(Received for publication, October 11, 1994; and in revised form, November 21, 1994)
From the
The catalytic sector, F, and the membrane sector,
F
, of the mitochondrial ATP synthase complex are joined
together by a 45-Å-long stalk. Knowledge of the composition and
structure of the stalk is crucial to investigating the mechanism of
conformational energy transfer between F
and F
.
This paper reports on the near neighbor relationships of the stalk
subunits with one another and with the subunits of F
and
F
, as revealed by cross-linking experiments. The
preparations subjected to cross-linking were bovine heart
submitochondrial particles (SMP) and F
-deficient SMP. The
cross-linkers were three reagents of different chemical specificities
and different lengths of cross-linking from zero to 10 Å.
Cross-linked products were identified after gel electrophoresis of the
particles and immunoblotting with subunit-specific antibodies to the
individual subunits
,
,
,
, OSCP, F
,
A6L, a (subunit 6), b, c, and d.
The results suggested that the two b subunits form the
principal stem of the stalk to which OSCP, d, and F
are bound independent of one another. Subunits b, OSCP, d, and F
cross-linked to
and/or
, but
not to
or
. The COOH-terminal half of A6L, which is
extramembranous, cross-linked to d but not to any other stalk
or F
subunit. No cross-links of subunits a and c with any stalk or F
subunits were detected. In
F
-deficient SMP, cross-linked b + b and d + F
dimers appeared, and the
extent of cross-linking between b and OSCP diminished greatly.
The addition of F
to F
-deficient particles
appeared to reverse these changes. Treatment of F
-deficient
particles with trypsin rapidly hydrolyzed away OSCP and F
,
fragmented b to membrane-bound 18-, 12-, and 8-9-kDa
antigenic fragments, which cross-linked to d and/or with one
another. Trypsin also removed the COOH-terminal part of A6L, but the
remainder still cross-linked to subunit d. Models showing the
near neighbor relationships of the stalk subunits with one another and
with the
and
subunits at a level near the proximal end
(bottom) of F
and at the membranematrix interface are
presented.
The bovine heart mitochondrial ATP synthase complex isolated in
our laboratory contains 13 well characterized subunits (Galante et
al., 1979; Hekman et al., 1991), exhibits completely
oligomycin-sensitive ATPase and ATP-P
exchange
activities (Stiggall et al., 1978; Galante et al.,
1979), and can drive via ATP hydrolysis a second mitochondrial proton
pump when coincorporated in liposomes (Eytan et al., 1990).
The polypeptide components of the ATP synthase are the
,
,
,
, and
subunits of the catalytic sector F
,
plus OSCP, a (subunit 6), b, c, d,
F
, and A6L, which together make up the stalk and the
membrane sector F
. Preparations of the ATP synthase complex
also contain substoichiometric amounts of the ATPase inhibitor protein,
IF
. The stoichiometry of F
subunits is
, and the
stoichiometry of OSCP, d, A6L, b, and F
,
as determined by radioimmunochemical techniques in well coupled SMP (
)(Hekman et al., 1991), is 1:1:1:2:2. Bovine ATP
synthase also contains multiple copies of subunit c (Graf and
Sebald, 1978; Sebald et al., 1979; Kiehl and Hatefi, 1980),
but the precise number remains to be determined. In intact
mitochondria, there is 1 mol of IF
/mol of F
(Hekman et al., 1991). By comparison, the Escherichia coli ATP synthase is composed of only eight
subunits, with the F
-F
stoichiometry
-ab
c
(Fillingame, 1990). Mitochondrial subunits
,
, and
have considerable sequence similarity to their E. coli counterparts. Mitochondrial OSCP and
are analogous to E.
coli
and
, respectively, and, largely on the basis of
hydropathy profiles, mitochondrial subunits a, b, and c are considered analogous to the E. coli a, b, and c, respectively (Fillingame, 1990).
