits micromolar apparent-affinity constants10,11. As
observed for mitochondrial adenylate
transport, movement of adenylates into
the lumen of enriched ER is associated
with counterexchange11,12. However, unlike the mitochondrial ADP/ATP transporter, the ER ATP/ADP-transport system
was not inhibited by carboxyatractylate10,11. This suggests that the two transporters exhibit substantial structural differences. ER vesicles enriched from a
bakers’-yeast mutant that lacks the ERmembrane-bound Sac1p protein exhibit
strongly reduced rates of ATP import12.
From this observation, it was logical to
assume that Sac1p is an adenylate transporter. However, because Sac1p possesses only one putative transmembrane domain13, one would have to
assume a rather unusual topology for
this to be a solute transporter. Indeed, a
detailed alignment of the amino acid sequence of Sac1p with those of other proteins suggested that Sac1p performs
a function similar to an inositol-5phosphate phosphatase14; no connection
between such a function and the ER ATP
transporter has been reported.
Bacterial adenylate transport
In 1976, Winkler15 reported that the bacterium Rickettsia prowazekii, a pathogenic invader of eukaryotic cell cytoplasm
and the etiologic agent of louse-borne typhus, transports ATP – the first report of a
strange intracytoplasmic creature’s benefiting from an adenylate-transport system. There is no net transport of adenylates into rickettsiae by this system;
rather, these bacteria obtain energy
through the obligate exchange of ATP for
ADP. The transport system exhibits a halfmaximal velocity at a substrate concentration of ~75 mM and is highly specific in
that deoxyribonucleotides, AMP and nonadenine-based ribonucleotides are not
substrates; specificity resides in the base,
the number of phosphates and the sugar.
In 1982, Hatch and co-workers16 showed
that the bacterium Chlamydia psittaci
possesses a similar ATP/ADP transport
system. Both of these bacterial species
are obligate intracellular parasites. R.
prowazekii can grow only within the cytoplasm of a eukaryotic host cell, and
chlamydiae can grow only within special
intracytoplasmic vacuoles. Thus, the evolutionary acquisition of such a transport
system in these bacteria allowed them
to parasitize the energy reserves of their
host. None of the genomic sequences
currently available encode a protein that
is homologous to that in mitochondria,
rickettsiae, chlamydiae or plastids (see
below).
Differences in preferred directions of
exchange
Mitochondrial transporters differ from
plastidic and rickettsial transporters in
their preferred direction of ATP transport (see Fig. 1). In the test tube, all
possible directions of exchange of ATP
and ADP are observed. However, in situ
there is a single preferred and productive direction. Rickettsiae and chlamydiae take ATP from the host cell cytoplasm, use the ‘high-energy phosphate’
and return ADP, which the host cell
recharges to ATP. By contrast, mitochondria provide the cytoplasm with ATP17.
Unlike mitochondrial ATP, the ATP
generated in chloroplasts through
photosynthesis is not available to the
cytoplasm but is totally consumed by
CO2 fixation and other anabolic processes
that take place within the interior of the
chloroplast. Transport studies on isolated
chloroplasts revealed that ATP import
occurs twice as fast as ADP uptake7. In the
case of heterotrophic pea-root plastids,
uptake of ATP and ADP occurs at similar
rates7, which indicates that the cytosolic
ATP:ADP ratio is important for control of
anabolic reactions in storage plastids.
Although strict control of adenylatetransport polarity might be limited to
mitochondria, in both rickettsiae and
plastids the ATP influx appears to be the
favoured direction, because cytosolic
ATP:ADP ratios are high.
Sequences of bacterial and plastidic
transporters
The gene that encodes the rickettsial
adenylate transporter (tlc) was sequenced in 1989; on the basis of the deduced amino acid sequence, we can predict that the rickettsial protein is twice as
large as (56.7 kDa), and shares no meaningful sequence homology with, its mitochondrial analogues, although both are
typical membrane proteins18. The rickettsial transporter has 12 predicted
transmembrane domains and, unlike the
mitochondrial transporter, has no repeating elements. Peptide-specific-antibodyaccessibility experiments place the
C-terminus in the rickettsial cytoplasm19.
