I04
BIOCHEMICAL SOCIETY TRANSACTIONS
Structure and genes of ATP synthase
JOHN E. WALKER, ALISON L. COZENS,
MARK R. DYER, IAN M . FEARNLEY,
STEPHEN J. POWELL and MICHAEL J. RUNSWICK
M . R. c‘. Luhorutory of Molecular Biology, Hills Road,
Cumbridge C B 2 2 Q H , U . K .
Recent studies in a cyanobacterium (Syncchoc0ci~u.s6301)
show that the genes for its ATP synthase are grouped
in two clusters, probably operons (Fig. 1) (Cozens &
Walker, 1982). The clusters contain genes that code for
homologues of all eight of the subunits of the E. coli
enzyme. They also suggest an increased complexity in the
ATP synthase (proton-translocating ATPase, H + -ATPase, cyanobacterial enzyme, since one of the gene clusters conF, F, ATPase) is a component of the cytoplasmic membrane tains two related genes which encode proteins with similar
of eubacteria, the thylakoid membrane of chloroplasts and structures to the E. c d i F,, component, subunit b. This
the inner membrane of mitochondria. The Escherichia coli implies that, rather than assembling two identical b subunits
enzyme the simplest so far defined, is a complex of eight as in E. coli, the cyanobacterial enzyme may contain one
different polypeptides; five of them, M , fi, 7, 6 and E , con- subunit per complex of each of the non-identical homologues
stitute the extrinsic membrane F, domain of the enzyme, b and b’. The E c d i b subunit has a characteristic structure
and the remainder, a, b and c, make up the membrane with an N-terminal hydrophobic domain which forms a
sector, F,,. Their stoichiometries are cc,&y, E , a , bzc,(k,z transmembrane segment, probably a-helical. The remainder
(Fillingame, 198 I ) . Gene and protein studies, described of the protein is hydrophilic and contains many charged
below, of the chloroplast enzyme and the related complex in amino acids; it lies outside the lipid bilayer and provides an
the cyanobacterium Synechococcus 6301, have shown that important structural link between the extramembranous F,
homologues of these eight bacterial proteins are present as sector and the intrinsic membrane sector, F,, (Walker rt a/.,
they are also in the bovine mitochondria1 enzyme. The latter 19826).
The cyanobacterial homologues b and b’ differ from E.
is considerably more complex and has at least five supernumerary protein components. Thus, these experiments c d i b in so far as their N-terminal hydrophobic domains are
establish a structural unity for the enzymes in eubacteria, considerably longer. This is reminiscent of the equivalent
chloroplasts and mitochondria which itself suggests that the subunit in chloroplast ATP synthase, subunit I . Here it has
enzymes function in a basically similar manner.
also been shown by sequence studies of its gene to have an
extra N-terminal hydrophobic extension relative to the E .
Euhuctcuial A T P synthuses
coli subunit (Bird rt a/., 1985). Sequence analysis of the gene
Much of the information concerning the structure of the product isolated from the chloroplast enzyme complex has
E. coli enzyme has been derived by a combination of protein demonstrated that the extra extension is removed in a postsequence studies of the subunits (Walker et al., 1 9 8 2 ~ ) translational processing event, although the functional
and of DNA sequence studies of the corresponding genes significance of this finding is unknown. However, it suggests
(reviewed by Walker et ul., 1984). In E. coli the genes are that the cyanobacterial homologues will be similarly proco-transcribed from the unc or utp operon (Gibson, 1982). cessed. One possibility is that the extra domain serves to
The operon contains two sub-clusters of genes, one of three direct the pre-protein to the thylakoid membrane in the
genes for F,, subunits (promoter proximal) followed by a chloroplast or to the specialized photosynthetic membrane
second of five genes for F, components (see Fig. I). In two of the cyanobacterium, although it should be emphasized
members of the Rhodospirillnceae or purple non-sulphur that this process is not thought to involve transfer of the
bacteria, R1iodopsc.uclomonu.s hlastica and Rhodospirillum complete protein across a membrane.
ruhrum, the five genes for F, components form an operon
divorced from F,, genes (Tybulewicz et a/., 1984; Falk et a/. The chloroplast enzyme
Gel electrophoresis in the presence of urea of ATP synthase
1985). However, the gene order for F, genes is maintained
isolated from spinach and bean chloroplasts indicates that
(Fig. I ) .