Bovine
and the rat F have been crystallized, and quaternary
structures, respectively, at 6.5 and 3.6 Å resolution have been
reported (Abrahams et al., 1993; Bianchet et al.,
1991). More recently, the structure of bovine F
containing
four molecules of AppNP, and one molecule of ADP has been published,
showing at 2.8 Å resolution the three-dimensional structures of
the
and
subunits plus about 50% of the
(Abrahams et al., 1994). However, structural information regarding the
remainder of the ATP synthase complex is limited. Subunit c is
considered to be shaped like a hairpin, with the hydrophobic arms
forming
-helices that traverse the membrane and the hydrophilic
hairpin bend protruding from the membrane on the F
side.
Recent two-dimensional NMR data for the isolated E. coli subunit c in a chloroform-methanol-water solvent are in
agreement with the hairpin model (Girvin and Fillingame, 1993, 1994).
Small-angle neutron scattering studies of bovine OSCP (molecular weight
20,967) have suggested an elongated shape of approximately 90
30
30 Å, with 43%
-helical structure as calculated
from circular dichroism measurements (Dupuis et al., 1983).
Other than hydropathy profiles, information regarding the structures of
other ATP synthase subunits is not available.
Because OSCP can be
readily and reversibly removed from SMP together with F (Steinmeier and Wang, 1979; Matsuno-Yagi and Hatefi, 1984), it is
considered to be a stalk component required for proper binding of
F
to F
. In addition, our previous data, based
on accessibility of ATP synthase subunits in SMP and mitoplasts
(mitochondria denuded of outer membrane) to proteases and
subunit-specific antibodies, indicated that OSCP, b, d, F
, and the COOH-terminal half of A6L were
accessible from the matrix but not from the cytosolic side of the inner
membrane, whereas subunits a and c were not
accessible to the above reagents from either side of the mitochondrial
inner membrane (Hekman et al., 1991). This paper presents the
results of cross-linking experiments involving the ATP synthase
subunits in SMP. The data are consistent with the results of our
earlier topography studies described above and provide information
regarding the near neighbor relationships of OSCP, b, d, F
, and A6L, which appear to contribute to the
formation of the stalk of the bovine ATP synthase complex.
For cross-linkings involving sulfhydryl groups, the photoactivable heterobifunctional MBP was used. MBP dissolved in dimethylformamide was added at 1 mM to SMP at l mg/ml in 20 mM HEPES, 0.25 M sucrose, pH 7.2, and the mixture incubated for 25 min at 20 °C in dim light. Unreacted MBP was either removed by centrifugation or first quenched by the addition of 10 mM dithiothreitol and then removed by centrifugation. After resuspension in HEPES/sucrose buffer, the particles were irradiated with an 8-watt longwave UV lamp at a distance of 5 cm for 10 min on ice, with occasional mixing. The particles were then prepared for gel electrophoresis by the addition of an equal volume of SDS-PAGE buffer as indicated above. For control, particles were preincubated with 5 mMN-ethylmaleimide for 20 min before the addition of MBP and UV irradiation. Under these conditions, no cross-linking was observed. Furthermore, SMP or ASU particles were treated with MBP and then subjected to UV irradiation at 1.0 as well as 0.1 mg/ml and analyzed for cross-linked products by SDS-PAGE and immunoblotting. There was no evidence of additional cross-linking at the higher particle concentration, but at 0.1 mg of particle protein/ml there was a higher yield of the cross-linked products, consistent with the less shielding effect of the particles against UV irradiation at the lower protein concentration.
The IgG
fractions used for immunoblotting were affinity purified according to
Olmsted(1981), using as a source of antigen homogeneous subunits or
subunits transferred to nitrocellulose membranes after SDS-PAGE of
F or purified ATP synthase complex. The affinity-purified
antibodies were stored in small aliquots at -20 °C.
Representative of the
cross-linking pattern observed with the reagents mentioned above are
the results shown in Fig. 1, where DST was the cross-linking
reagent. Fig. 1A exhibits immunoblots of a 15% SDS gel,
which favors transfer to nitrocellulose molecular mass species <80
kDa, and Fig. 1B shows immunoblots of a 10% SDS gel,
which contains cross-linked products of higher molecular mass. The top of each lane states the antibody with which that
nitrocellulose strip was blotted. In these and subsequent figures, the
fastest moving (lowest) protein band is the uncross-linked subunit,
except that in Fig. 1B all of the uncross-linked
F and parts of uncross-linked OSCP and d ran out
of the gel. It might also be noted that there are no immunoreactive
protein bands below uncross-linked b, OSCP, and d in Fig. 1A and below
and
in Fig. 1B. This indicates the absence of immunoreactive
fragments (e.g. due to proteolysis) of these subunits in the
SMP preparations used, which is an important consideration in the
identification of cross-linked products.
