Bovine Coupling Factor 6, with Just 14.5% Shared Identity,
Replaces Subunit h in the Yeast ATP Synthase*
Received for publication, September 6, 2000, and in revised form, November 6, 2000
Published, JBC Papers in Press, November 16, 2000, DOI 10.1074/jbc.M008123200
Jean Velours‡§, Jacques Vaillier‡, Patrick Paumard‡¶, Vincent Soubannier‡¶,
Jie Lai-Zhang?, and David M. Mueller?
From the ‡Institut de Biochimie et Ge ´ne ´tique Cellulaires du CNRS, Universite ´ Victor Se ´galen, Bordeaux 2,
1 rue Camille Saint Sae ¨ns, 33077 Bordeaux, cedex France and ?Department of Biochemistry and Molecular Biology,
Chicago Medical School, North Chicago, Illinois 60064
The mammalian mitochondrial ATP synthase is com-
posed of at least 16 polypeptides. With the exception of
coupling factor F6, there are likely yeast homologs for
each of these polypeptides. There are no obvious yeast
homologs of F6, as predicted from primary sequence
comparison of the putative peptides encoded by the
open reading frames in the yeast genome. In this manu-
script, we demonstrate that expression of bovine F6
complements a null mutant in ATP14 gene in yeast Sac-
charomyces cerevisiae. Subunit h of the yeast ATP syn-
thase is encoded by ATP14 and is just 14.5% identical to
bovine F6. Expression of bovine F6in an atp14 null mu-
tant strain recovers oxidative phosphorylation, and the
ATP synthase is active, although functioning with a
lower efficiency than the wild type enzyme. Like sub-
unit h, bovine F6is shown to interact mainly with sub-
unit 4 (subunit b), a component of the second stalk of the
enzyme. These data indicated the subunit h is the yeast
homolog of mammalian coupling factor F6.
The F0F1-ATP synthase is the major enzyme responsible for
the aerobic synthesis of ATP. It exhibits a tripartite structure
consisting of a head piece (F1catalytic sector), base piece (F0,
membrane sector), and two connecting stalks. F1is a water-
soluble unit retaining the ability to hydrolyze ATP. F0is em-
bedded in the membrane and is mainly composed of hydropho-
bic subunits forming a specific proton conducting pathway. The
connecting stalks are composed of components from both F1
and F0. When F1and F0are coupled, the enzyme functions as
a reversible H?-transporting ATPase or ATP synthase (1, 2).
The establishment of the crystal structure of the major part
of the bovine F1(3) allowed experiments that demonstrated
that the enzyme is a molecular rotary motor, as shown by the
ATP-dependent rotation of the ?-subunit (4, 5) and consistent
with the binding site hypothesis of Boyer (6). In Escherichia
coli, F1and F0are linked by two stalks, one of which is made of
subunits ? and ?, and these also constitute a part of the rotor
(7). Three other subunits, ? of F1and the two b-subunits of F0,
are also involved in the binding of F1to F0and are thought to
form the second stalk of the stator. The stator is thought to fix
the ?3?3oligomer to the a-subunit, thus allowing rotation of the
c-subunit oligomer together with the ?- and ?-subunits. (8–10)
while holding the head piece in place.
The E. coli ATP synthase and the bovine enzyme are com-
posed of 8 and 16 different subunits, respectively (11). In the
case of Saccharomyces cerevisiae, the F0F1-ATP synthase is
composed of at least 13 different subunits involved in the
structure of the enzyme; the disruption of any of the corre-
sponding structural genes leads to a lack of assembly of the
holo-complex (12). Recently, the establishment of the structure
of the yeast enzyme at 3.9 Å resolution revealed the structure
of the F1and the subunit c oligomer of F0(13).
Among the supernumerary subunits of the yeast ATP syn-
thase F0, subunit h has been described as an essential compo-
nent because inactivation of the ATP14 gene led to a lack of
oxidative phosphorylations (14). Recently cross-linking experi-
ments (15) have positioned this hydrophilic and acidic compo-
nent of 10,408 Da close to subunit 4 (subunit b), a component of
the second stalk of the ATP synthase. In this paper we report
the complementation of a yeast strain devoid of the yeast ATP
synthase subunit h by a single copy vector bearing a DNA
sequence encoding the bovine coupling factor 6. This is a rather
remarkable result because subunit h and bovine F6share only
14.5% sequence identity.