the chloroplast enzyme is slightly more complicated than the
Abbreviations used: oscp, oligomycin-sensitivity conferral protein;
E. coli enzyme, being a complex of nine different polyDCCD. dicyclohexylcarbodi-imide.
peptides (Pick & Racker, 1979; Westhoff ct d.,1985; Suss,
1987
619th M E E T I N G . C A M B R I D G E
Table 1 . ('ornptrrison niotricvs ,/iw r . /j und c subunits o/' A TP .s~~ntlirrses
References: (i) Walker cJt 01. (1985), Isaac e t ul. (1985); (ii) Walker 1'1 a / . (1985). Boutry
& Chua (1985); (iii) Sebald & Hoppe (1981), Dewey e t a / . (1985).
( i ) z-subunits
( I ) Wheat chloroplast
( 2 ) . ~ l ~ r l c ~ c ~ / r o c . o c . ~630
~ r i . I\
( 3 ) E. c d i
(4) Rsp. rrrhrimr
( 5 ) Rps. h/us/icu
(6) Bovine mitochondrion
( 7 ) Maiie mitochondrion
( i i ) /]-subunits
( I ) Tobacco chloroplast
( 2 ) ~ ~ . , l c , c ~ / r o t ~ o c630
~ [ ~ II i . v
( 3 ) E. c , o / r
(4) Rsp. rrrhrunr
( 5 ) R p . h/trs/ic~tr
(6) Bovine mitochondrion
( 7 ) Tobacco mitochondrion
(111)
I00
71
59
57
57
57
(1)
100
53
61
59
59
58
(2)
I00
75
71
70
(4)
I00
79
62
64
64
66
67
(1)
I00
63
65
65
63
64
(3)
100
72
72
71
(4)
52
I00
(7)
I00
(7)
c-subunits
( I ) Wheat chloroplast
I00
( 3 ) . s , 1 . , l c ~ ~ / r , ~630
~~~
I ~ ~ ~ ~ ~ r r88
.\
40
( 3 ) Bwi//u.s ucidoc~u/cluriu.s
(4) PS3
36
( 5 ) E. d
i
29
( 6 ) Rsp. rrihrrini
34
(7) Yeast mitochondrion
28
( X ) Bovine mitochondrion
33
( 9 ) Tobacco mi t ochondrion
20
(1)
-
I00
40
41
33
39
73
29
20
(2)
;'.
Mitochondria1 A TP s.twtliuse
Sequence analysis of the five constituent polypeptides of
F, -ATPase from bovine heart mitochondria has shown that
the M . /I. and 1' subunits are homologous with bacterial
homonyms, and that (confusingly) the bovine 6 and E subunits are not (Walker et al., 1985). Rather, bovine 6 is
equivalent to bacterial c and bovine I: has n o bacterial (or
Vol. 15
40
25
25
2X
23
(4)
~
1986). Five of them can be identified as the five components
of coupling factor CF, r , /I.
(5 and I : . and are equivalent
to their bacterial homonyms. Two further components are
known to be the homologues of bacterial subunits b (known
as subunit I in choloroplasts; Bird ct u/:, 1985) and c
(chloroplast subunit 111). An additional component with a
molecular mass of 20 kDa, known as subunit X, has been
proposed to be the equivalent of bacterial subunit a (Cozens
et a/.. 1986). The ninth component, subunit I I . has a molecular mass of I5 kDa and is likely to be the equivalent of
the Synechococcus subunit. b' (athough this suggestion lacks
protein chemical support a t present).
Indeed. the body of available evidence suggests a rather
close similarity between chloroplast and cyanobacterial
A T P synthases. This is illustrated, for example, by binary
comparisons of sequences of a, /I and c subunits from
eubacteria, mitochondria and chloroplasts. This clearly demonstrates that the cyanobacterial and chloroplast subunits
are more closely related to each other than to homologues
from other sources (Table I ) . Their close evolutionary
relationship is further emphasized by the similarity of the
arrangement of the six genes for A T P synthase subunits that
are found in chloroplast D N A with the two clusters in the
genome of Syncdiococcus 630 I (Fig. 1 ). The three remaining
subunits, 7 , 6 and X, are encoded in nuclear genes that have
not been characterized.
.