Figure 1: Cross-linking of SMP with DST. SMP at 1 mg/ml were incubated with 1 mM DST for 30 min at 20 °C. After termination of the reaction, samples were subjected to a 15% (panel A) and a 10% (panel B) SDS-PAGE, and proteins were electrotransferred to nitrocellulose membranes. Strips of nitrocellulose containing identical cross-linked products were blotted with the affinity-purified antibodies indicated on the top of each lane. The immunoreactive bands were visualized with the Enhanced Chemiluminescence detection system. For details, see ``Experimental Procedures.''
In Fig. 1A,
the cross-linked products of b + OSCP, b +
F, and d + A6L are marked. Also marked are
two species of b + d, which are better separated
in Fig. 1B. These could be the result of two types of
cross-linking between d and the same molecule of b or
of different types of cross-linking between d and the two
molecules of b. The unlabeled top bands in Fig. 1A are cross-linked products of each subunit with
/
, which are also clearer in Fig. 1B. These
species appear to be
+
,
+ b,
+ OSCP,
+ OSCP,
+ b,
+ d, and
+ F
. The unmarked cross-linked
bands seen in the upper parts of the lanes in Fig. 1B are presumably cross-linked trimers and more
complex products. Essentially a similar cross-linking pattern was
obtained when SMP were treated with EDC in the presence of NHS. The
product of d + A6L was particularly favored under the
latter conditions, and the major cross-linked products between the
stalk and F
subunits were
+ b,
+ OSCP,
+ F
, and
+ d (data not shown). Although not shown in Fig. 1, SMP
cross-linked with either DST or EDC + NHS and immunoblotted to
anti-a or anti-c antibodies showed no cross-linked
products involving these subunits.
A close scrutiny of Fig. 1, A and B, suggests the absence of
cross-linked products involving the stalk subunits and the small
subunits of F. For example, one would expect the
cross-linked products of the
subunit of F
with b, OSCP, and d to produce a protein band
immunoreactive to anti-b, anti-OSCP, or anti-d in the M
region of 50-60 kDa. However, as is clear
in Fig. 1B, this region of these lanes is
devoid of any such protein band. To investigate this issue further, SMP
and purified F
-ATPase were separately treated with DST and
subjected to SDS-gel electrophoresis and immunoblotting with
affinity-purified antibodies to the individual F
subunits
,
,
, and
. The results are shown in Fig. 2.
The paired lanes compare the cross-linked products of the
F
subunits as marked in the figure in SMP and isolated
F
-ATPase. In the first and thirdlanes from the left, the bands not seen in the second and fourth lanes are the cross-linked
products, respectively, of
and
with the stalk subunits (see Fig. 1B). Other bands common in the first four
lanes are the cross-linked products of
+
and
+
plus one or another small subunit of F
.
The important message of Fig. 2is contained, however, in the last four lanes. It is seen that in SMP or isolated F
the cross-linked products of
and
are essentially the
same in each case, even though the yields of the cross-linked products
of
appear to be less in SMP (perhaps due to shielding of
)
than in isolated F
. These results suggest, therefore, that
in SMP
and
do not cross-link to any stalk subunit in the
presence of DST as the cross-linking agent. Similar results were
obtained when EDC + NHS were used. However, since
and
produced a number of cross-linked products common to SMP and isolated
F
, it is clear that they are accessible to cross-linkers in
SMP. The question, therefore, is how
and
are located in SMP
in relation to b, OSCP, d, F
, and A6L.
Figure 2:
Comparison of the cross-linking patterns
of F subunits in purified F
and SMP treated
with DST. F
(0.2 mg/ml) and SMP (1 mg/ml) were cross-linked
with DST as in Fig. 1. Samples of cross-linked SMP and F
were subjected to 15% SDS-PAGE in parallel, and proteins were
electrotransferred to nitrocellulose membrane and blotted with the
affinity-purified antibodies indicated on top of the paired lanes. Immunoblots were developed as indicated in Fig. 1. The positions of prestained molecular mass markers are
shown in kDa on the left of the
figure.