Yeast Strains and Nucleic Acid Techniques—The S. cerevisiae strain
D273–10B/A/H/U (MAT?, met6, ura3, his3) (16) was the wild type
strain. The strain with the null mutation in ATP14 (MAT?, met6, ura3,
his3, ATP14::URA3) has been described (14). The ?ATP14 strain con-
taining the plasmid pbF6, was obtained by transformation of the null
mutant in ATP14 gene by the nonintegrative single copy vector,
pRS313, which contains the coding region of mature bF6,1the leader
peptide of the ?-subunit of the yeast ATP synthase, and the upstream
and downstream transcriptional controlling elements of the ATP2 gene.
The expression plasmid for expression of bF6, pbF6, was made essen-
tially as described (17). In this scheme, the coding region of mature bF6
replaces the coding region of the gene encoding the ?-subunit of the ATP
synthase, ATP2. This allows the expression of bF6to be under the same
controls as that of the ATP2 gene. The coding region of bF6is amplified
by PCR, and this PCR fragment is used to directly replace the coding
region of the ATP2 gene. The replacement occurs by site-specific ho-
mologous recombination effected in yeast. In addition to the bases
required to direct the synthesis of the coding region of bF6, the PCR
* This work was supported by the Center National de la Recherche
Scientifique, the Ministe `re de la Recherche et de l’Enseignement Su-
pe ´rieur, the Universite ´ Victor Se ´galen, Bordeaux 2, and the Etablisse-
ment Public Re ´gional d’Aquitaine and by National Institutes of Health
Grant GM44412 (to D. M. M.). The costs of publication of this article
were defrayed in part by the payment of page charges. This article must
therefore be hereby marked “advertisement” in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Institut de Bio-
chimie et Ge ´ne ´tique Cellulaires du CNRS, Universite ´ Victor Se ´galen,
Bordeaux 2, 1, rue Camille Saint Sae ¨ns, 33077 Bordeaux cedex France.
Tel.: 33-5-56-99-90-48; Fax: 33-5-56-99-90-51; E-mail: jean.
¶ Recipients of a research grant from the Ministe `re de la Recherche et
de la Technologie.
1The abbreviations used are: bF6, bovine coupling factor 6; CCCP,
carbonyl cyanide m-chlorophenylhydrazone; DCCD, dicyclohexylcarbo-
diimide; DSP, dithiobis[succinimidylpropionate]; OSCP, oligomycin-
sensitivity-conferring protein; PCR, polymerase chain reaction.
THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 276, No. 11, Issue of March 16, pp. 8602–8607, 2001
Printed in U.S.A.
This paper is available on line at http://www.jbc.org
quence alignment of both subunits shows a very low sequence
identity of just 14.5% and, when allowing for amino acid re-
placements, a 54% similarity (Fig. 6). This low level of sequence
identity and homology is at the level seen between two random
peptides. In contrast, most of the remaining subunits of the
ATP synthase demonstrate a high degree of identity (31). For
instance, the ?-and ?-subunits of the yeast ATP synthase are
highly conserved with percent identities of about 72 and 75%,
respectively. Thus, it is surprising that bF6is able to replace
subunit h and form a functional enzyme, even now, knowing
that they are functional homologs.
The biochemical studies here demonstrate that the comple-
mentation by bF6is due to the direct replacement of subunit h
with bF6and not due to a secondary mechanism. The biochem-
ical studies indicate that bF6occupies that same spatial rela-
tionship in the yeast enzyme as subunit h. Cross-linking prod-
ucts involving subunit h and subunit 4, a component of the
second stalk, were obtained from positions K98C (15) and
Q203C of the latter subunit,2which are two positions located in
the hydrophilic part of subunit 4, thus suggesting that subunit
h also participates to form the second stalk or stator. Bovine F6
is a component of F0(11, 32), and it is associated with the stalk
as shown by reconstitution experiments (33). Nearest neigh-
bors relationships have been demonstrated, by cross-linking
experiments, between F6and the b-subunit (subunit 4) (34) and
bF6with the ?- and ?-subunits (35). The data in this study
indicate that subunit 4 is the major peptide cross-linked with
bF6using DSP within the yeast enzyme missing subunit h.
