biochem

biochem;

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Read the attached article: “
A Mitochondrial Pyruvate Carrier Required for Py
ruvate Uptake in Yeast,
Drosophila, and Humans,”
Science
, 337
, p.
96

100
and answer the following questions.
The authors describe experiments performed to identify the carrier protein involved in pyruvate uptake
into the mitochondria.
1)
How did the authors demonstrate that the
Mpc1 protein was located in the mitochondrial inner
membrane?
2) Describe how
the authors demonstrate
d
that Mpc1 protein function was conserved through
evolution.
3) The Drosophila that lacked dMPC1 (dMPC1

) died q
uickly on a sugar only diet (Figure 2A). What does
this suggest about sugar metabolism in these flies?
How did measurement of metabolites in Figure 2F,
3A and 3B help the authors understand why the dMPC1

flies were dying?
4) Even without a pyruvate carri
er
to transport pyruvate from the cytosol to the matrix, the
mitochondri
a can still have some pyruvate in the matrix.
Describe one way that pyruvate can be
generated in the mitochondrial matrix
in the absence of Mpc1.
5)
Describe the experiment in this pa
per that most directly demonstrates that Mpc1 is a pyruvate
carrier.
PDH activity was almost normal in
mpc1
D
cells
grown in rich medium [when the E2 subunit was
lipoylated (Fig. 2C)]. Thus, the MPC proteins ap-
peared to act upstream of PDH and may function
in the transport of pyruvate into mitochondria.
We therefore measured uptake of
14
C pyruvate
in mitochondria isolated from WT,
mpc1
D
,
mpc2
D
,
mpc3
D
,and
mpc2
D
mpc3
D
cells grown in lactate
medium (Fig. 3A). The specificity of uptake was
assessed by the use of UK5099, an inhibitor of
the mitochondrial pyruvate carrier (
14
). Uptake of
pyruvate in WT mitochondria was sensitive to
the proton ionophore carbonyl cyanide m-chloro
phenyl hydrazone (CCCP) (Fig. 3B). Mitochon-
dria from
mpc1
D
and
mpc2
D
mpc3
D
cells showed
decreased pyruvate uptake (Fig. 3, A and B), de-
spite a normal mitochondrial membrane poten-
tial (fig. S5). Surprisingly, deletion of
MPC3
alone
impaired pyruvate uptake in mitochondria, where-
as mitochondria from the
mpc2
D
mutant trans-
ported pyruvate normally. Because this result did
not correlate with the phenotypes of
mpc2
D
and
mpc3
D
single mutants grown in SD, we inves-
tigated the expression of Mpc2 and Mpc3 in SD
and lactate media. In SD, yeast expressed mainly
Mpc2, whereas in lactate medium, they mainly
expressed Mpc3 (Fig. 3C). This expression pat-
tern could be explained, at least in part, by the
presence of binding sites for Gcn4 (a transcrip-
tion factor activated by amino acid starvation)
upstream of
MPC2
(
15
). This raises the possi-
bility that under certain growth conditions, these
two proteins might have specific, nonredundant
functions.
We next assessed whether mouse MPC1
(mMPC1) and MPC2 (mMPC2) could restore
growth of yeast cells lacking a functional pyru-
vate transporter (Fig. 4, A and B). mMPC1 alone
restored growth of
mpc1
D
cells, but mMPC2 failed
to restore growth of the double-deletion strain
of its orthologous genes
MPC2
and
MPC3
.
However, growth of the triple-deletion strain
mpc1
D
mpc2
D
mpc3
D
or of
mpc2
D
mpc3
D
cells
was restored by coexpression of both mMPC1
and mMPC2 (Fig. 4A). Thus, mMPC1 and
mMPC2 together functionally complement the
absence of pyruvate transport. We next expressed
mMPC1 and mMPC2, alone and in combination,
in the bacterium
Lactococcus lactis
(Fig. 4C),
which has been successfully used to express and
characterize mitochondrial transporters (
16
). No
pyruvate uptake was observed in bacteria ex-
pressing either protein alone compared with the
empty vector control. However, a fourfold in-
crease in pyruvate uptake was detected when
mMPC1 and mMPC2 were coexpressed (Fig. 4,
D and E). This uptake was sensitive to the mito-
chondrial pyruvate carrier inhibitor UK5099 and
to 2-deoxyglucose, which
collapses the proton elec-
trochemical gradient (Fig. 4E) (
17
). Moreover,
artificially increasing the membrane potential
by lowering the pH in the import buffer from
7.2 to 6.2 significantly increased pyruvate uptake
(two-tailed
t
test,
P
< 0.05) (Fig. 4E). Thus, coex-
pression of mMPC1 and mMPC2 in bacteria is
sufficient to allow import of pyruvate with similar
properties to the mitochondrial pyruvate carrier
(
3
). We therefore conclude that the mitochondrial
pyruvate carrier is composed of Mpc1 and either
Mpc2 or Mpc3 in yeast and of MPC1 and MPC2
in mammals.
References and Notes
1. J. K. Hiltunen, Z. Chen, A. M. Haapalainen,
R. K. Wierenga, A. J. Kastaniotis,
Prog. Lipid Res.
49
, 27 (2010).
2. L. J. Reed,
J. Biol. Chem.
276
, 38329 (2001).
3. A. P. Halestrap,
Biochem. J.
148
, 85 (1975).
4. S. Da Cruz
et al
.,
J. Biol. Chem.
278
, 41566
(2003).
5. D. K. Bricker
et al
.,
Science
337
, 96 (2012).
6. J. R. Dickinson, I. W. Dawes,
J. Gen. Microbiol.
138
,
2029 (1992).
7. J. R. Dickinson, D. J. Roy, I. W. Dawes,
Mol. Gen. Genet.
204
, 103 (1986).
8. R. A. Harris, M. Joshi, N. H. Jeoung, M. Obayashi,
J. Nutr.
135
(suppl.), 1527S (2005).
9. M. S. Schonauer, A. J. Kastaniotis, V. A. Kursu,
J. K. Hiltunen, C. L. Dieckmann,
J. Biol. Chem.
284
,
23234 (2009).
10. J. E. Lawson, R. H. Behal, L. J. Reed,
Biochemistry
30
,
2834 (1991).
11. K. M. Humphries, L. I. Szweda,
Biochemistry
37
, 15835
(1998).
12. H. Wada, D. Shintani, J. Ohlrogge,
Proc. Natl. Acad. Sci.
U.S.A.
94
, 1591 (1997).
13. U. Hoja
et al
.,
J. Biol. Chem.
279
, 21779 (2004).
14. A. P. Halestrap, R. M. Denton,
Biochem. J.
148
,97
(1975).
15. K. D. MacIsaac
et al
.,
BMC Bioinformatics
7
, 113 (2006).
16. E. R. Kunji, D. J. Slotboom, B. Poolman,
Biochim.
Biophys. Acta
1610
, 97 (2003).
17. E. R. Kunji, E. J. Smid, R. Plapp, B. Poolman, W. N. Konings,
J. Bacteriol.
175
, 2052 (1993).
Acknowledgments:
We are grateful to R. Loewith and F. Stutz
for strains and technical help, L. Szweda for antibodies,
A. Kastaniotis for technical help on lipoic acid determination,
Y. Que for erythromycin resistance cassette, and H. Riezman,
A. Jourdain, and the Martinou lab for fruitful discussions.
This work was supported by Novartis Science Foundation
(S.H.), the Swiss National Science Foundation (subsidy
31003A-141068/1 to J.-C.M.), and the state of Geneva.
Supplementary Materials
www.sciencemag.org/cgi/content/full/science.1218530/DC1
Materials and Methods
Supplementary Text
Figs. S1 to S5
Table S1
References (
18