Figure 3: Cross-linking of ATP synthase subunits in SMP with the heterobifunctional photoactivable sulfhydryl reagent, MBP. SMP at 1 mg/ml were incubated with 1 mM MBP for 25 min at 20 °C. Particles were sedimented by centrifugation, resuspended in buffered sucrose solution, and UV irradiated for 10 min or kept on ice in the dark. Samples were subjected to 15% SDS-PAGE, and proteins were electrotransferred to nitrocellulose and probed with the affinity-purified antibodies indicated on the top of the lanes. Immunoreactive bands were visualized as in Fig. 1. Lanes 1, 4, and 7, sample of SMP UV irradiated for 10 min; lanes 2, 5, and 8, MBP-treated, nonirradiated SMP; lanes 3, 6, and 9, MBP-treated and UV-irradiated SMP. The positions of prestained molecular mass markers are shown in kDa on the right of the figure.
It was of interest to see whether the removal of
F from SMP might alter the cross-linking of stalk subunits.
F
-depleted ASU particles were prepared essentially
according to Racker and Horstman(1967). These preparations are depleted
with respect to F
and suffer partial loss of OSCP. The
addition of F
+ OSCP reconstitutes particles with high
ATP synthase activity (Matsuno-Yagi and Hatefi, 1984). The remainder of
OSCP can be removed by further alkaline extraction of ASU particles,
but this was not done here because our aim was to investigate the
effect of F
removal. When treated with MBP, ASU particles
produced two major cross-linked products among the cysteine-containing
subunits of the F
-depleted ATP synthase. One was b + F
as in Fig. 3, the other was a band at
about 44 kDa, whose mobility and singular reactivity with only
anti-b IgG suggested that it is a cross-linked b + b dimer (Fig. 4, lane 2). There
were also detectable amounts of b + OSCP (Fig. 4, lane 6). The addition to ASU particles of F
(2 and
6 mol/mol F
, respectively, in the third and fourth lanes of each set in Fig. 4) increased the yield
of b + OSCP dimer and diminished the yield of the
putative b + b dimer. There are two possible
explanations for these results: (i) removal of F
caused a
partial separation of b and OSCP so that b-MBP was
too distant from OSCP to cross-link upon photoactivation or (ii)
removal of F
resulted in masking of the thiol group of
OSCP, making it unable to form OSCP-MBP to cross-link with b upon photoirradiation. We prefer the first possibility because
structural perturbation of the stalk may also bring the two
MBP-modified b subunits closer to one another for
cross-linking after photoactivation of their benzophenones. As seen in Fig. 4, the cysteine-containing subunit d in ASU
particles (or in SMP) did not form any MBP-mediated cross-linked
products. As was shown previously, subunit d in SMP and ASU
particles is resistant to trypsinolysis. It is possible, therefore,
that much of subunit d, including its cysteine residue, is
shielded by other ATP synthase subunits in SMP and ASU particles.
Figure 4:
Cross-linking of F-deficient
ASU particles with MBP in the absence and presence of added
F
. ASU particles (0.5-1.0 mg/ml) were incubated with
1 mM MBP for 25 min at 20 °C, and excess reagent was
removed by centrifugation. MBP-modified particles were incubated
without added F
(lanes 1, 2, 5, 6, 9, and 10) or with 325 µg of
F
/mg of particles (lanes 3, 7, 11) or 1.0 mg of F
/mg of particles (lanes
4, 8, and 12), each for 30 min at 37 °C.
Particles were reisolated by centrifugation, washed, resuspended in 20
mM Tris-HCl, pH 7.5, containing 0.25 M sucrose and UV
irradiated for 10 min on ice. Samples were subjected to 15% SDS-PAGE,
and proteins were electrotransferred to nitrocellulose and blotted with
the affinity-purified antibodies indicated on the top of the lanes. Immunoreactive bands were visualized as in Fig. 1. Lanes 1, 5, and 9,
MBP-treated, nonirradiated ASU particles; other lanes,
MBP-treated ASU particles UV irradiated for 10 min on ice. The
positions of prestained molecular mass markers are shown in kDa on the right of the figure.