In 1995, Kampfenkel et al.20 identified an
Arabidopsis thaliana cDNA that encodes a
protein that shares .66% similarity with
the rickettsial ATP/ADP transporter but
lacks any sequence relationship with mitochondrial adenylate transporters. The
surprisingly high degree of similarity to
a prokaryotic protein indicated that this
was a new type of eukaryotic adenylate
transporter; the transporter was termed
ATP/ADP transport protein (A. thaliana)
[AATP(At)]. AATP(At) has an N-terminal
amino acid extension that is not present
in the bacterial homologues. This extension has a structure typical of four other
proteins that reside in the inner membrane of the plastid envelope, which suggests that AATP(At) is also a plastidic
protein20. As predicted, the gene product of in-vitro-translated AATP cDNA is
incorporated into the inner membrane
of chloroplasts21. Similar cDNAs for plastidic ATP/ADP transporters are present
in maize and potato, and partial sequences from several other plant cDNA
libraries have been identified.
Southern-blot analysis of genomic DNA
extracted from two A. thaliana ecotypes
revealed another isoform of the plastidic
ATP/ADP transporter, AATP2(At) (Ref. 20).
We have cloned and sequenced
AATP2(At), which is very similar to
AATP1(At) (Ref. 22). Sequencing of the
complete genomes of rickettsia and
chlamydia has revealed that there are
four homologues of the tlc gene in rickettsiae and two in chlamydiae. Figure 2
shows the homologies in a selected region of all the currently identified nonmitochondrial ATP/ADP transporters. The
rickettsial and plastidic genes are known
to be expressed, because they were discovered through cDNAs from plastids,
and mRNAs for all five homologous genes
are found in rickettsiae (S. G. E. Andersson
et al., unpublished). However, these
homologues may well exhibit different
biochemical properties and might have
different substrate specificities.
Expression in heterologous systems
A plant transporter, AATP(At), was recently expressed and inserted into the
Escherichia coli cytoplasmic membrane
for the first time23 (in an E. coli strain24
suitable for the heterologous expression
of membrane-bound proteins). The two
heterologously expressed plastidic
ATP/ADP transporters, AATP1(At) and
AATP2(At), mediate the counterexchange
mode of transport and exhibit similar
affinities for ATP and ADP22,23. The difficulty of growing and purifying obligate
intracellular bacteria also makes heterologous expression of their genes in E.
coli highly desirable. We have functionally expressed the rickettsial tlc gene25,26
and both chlamydial genes (H. E. Neuhaus
and H. H. Winkler, unpublished) in
E. coli cytoplasmic membranes. We have
65
REVIEWS
TIBS 24 – FEBRUARY 1999
Figure 2
Similarity of non-mitochondrial adenylate-transport systems and their homologues. The
aligned fragments include the transmembrane helix 4, cytoplasmic loop 5, and part of
transmembrane helix 5 in Rickettsia prowazekii (Rp). Vertical bars indicate residues that
are identical. The bottom three rows indicate residues that are identical in the rickettsial,
chlamydial or plastidic groups. Ct, Chlamydia trachomatis; Ma, maize plastid; Po, potato
plastid; Rr, Rickettsia rickettsii.
also functionally reconstituted both the
plant AATP and the rickettsial tlc gene
products in proteoliposomes.
Regulation and the ATP/ADP transporter
Anabolic metabolism in heterotrophic
plastids depends upon a supply of cytosolic ATP. An analysis of fatty acid and starch
biosynthesis in enriched cauliflowerbud amyloplasts indicated that both
metabolic pathways compete for stromal
ATP27. The rate of end-product synthesis
is dependent on the activity of the ATP/
ADP transporter, is inhibited when other
ATP-consuming reactions are active at
the same time, and does not occur if ATP
is absent from the incubation medium.
The plastidic ATP/ADP transporter has
a high affinity for both ATP and ADP7,22,23;
both metabolites should therefore compete for binding to the transporter in vivo.
Indeed, an increase in the ADP:ATP ratio in
the incubation medium strongly reduces
the rate of starch biosynthesis in isolated
cauliflower-bud amyloplasts8. Hence, the
plastidic ATP/ADP transporter might help
to regulate the rate of end-product synthesis in storage tissues. Moreover, supply of
high levels of carbohydrates to storage
tissues correlates with elevated rates of
respiration and thus a high cytosolic
ATP:ADP ratio28. The increased energy
charge in the cytosol is reflected in the
amyloplast, where it stimulates the incorporation of carbon into starch.