23
)
29 December 2011; accepted 11 May 2012
Published online 24 May 2012;
10.1126/science.1218530
A Mitochondrial Pyruvate Carrier
Required for Pyruvate Uptake in Yeast,
Drosophila
, and Humans
Daniel K. Bricker,
1
*
Eric B. Taylor,
2
*
John C. Schell,
2
*
Thomas Orsak,
2
*
Audrey Boutron,
3
Yu-Chan Chen,
2
James E. Cox,
4
Caleb M. Cardon,
2
Jonathan G. Van Vranken,
2
Noah Dephoure,
5
Claire Redin,
6
Sihem Boudina,
7
Steven P. Gygi,
5
Michèle Brivet,
3
Carl S. Thummel,
1
Jared Rutter
2

Pyruvate constitutes a critical branch point in cellular carbon metabolism. We have identified
two proteins, Mpc1 and Mpc2, as essential for mitochondrial pyruvate transport in yeast,
Drosophila
, and humans. Mpc1 and Mpc2 associate to form an ~150-kilodalton complex in the
inner mitochondrial membrane. Yeast and
Drosophila
mutants lacking
MPC1
display impaired
pyruvate metabolism, with an accumulation of upstream metabolites and a depletion of
tricarboxylic acid cycle intermediates. Loss of yeast Mpc1 results in defective mitochondrial
pyruvate uptake, and silencing of
MPC1
or
MPC2
in mammalian cells impairs pyruvate oxidation.
A point mutation in
MPC1
provides resistance to a known inhibitor of the mitochondrial
pyruvate carrier. Human genetic studies of three families with children suffering from lactic
acidosis and hyperpyruvatemia revealed a causal locus that mapped to
MPC1
, changing single
amino acids that are conserved throughout eukaryotes. These data demonstrate that Mpc1
and Mpc2 form an essential part of the mitochondrial pyruvate carrier.
P
yruvate occupies a pivotal node in the reg-
ulation of carbon metabolism as it is the
end product of glycolysis and a major sub-
strate for the tricarboxylic acid (TCA) cycle in
mitochondria. Pyruvate lies at the intersection
of these catabolic pathways with anabolic path-
ways for lipid synthesis, amino acid biosynthesis,
and gluconeogenesis. As a result, the failure to
correctly partition carbon between these fates
lies at the heart of the altered metabolism evi-
dent in diabetes, obesity, and cancer (
1
,
2
). Owing
to the fundamental importance of pyruvate, the
mitochondrial pyruvate carrier (MPC) has been
studied extensively (
3
,
4
). This included the dis-
covery that
a
-cyanocinnamate analogs, such as
UK-5099, act as specific and potent inhibitors
of carrier activity (
5
). In spite of this character-
ization, however, the gene or genes that encode
the mitochondrial pyruvate carrier remain un-
known (
6
,
7
).
As part of an ongoing effort to characterize
mitochondrial proteins that are conserved through
evolution, we initiated studies of the MPC protein
family (originally designated BRP44 and BRP44L
6 JULY 2012 VOL 337
SCIENCE
www.sciencemag.org
96
REPORTS
on November 5, 2012
www.sciencemag.org
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