100
chloroplast) counterpart (Walker c't u/., 1982~).The equivalent subunit of bacterial 6 is another subunit of bovine
ATP-synthase, the oscp (oligomycin-sensitivity conferral
protein), which is not released from the membrane complex
as a component of the F, -ATPase. It and a second protein,
F,, are required for correct binding of F, to the membrane
sector. Thus, oscp appears to play a functionally similar role
to bacterial 6, as the homology would imply.
Homologues o f bacterial F, subunits a, b and c have also
been characterized in the bovine mitochondrial enzyme.
Two of them, ATPase-6 (equivalent to bacterial a ) and
bovine c [the dicyclohexylcarbodi-imide (DCCD)-reactive
proteolipid; Sebald & Hoppe, 19811. are soluble in mixtures
of chloroform and methanol and can be extracted with the
organic solvent from both mitochondria and the purified
A T P synthase complex (Fearnley & Walker, 1986). Subunit
b has the characteristic hydrophobic N-terminal domain
( J . E. Walker & M . J . Runswick, unpublished work). T w o
additional components have been isolated and sequenced
from the membrane sector of mitochondrial A T P synthase.
The larger, M , 19000, called subunit d is not particularly
hydrophobic but remains with the membrane sector when
F, is released (J. E. Walker & M . J. Runswick, unpublished
work). The smaller, known as A6L, is the third component
of the enzyme complex to be isolated from the chloroform/
methanol extract of mitochondria (Fearnley & Walker,
1986). Its function is unknown, but it may be analogous
(although it is not significantly homologous) t o the aapl
protein characterized from yeast mitochondrial A T P synthase (Velours et ul., 1984). The fungal protein is required
for correct assembly of the enzyme complex (Macreadie
et al., 1983). The final component to have been characterized from the bovine A T P synthase is the inhibitor
protein. a small basic protein that binds to the F, sector
I06
(Pullman & Monroy, 1963). It may be important in the
physiological regulation of the enzyme (Ernster et ul., 1979;
Pedersen et ul., 1981).
As in the chloroplast, genes for mitochondrial ATP
synthase subunits are found both in the nucleus and in the
organellar DNA. In mammals and invertebrates two of
them, A6L and ATPase-6, are encoded by overlapping
genes in mitochondrial DNA (Fearnley & Walker, 1986),
and the remainder are nuclear gene products. A similar
arrangement persists in fungi where both ATPase-6 and
uupl are mitochondrial gene products. The notable exception
to this pattern is yeast in which subunit c (the DCCD-reactive
proteolipid) is a mitochondrial gene product (Macino &
Tzagoloff, 1979) in addition to ATPase-6 and uupl. In all
other species studied it is encoded in the nucleus (summarized) by Walker & Tybulewicz, 1985).
cDNAs corresponding to 10 of the 11 nuclear-encoded
subunits of the bovine ATP synthase have been characterized (the exception is the &subunit). These c-DNAs have
been isolated from a bovine cDNA library (Gay & Walker,
1 9 8 5 ~ using
)
mixed synthetic oligonucleotides 17 bases in
length, designed on the basis of known protein sequences in
the subunits (J. E. Walker, N. J . Gay, S. J. Powell & M. J.
Runswick, unpublished work).
A surprising finding from these studies was that the
DCCD-reactive proteolipid has two different cDNAs. They
encode exactly the same mature proteolipid but differ in
silent positions of codons and in the 3' non-coding regions.
They differ also in their N-terminal precursor sequences that
serve to direct the proteins from the cytoplasm to the
mitochondrion and that are removed from the precursors in
the process (Gay & Walker, 19856). The two genes, named
PI and P2, appear to be expressed differently in various
bovine tissues.
So far no evidence has been obtained that other ATP
synthase genes are similarly duplicated. However, recently
another bovine mitochondrial membrane component, the
ADP/ATP translocase, has been shown to have two cDNAs
that encode very similar but slightly different proteins.
These also are expressed in a tissue-specific manner
(S. J . Powell & J . E. Walker, unpublished work).
The cDNAs for ATP synthase subunits are being used in
turn to isolate the corresponding genes from a genomic
library. In this way, for example, the gene for the precursor
of the y-subunit has been isolated and shown to be divided
into at least nine exons distributed over about 15 kilobases
of DNA (M. R. Dyer & J. E. Walker, unpublished work).
BIOCHEMICAL SOCIETY TRANSACTIONS
Bird. C. R., Koller. B., Auffret. A. D.. Huttly. A. K., Howe. C. J..