It is generally assumed that the ATP
required for photosynthetic CO2 fixation
derives from the ATP synthase driven by
66
the pH gradient across the thylakoid
membrane. However, there are several
indications that, at least under certain
conditions or in certain types of chloroplast, the uptake of cytosolic ATP supplements anabolic reactions. Such an
ATP uptake would be required when
photosynthesis does not deliver sufficient ATP for CO2 fixation and other anabolic reactions. The plastidic ATP/ADP
transporter should be responsible for
translocating ATP generated in the mitochondria from cytosol to stroma in these
circumstances (see Fig. 3).
Depending on the rate of ATP uptake,
the transport of ATP might contribute
to carbon fixation either by only a few
percent or substantially. Import of ATP
ranges from 5 micromoles per milligram
of chlorophyll per hour (in spinach
chloroplasts)5 to .40 micromoles per
milligram of chlorophyll per hour (in
Digitaria sanguinalis chloroplasts)29.
Finally, in isolated chloroplasts from
sweet-pepper fruits, the addition of ATP
results in the highest observed rates of
starch biosynthesis in the light30. In conclusion, photosynthetic carbon metabolism in several organisms is obviously
positively influenced by exogenous ATP;
the plastidic ATP/ADP transporter is
thus important in both heterotrophic
plastids (i.e. amyloplasts) and photoautotrophic plastids (i.e. chloroplasts).
Transcriptional regulation of the
ATP/ADP transporter is seen in R.
prowazekii31,32. Transcription of mRNA
from the tlc gene is downregulated when
rickettsiae accumulate in large numbers
in the host cell and when rickettsiae are
grown in respiratory-deficient host cells
in low-glucose medium. We suggest that,
as the energy charge in the host cells
falls, fewer ATP/ADP transporters are
synthesized by rickettsiae. Rickettsiae
not only import ATP but also synthesize
ATP by oxidative phosphorylation. In
this sense, they are more like the
photoautotrophic chloroplasts than the
heterotrophic plastids. Thus, if rickettsiae have a higher energy charge than
their, damaged, host cells, there will
be an efflux of ATP from the parasite.
Downregulation of tlc transcription would
therefore be a reasonable way for
rickettsiae to minimize this adverse
exchange of adenylates.
Evolution
Perhaps the most exciting, but speculative, conclusion that can be drawn
from these studies involves the endosymbiotic origins of organelles. According to the endosymbiont hypothesis,
mitochondria evolved from a primitive
eubacterium that exhibited oxidative
metabolism and invaded, and remained
within, a primitive nucleated, nonoxidative cell. This particular symbiotic
relationship gave rise to the modern eukaryotic cell. (See Martin and Müller33
for an alternative view, in which the
host cell was even more primitive.)
What kind of bacterium became a
mitochondrion? Because present-day
mitochondria have retained a small
part of their original bacterial genome,
the sequences and organization of
these retained genes in modern mitochondrial DNA can be compared with a
variety of modern bacteria. Such comparisons suggest that mitochondria
are most closely related to the rickettsial branch of the a division of the
proteobacteria34–38.
We speculate that this obligate-intracytoplasmic precursor of both mitochondria and modern rickettsiae carried out oxidative phosphorylation, was
a dangerous intracytoplasmic parasite
because it could grow faster than its
host cell, and was more useful to its
host than are contemporary rickettsiae.
These rickettsiae could have supplied a
nutrient to the host and/or detoxified
the host’s cytosol by feeding on the
host’s metabolic end products. Although
a non-oxidative host cell would have
been inefficient at converting foodstuffs
to energy, such a cell would still have
had a high energy charge and could
have supplied rickettsiae with ATP.