Figure 5:
Cross-linking of ATP synthase subunits in
F-deficient ASU particles treated with trypsin. ASU
particles (3.5 mg/ml) were incubated with trypsin (25:1 ratio by
weight, panel A, or 5:1 ratio by weight, panel B) for
1 h at 37 °C. Proteolysis was arrested by the addition of excess
soybean trypsin inhibitor plus 1 mM PMSF, and particles were
reisolated by centrifugation, washed, and cross-linked with 1 mM DST or 5 mM EDC in the presence of 5 mM NHS.
Samples were subjected to 15% SDS-PAGE, and proteins were
electrotransferred to nitrocellulose membrane and blotted with the
affinity-purified antibodies indicated on top of the lanes. Immunoblots were developed as described in Fig. 1. The tables of plus and minus signs on top of the immunoblots show the treatments the ASU particle of
each lane had received. Arrowheads are described
under ``Results.'' The designations b(18), b(12), and b(8) are for membrane-bound tryptic
fragments of subunit b with approximate M
values of 18, 12, and 8, respectively. The positions of
prestained molecular mass markers are shown in kDa on the left of panels A and B.
The effect of trypsin (1:25 weight ratio of trypsin to
particle protein) on subunits b and d agreed with the
results of others (Collinson et al., 1994a; Walker and
Collinson, 1994), which showed that b was hydrolyzed at
Arg-Gly
,
Lys
-Arg
, and
Arg
-His
, and d at
Lys
-Leu
(see Fig. 5A, lanes
2-4 and 8-10). Hydropathy analysis of subunit b shows two hydrophobic clusters of about 20 residues each
near the NH
terminus followed by a 130-residue-long
hydrophilic region to the COOH-terminal end. This suggests that the
molecule is anchored to the membrane via its two hydrophobic clusters
and that its short hydrophilic NH
terminus
30
residues) and its 130-residue-long hydrophilic tail are extramembranous
on the F
side. According to this picture, the tryptic
cleavages at Arg
-Gly
and at
Lys
-Arg
(or
Arg
-His
) would produce two membrane-bound
fragments, respectively, about 18-19 and 12-14 kDa. These b fragments, which still reacted with our polyclonal antibody
preparation, are seen in lanes 2-4 of Fig. 5A. They cross-linked in the presence of DST or
EDC + NHS with subunit d to produce the two bands marked
with arrowheads in lanes3, 4, 9, and 10 of Fig. 5A. It may also be
noted that the yield of the slower moving cross-linked product was
greater when DST was the cross-linking reagent, and the yield of the
faster moving cross-linked product was greater when EDC + NHS were
used for cross-linking. Other cross-linked products recognized and
marked by arrowheads in Fig. 5A are the b + b dimer in lane 6 and d +
F
in lane 11. We suspect that the band marked d + A6L, d + x is composed of more than one
cross-linked product of subunit d. However, additional
information is not available at this time.
The data in Fig. 5B are from an aliquot of ASU particles that was
treated with the higher concentration of trypsin (1:5 weight ratio of
trypsin to particle protein). It is seen in lanes 1 and 2 that subunit b was further degraded to produce an
8-9-kDa antigenic fragment, which was still membrane-bound.
Considering that there are about 70 amino acids from the NH terminus to the end of the second hydrophobic cluster of subunit b, the tryptic cleavage site to produce an 8-9-kDa
membrane-bound fragment must be very close to where the COOH-terminal
hydrophilic tail of b exits the membrane. If we assume that
the hydrophobic clusters of b are nonantigenic, then it is
likely that our anti-b IgG recognizes the 30-residue-long
NH
terminus of subunit b, which is probably
extramembranous. As seen in lane 3 of Fig. 5B,
this 8-9-kDa fragment disappeared in the presence of EDC +
NHS and produced the cross-linked products marked with arrowheads. The uppermost band marked with an arrowhead also appeared to form in lower yield when DST was
the cross-linking reagent (Fig. 5B, lane 2).