Thus, bF6and subunit h appear to occupy the same spatial
arrangement in the enzyme, and bF6can directly replace sub-
unit h in the yeast enzyme.
Despite the lack of primary structural similarity, subunit h
and bF6must share some structural features that define them
as functional homologs, and indeed, there are some features
that are conserved. First, they are both relatively small pep-
tides with calculated molecular masses of 10,408 and 8,958 Da
for subunit h and bF6, respectively. Second, both subunit h and
bF6are acidic proteins showing pI of 4.06 and 5.23, respec-
tively. Third, secondary structural computer analyses predict
two ?-helix regions for both subunits: the first ?-helix formed
by residues 6–23 for bF6and by residues 2–13 for subunit h
and the second ?-helix formed by residues 34–50 for bF6and
residues 47–64 for subunit h. Fourth, they both have an acidic
tail, although it is slightly longer in subunit h. Thus, these
conserved features may form some of the basis required for the
functional replacement of bF6for subunit h. Of course, beyond
these features, it is possible that despite low primary sequence
identity, that these peptides fold into similar three-dimen-
The partial complementation of the null mutant atp14 by the
bovine coupling factor 6 is more consistent with the fact that F6
or h are important for stabilizing the ATP synthase than for a
mechanistic role. Of all the F0components, bF6and subunit h
are the only acidic proteins. One possible hypothesis is that
these highly negative charged proteins help in the association
of other positively charged components of the second stalk,
such as subunits 4, d, and OSCP, three proteins with calculated
pIs of 7.83, 8.92, and 9.3, respectively.
Expressions of other subunits of the mammalian ATP syn-
thase have been demonstrated to complement the correspond-
ing null mutations in yeast. Expression of bovine ?-, ?-, ?-, and
?-subunits (17) and rat OSCP (36) have all complemented the
corresponding null mutant strains in yeast. However, although
some of these homologous peptides did not show a large amount
of identity, it was always high enough to suggest them as
homologs by simple primary structural analysis. Subunit h and
bF6are so divergent that even a one on one comparison of their
primary structure provided no clue that they were indeed ho-
mologs. The results of this study are even more startling be-
cause these peptides are not the sole peptide in an enzyme
complex, but must interact within a heterosubunit multimeric
enzyme complex. The implications of this are significant be-
cause they indicate that primary structural analysis cannot be
used as the sole evidence that functional peptide homologs do
not exist between two species. This is true even when the
peptide is within a multimeric peptide complex that otherwise
might be highly conserved.
Acknowledgments—We thank Dr. D. Bre `thes and Dr. P. V. Graves
for helpful discussions and for critical reading this manuscript.