REVIEWS
TIBS 24 – FEBRUARY 1999
These primitive rickettsiae might have
had an ATP/ADP transporter that preferentially took ATP from the host cytosol,
or a transporter that had no preference
for ADP or ATP. Alternatively, they could
have lacked an ATP/ADP transporter:
the transporter might have appeared
later. However, the lack of an ATP/ADP
transporter must be reconciled with the
observation that chlamydiae, which diverged from the proteobacteria early
on, have a homologous transport system. In any case, we can be sure that the
primitive rickettsia would not have had
a mitochondrial-type transporter that
supplied ATP to the host cytosol.
As protomitochondria evolved from
these rickettsiae, there would have been
a shift from parasitism to symbiosis.
Many genes would have become
nonessential and been lost from, or
modified within, the rickettsial genome.
Rickettsial genes would have been
placed under host-cell control by being
moved to the nucleus, and leader sequences would have evolved in some
products of nuclear genes, both of rickettsial and of non-rickettsial origin, so
that these proteins could be translocated correctly from cytosolic ribosomes to the protomitochondria.
Changes in the rickettsial genome that
made it grow more slowly and less independently could have allowed the modified rickettsiae to remain in the cell
without causing host-cell lysis.
The origin of the nuclear gene that encodes the mitochondrial ADP/ATP transporter is unknown. We can assume that
it did not evolve from a rickettsial transporter, because the two transporters
lack homology. Subsequent evolution of
the animal mitochondrion required only
a massive transfer of genes to the nucleus and a loss of genes that were redundant or useless, such as the rickettsial ATP/ADP transporter. But the
plant cell was just beginning its unique
endosymbiotic relationship39.
Analysis of the residual genome of the
modern chloroplast suggests that
the ancestors of cyanobacteria formed
the primordial chloroplast. Cyanobacteria, if we take Synechocystis sp. as
representative, do not have a homologue of the ATP/ADP transporter. This
is as predicted, because they are not
intracellular bacteria and, thus, no external ATP would be available to them.
However, even without an ATP/ADP
transporter, the eukaryotic cell that
acquired a protochloroplast would
have had a great advantage because it
would have been able to fix CO2.
Oxygen
Water
Light
CO2
ATP
ADP
+
Fuel
Triose phosphate
H
H
+
Metabolites
Mitochondrion
Cytosol
Chloroplast
Figure 3
Flow of ATP (through ATP/ADP transporters) and nutrients between mitochondrion and
cytosol, and between cytosol and chloroplast in a plant cell.
However, as discussed above, if the
only ATP available was derived from
photophosphorylation, such plastids
would have had limited synthetic
ability. The plastid that acquired a
rickettsia-like ATP/ADP transporter
had an advantage.
How do we account for the homology
of the rickettsial and the plastidic transporters? One possible answer is that
the ATP/ADP transporter gene of the
primitive rickettsia was unused and
extraneous in the nucleus of the eukaryotic cell, and was the perfect gene to
be commandeered by the primitive plastid. (Indeed, there are several suggestions that genes that were previously in
the early mitochondrion were transferred to the nucleus and that the gene
products encoded by these genes were
then targeted to plastids39.) Alternatively, the rickettsial gene might by
this time have been lost from the eukaryotic cell, or such a gene might
never have existed in primitive rickettsiae. If so, then there must have been
a second rickettsiae interaction, an
interaction between a more evolved
rickettsiae and a eukaryotic cell that
already had mitochondria, to make this
gene available to the plastid. In either
case, the formerly rickettsial gene
would have been modified to code for a
product that functioned optimally as
a plastidic protein and could be targeted for insertion into the inner membrane of the chloroplast. Such scenarios perhaps explain the curious
fact that, across kingdoms, the plant
and the rickettsial genomes share,
with each other but not with animal
genomes, descendants of a gene that
encodes an ATP/ADP transporter.
Acknowledgements
This work was supported by Public
Health Service grant AI-15035 from the
National Institute of Allergy and
Infectious Diseases.
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Oncogenic alterations of
metabolism
Chi V. Dang and Gregg L. Semenza
Over seven decades ago, classical biochemical studies showed that
tumors have altered metabolic profiles and display high rates of glucose
uptake and glycolysis. Although these metabolic changes are not the
fundamental defects that cause cancer, they might confer a common advantage on many different types of cancers, which allows the cells to survive and invade. Recent molecular studies have revealed that several of
the multiple genetic alterations that cause tumor development directly
affect glycolysis, the cellular response to hypoxia and the ability of tumor
cells to recruit new blood vessels.