The proteolysis pattern of bovine b with trypsin as shown in Fig. 5(see also Collinson et al., 1994a; Walker and
Collinson, 1994) is similar to that of E. coli subunit b (Steffens et al., 1987), which is a further indication of
the analogy between them.
Considerable information is now available regarding the
mechanisms of ATP hydrolysis (Al-Shawi et al., 1990; Penefsky
and Cross, 1991; Boyer, 1993) and synthesis (Matsuno-Yagi and Hatefi,
1990; Hatefi, 1993) at the level of the catalytic sector,
F, of the ATP synthase complex. Also, the crystal structure
of a form of bovine heart F
containing four molecules of
AppNP and one molecule of ADP has been solved recently at 2.8 Å
resolution (Abrahams et al., 1994). In addition, it is known
that transmembrane protons pass only through the membrane sector,
F
, of the ATP synthase complex, and energy communication
between F
and F
takes place via coupled
conformation changes of subunits (for review, see Hatefi, 1993). At the
level of F
, protein-bound ATP is synthesized from
protein-bound ADP and P
without energy utilization, but the
binding of P
and ADP to F
and the release of
ATP from F
are the energy-requiring processes, which are
apparently accomplished by appropriate conformation changes of the
subunits. The crystal structure of bovine F
(Abrahams et al., 1994) and the cryoelectron microscopy studies of
Capaldi and co-workers Wilkins and Capaldi, 1994, and references
therein) on the E. coli F
suggest that subunits
and
are involved in conveying conformation changes to and
from F
. However, in both the bovine and E. coli ATP synthases there is a 45-Å-long stalk between F
and F
(Soper et al., 1979; Capaldi et
al., 1992; see also Dubachev et al., 1993), whose
structure and role in energy communication between F
and
F
are not known. If one assumes that the ATP synthase
subunits that are not components of F
but are entirely or
partly extramembranous (Hekman et al., 1991) contribute to the
composition of the stalk, then subunits b, OSCP, d,
F
, and A6L would qualify as candidates. As possible
components of the stalk, these subunits would be directly or indirectly
involved in conformational energy transfer between F
and
F
. Therefore, information regarding their relative
positions in the stalk and their spatial interaction with one another
as well as with F
and F
subunits would be
crucial to investigating the mechanism of energy transfer within the
ATP synthase complex.
As mentioned earlier, OSCP is easily removable
from SMP (Senior, 1979) or preparations of the ATP synthase complex
(Galante et al., 1981). Removal of OSCP also results in the
removal of F, but both can be added back to F
to reconstitute oligomycin-sensitive ATPase activity (Galante et al., 1981) or to depleted SMP to reconstitute oxidative
phosphorylation at high rates (Matsuno-Yagi and Hatefi, 1984). In
addition, it is known that OSCP binds to bovine F
(Hundal et al., 1983; Dupuis et al., 1985) and that the
F
-OSCP complex can bind subunits b, F
,
and d (Walker and Collinson, 1994). Evidence that F
can be reversibly extracted from SMP is also available
(Fessenden-Raden, 1972). These data are consistent with our earlier
studies showing that b, OSCP, d, and F
are accessible to subunit-specific antibodies in well coupled
SMP, but not in mitoplasts, and that in SMP subunits b, OSCP,
F
, and the COOH-terminal antigenic part of A6L could be
degraded by trypsin (Hekman et al., 1991). These studies also
indicated certain subunit accessibility differences between SMP and the
purified ATP synthase complex, which decided our choice of material for
determining the stoichiometry of subunits in F
and the
stalk (Hekman et al., 1991) as well as for our studies
reported here on the near neighbor relationships among the stalk
subunits and between the latter and the subunits of F
and
F
.
Table 1summarizes the results obtained in SMP
and F-depleted SMP with three cross-linking reagents (DST,
MBP, EDC) of different chemical reactivities and different effective
distances for cross-linking. Considering first the near neighbor
relationships of the stalk subunits, it is clear that OSCP cross-linked
only to b, and A6L only to d. The NH
terminus of A6L contains a short hydrophobic cluster (
20
residues), which appears to anchor A6L to the membrane. The remainder
of the molecule is hydrophilic and apparently extramembranous. Trypsin
cleaves off the COOH-terminal antigenic part of A6L (Hekman et
al., 1991), but the remainder of the molecule is still close
enough to subunit d to form a cross-linked product in the
presence of DST (Fig. 5, lane 9, bottom band marked with an arrowhead). This also suggests that
subunit d is present near the membrane. Indeed, the NH
terminus of d contains a short hydrophobic cluster of
amino acids, which may be intercalated into the membrane.