Dyer, T. A. & Gray. J. C. (1985) EMBO J . 4. 1381 1388
Boutry. M . & Chua. N.-H. (1985) EMBO J . 4, 2159 2166
Cozens, A. L. & Walker, J. E. (1987) J. Mol. Biol. in the press
Cozens, A. L.. Walker. J. E.. Phillips. A. L.. Huttly, A. K . &
Gray. J. C. (1986) E M B O J . 5. 217 222
Dewey. R. E.. Schuster. A. M., Lering. C. S., 111 & Timothy, D. H.
(1985) Proc. Nu//. A(,ud. Sci. U . S . A . 82, 1015 1019
Ernster, L.. Carlsson. C.. Hundal. T. & Nordenbrand. K. (1979)
Merhvds Enzymol. 55, 399 407
Falk, G., Hampe, A. & Walker, J. E. (1985) Biochcm. J . 228. 391 407
Fearnley. 1. M. & Walker, J. E. (1986) E M B O J . 5. 2003 2008
Fillingame, R. H . (1981) C u r r . Top. Biomerg. 11, 35 106
Gay. N. J. & Walker. J. E. (1981) Nuclc+ Acids Res. 9. 3919 3926
Gay. N. J. & Walker. J. E. ( 1 9 8 5 ~ )Biochcwi. J . 225. 707 712
Gay. N. J. & Walker. J. E. (198%) EMBO J. 4. 3519-3524
Gibson, F. (1982) Pro(.. R. Sot. London Scv. B 215, 1-18
Isaac. P. G.. Brennicke. A.. Dunbar. S. M. & Leaver. C. J. (1985)
Curr. Gcnet. 10. 321 328
Macino. G . & Tzagoloff, A. (1979) J. BIol. Chm. 245, 4617 4623
Macreadie. I. G.. Novitski. C. E.. Maxwell, R. J.. John. U.. Ooi.
B. G.. McMullen, G . L.. Lukins. H. B., Linnane. A. W. & Nagley, P. (1983) Nuclc,ic, A d s Rcs. 11. 4435-3351
Pedersen. P. L.. Schwerzmann. K. & Cintron, N. (1981) Curr. Top.
Bioenerg. 1 I. 149 199
Pick, U. & Racker, E. (1979) J. B i d . Chmi. 254, 2793 2799
Pullman, E. & Monroy, G . C. (1963) J . Biol. Chetri. 238, 3762 3769
Sebald, W. & Hoppe. J. (1981) Curr. Top. Bioenerg. 12, 2 64
Suss, K. H. (1986) FEBS Lc,//.201. 65 68
Tybulewicz, V. L. J.. Falk. G . & Walker, J. E. (1984)J. Mol. B i d . 179.
185-214
Velours, J.. ESpdrTd. M.. Hoppe. J., Sebald, W. & Guerin, B. (1984)
EMBO J . 3, 207-2 I2
Walker. J. E. & Tybulewicz, V. L. J . (1985) in The Molecular Biology
cfthcJ Pho/o.sjvrhr/ic Appurutus (Arntzen. C.. Bogorad. L.. Bonitz. S.
& Steinback. K.. eds.), pp. 141 153, Cold Spring Harbor Laboratory, Cold Spring Harbor. NY
Walker, J. E.. Auffret. A. D.. Carnc. A,. Gurnett. A.. Hanisch. P..
Hill. D. & Saraste. M. ( 1 9 8 2 ~Eur.
) J. Biochcvn. 123, 253 260
Walker. J. E.. Saraste. M. & Gay. N. J. (1982h) Nuturc, (London) 298.
867-869
Walker, J. E., Runswick, M. J. & Saraste. M. (19824 FEBS Lctr. 146.
393-396
Walker, J. E.. Saraste, M . & Gay. N. J. (1984) Biochim. f3iophy.s. Aciu
768, 164-200
Walker, J . E.. Fearnley, I . M., Gay, N. J.. Gibson. B. W.. Northrop,
F. D.. Powell, S. J., Runswick, M. J.. Saraste. M. & Tybulewicz.
V. L. J. (1985) J . MfJ/.B b l . 184, 677 701
Westhoff, P.. All, J., Nelson. N. & Herrman, R. G . (1985) Mol. Gcn.
Gene/. 199. 290 299
Received 27 August 1986
1987