A GENETICALLY ALTERED neoplastic
cell has special metabolic requirements
for its development into a three-dimensional tumor mass. Monolayer cultures
do not reflect the three-dimensional cellular growth of an avascular tumor,
which can be mimicked in soft-agar
anchorage-independent-growth assays.
When a tumor has grown to a detectable size, the local environment of
the cancer cells often becomes heterogeneous1. Small (,1 mm diameter)
tumor nodules, as well as microregions
of larger tumors, often have microecological niches that display significant
gradients of critical metabolites such as
oxygen, glucose and other nutrients
or growth factors (Fig. 1a). Tumors, in
contrast to normal tissue, exist in acidic
environments that result from production
C. V. Dang is at the Depts of Medicine,
Oncology, Pathology, and Molecular Biology
and Genetics, Johns Hopkins University
School of Medicine, Baltimore, MD 21205,
USA; and G. L. Semenza is at the Depts of
Pediatrics and Medicine, and the Institute of
Genetic Medicine, Johns Hopkins University
School of Medicine, Baltimore, MD 21205,
USA.
Email: cvdang@welchlink.welch.jhu.edu
68
of lactate and other acids. The cytosolic
pH of tumor cells, however, is maintained
as it is in normal cells.
Hypoxia occurs in tumor tissue that
is .100–200 mm away from a functional
blood supply2. Thus, tumor survival depends, in part, on the ability to recruit
new blood microvessels through angiogenic factors (Fig. 1b). Hypoxia tends to
be widespread in solid tumors, however, because cancer cells are more prolific than the invading, recruited endothelial cells, which commonly form a
new, disorganized blood supply. Human
tumors endure profound hypoxia,
which indicates that adaptation to hypoxic conditions is a crucial step in
tumor progression. The anaerobic use
of glucose as an energy source through
glycolysis (Fig. 2a) is, therefore, a
feature common to most solid tumors.
A better understanding of cancers at
the molecular level has provided insights into the causes of altered
metabolism in oncogenesis. Various
metabolic changes have been observed
in tumors. Here, however, we emphasize
changes in glucose metabolism and cellular responses to hypoxia, and discuss
three specific areas: (1) physiological
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responses used by tumor cells to adapt
to hypoxia; (2) oncogenic changes that
affect glucose metabolism; and (3)
tumor metabolism and apoptosis.
Hypoxia in tumors and normal tissues:
activation of genes that encode glycolytic
enzymes and vascular endothelial growth
factor
Normal tissue displays an oxygen
gradient across a distance of 400 mm
from a blood supply. By contrast, in situ
measurements of oxygen tension in
human tumors and tumor xenografts revealed significant hypoxia: cells adjacent to capillaries displayed a mean
oxygen concentration of 2%, and cells
located 200 mm from the nearest capillary displayed a mean oxygen concentration of 0.2% (Ref. 2). The profoundly
hostile environment selects for cells
that are adapted to chronic hypoxia. In
normal cells, a critical response to hypoxia is the induction of the hypoxiainducible transcription factor HIF-1, a
basic–helix-loop-helix (bHLH) transcription factor that consists of two
subunits, HIF-1a and HIF-1b (Ref. 3).
HIF-1b is also known as the arylhydrocarbon-receptor nuclear translocator
(ARNT)3. HIF-1 binds to the DNA sequence 59-RCGTG-39 and increases the
expression of genes that encode glycolytic enzymes, including aldolase A,
enolase 1, lactate dehydrogenase A
(Fig. 2b), phosphofructokinase L, phosphoglycerate kinase 1 and pyruvate
kinase M, as well as the vascular
endothelial growth factor (VEGF) gene,
which is important for angiogenesis4–8
(Fig. 3). In addition to alterations in oxygen tension, changes in glucose concentration also activate many glycolytic
enzyme genes through the carbohydrate-response element (ChoRE; 59CACGTG-39), which matches the consensus binding-site sequences for MYC
and HIF-1 (Fig. 2b)9,10. Studies of knockout mice have implicated HIF-1 and the
HLH–leucine-zipper transcription factor
USF2, which also binds to the 59CACGTG-39 sequence, in the regulation
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