1. Pedersen, P. L. (1996) J. Bioenerg. Biomembr. 28, 389–395
2. Weber, J., and Senior, A. E. (1997) Biochim. Biophys. Acta 1319, 19–58
3. Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature
4. Duncan, T. M., Bulygin, V. V., Zhou, Y., Hutcheon, M. L., and Cross, R. L.
(1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10964–10968
5. Noji, H., Yasuda, R., Yoshida, M., and Kinosita, K. (1997) Nature 386, 299–302
6. Boyer, P. D. (1993) Biochim. Biophys. Acta 1140, 215–250
7. Aggeler, R., Ogilvie, I., and Capaldi, R. A. (1997) J. Biol. Chem. 272,
8. Zhou, Y., Duncan, T. M., and Cross, R. L. (1997) Proc. Natl. Acad. Sci. U. S. A.
9. Sambongi, Y., Iko, Y., Tanabe, M., Omote, H., Iwamoto-Kihara, A., Ueda, I.,
Yanagida, T., Wada, Y., and Futai, M. (1999) Science 286, 1722–1724
10. Panke, O., Gumbiowski, K., Junge, W., and Engelbrecht, S. (2000) FEBS Lett.
11. Collinson, I. R., Runswick, M. J., Buchana, S. K., Fearnley, I. M., Skehel, J. M.,
van Raaij, M. J., Griffiths, D. E., and Walker, J. E. (1994) Biochemistry 33,
12. Velours, J., Paumard, P., Soubannier, V., Spannagel, C., Vaillier, J., Arselin,
G., and Graves, P. V. (2000) Biochim. Biophys. Acta 1458, 443–456
13. Stock, D., Leslie, A. G. W., and Walker, J. E. (1999) Science 286, 1700–1705
14. Arselin, G., Vaillier, J., Graves, P. V., and Velours, J. (1996) J. Biol. Chem.
15. Soubannier, V., Rusconi, F., Vaillier, J., Arselin, G., Chaingnepain, S., Graves,
P. V. Schmitter, J. M., Zhang, J. L., Mueller, D., and Velours, J. (1999)
Biochemistry 38, 15017–15024
16. Paul, M. F., Gue ´rin, B., and Velours, J. (1992) Eur. J. Biochem. 205, 163–172
17. Lai-Zhang, J., and Mueller, D. M. (2000) Eur. J. Biochem. 267, 2409–2418
18. Sikorski, R. S., and Hieter, P. (1989) Genetics 122, 19–27
19. Arselin de Chateaubodeau, G., Gue ´rin, M., and Gue ´rin, B. (1976) Biochimie
(Paris) 58, 601–610
20. Gue ´rin, B., Labbe, P., and Somlo, M. (1979) Methods Enzymol. 55, 149–159
21. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol.
Chem. 193, 265–275
22. Rigoulet, M., and Gue ´rin, B. (1979) FEBS Lett. 102, 18–22
23. Emaus, R. K., Grunwald, R., and Lemasters, J. J. (1986) Biochim. Biophys.
Acta 850, 436–448
24. Somlo, M. (1968) Eur. J. Biochem. 5, 276–284
25. Scha ¨gger, H., and Von Jagow, G. (1987) Anal. Biochem. 166, 368–379
26. Risler, J. L., Delorme, M. O., Delacroix, H., and Henaut, A. (1988) J. Mol. Biol.
27. Fessenden-Raden, J. M. (1972) J. Biol. Chem. 247, 2351–2357
28. Joshi, S., and Pringle, M. J. (1989) J. Biol. Chem. 264, 15548–15551
29. Sandri, G., Wojtczak, L., and Ernster, L. (1985) Arch. Biochem. Biophys. 239,
30. Ko, Y. H., Hullihen, J., Hong, S., and Pedersen, P. L. (2000) J. Biol. Chem. 275,
31. Walker, J. E., Fearnley, I. M., Gay, N. J., Gibson, B. W., Northrop, F. D.,
Powell, S. J., Runswick, M. J., Saraste, M., and Tybulewicz, V. L. (1985) J.
Mol. Biol. 184, 677–701
32. Collinson, I. R., Skehel, J. M., Fearnley, I. M., Runswick, M. J., and Walker,
J. E. (1996) Biochemistry 35, 12640–12646
33. Collinson, I. R., van Raaij, M. J., Runswick, M. J., Fearnley, I. M., Skehel,
J. M., Orriss, G. L., Miroux, B., and Walker, J. E. (1994) J. Mol. Biol. 242,
34. Joshi, S., and Burrows, R. (1990) J. Biol. Chem. 265, 14518–14525
35. Belogrudov, G. L., Tomich, J. M., and Hatefi, Y. (1995) J. Biol. Chem. 270,
36. Prescott, M., Higuti, T., Nagley, P., and Devenish, R. J. (1995) Biochem.
Biophys. Res. Commun. 207, 943–949
2V. Soubannier and J. Velours, unpublished observation.
Coupling Factor 6 of the Yeast ATP Synthase