As seen in Table 1, all of the stalk subunits, except A6L, cross-link to b in SMP. As mentioned above, the hydropathy analysis of b shows two hydrophobic clusters of 20 residues each located
30 residues from the NH
terminus. Therefore, one might
expect that the two hydrophobic clusters intercalate into the membrane,
with the short NH
-terminal segment and the long (
130
residues) COOH-terminal portion beyond the second hydrophobic cluster
being extramembranous on the F
side (Walker and Collinson,
1994). Furthermore, our study of the stoichiometry of F
stalk subunits indicated that there are two molecules of b/ATP synthase (Hekman et al., 1991), which agreed
with the stoichiometry of the structurally analogous subunit b of the E. coli ATP synthase (Fillingame, 1990). In
further agreement with this stoichiometry, we found in the present
study two cross-linked species of b + d in the
presence of either DST or EDC, even when b was partially
degraded by trypsin to form membrane-bound fragments of about 18 and 12
kDa (Fig. 5, lanes 3, 4, 9, and 10). We also found in F
-depleted SMP, treated with
either DST or EDC, a band that reacted only with anti-b IgG
and exhibited on SDS gels a mobility consistent with that of a
cross-linked b + b dimer.
In addition to
cross-linking of b to OSCP and F effected by DST
or EDC, b + OSCP and b + F
dimers were also
produced when the cross-linking reagent was the heterobifunctional
photoactivable sulfhydryl reagent, MBP (Fig. 3). Because F
is devoid of cysteine residues, the cross-link between b and F
must involve Cys
of b near its COOH terminus. The same might be true in the case of the
MBP-induced cross-link between b and OSCP, or the cysteine in
this case might be Cys
of OSCP. However, the fact that
the b-MBP adduct did not cross-link upon photoactivation to
,
,
, or
may mean that the COOH-terminal portion
of b, which contains Cys
, is folded back toward
F
and away from F
. Subunit b of E.
coli ATP synthase is also thought to have a folded conformation
(Fillingame, 1990; Penefsky and Cross, 1991). It might be added
parenthetically that treatment of SMP or ATP synthase preparations with
various mono- and dithiol modifiers results in uncoupling (Yagi and
Hatefi, 1984), and the studies of Papa and co-workers (Zanotti et
al., 1988) implicate Cys
of subunit b in
this type of uncoupling. Although not shown in Fig. 3, we
checked by immunoblotting for MBP-induced cross-links of
,
, d, and c, all of which contain cysteine, but none was
found. The cysteine of subunit c is within the membrane and
may not react with MBP (although it appears to react with p-chloromercuribenzoate). (
)Apparently, the
cysteines of
,
, and d are also unreactive with or
inaccessible to MBP.
As seen in Table 1, b, OSCP, d, and F, but not A6L, form cross-linked products
in SMP with
and/or
. Furthermore, in SMP, OSCP, d,
and F
can each cross-link to b but not to one
another. Therefore, it seems reasonable to assume that the two
molecules of subunit b, which extend from the membrane to
F
, form the principal stem of the stalk to which OSCP, d, and F
bind independently of one another. These
considerations are summarized in Fig. 6, which shows the near
neighbor relationships of the stalk subunits in cuts parallel to the
membrane at the proximal end (bottom) of F
(A) and
at the membrane-matrix interface (B). In A, subunits b, OSCP, d, and F
are all present because
they all cross-link to
and/or
(the parallelogram labeled
/
). They also cross-link to one another wherever
the circles representing each subunit overlap. In B (the bottom of the stalk), A6L protrudes from the membrane near d, and the two b subunits enter the membrane.
However, whether OSCP and F
extend the length of the stalk
from F
to F
to impinge upon the membrane is
unclear; therefore, they have not been included. In
F
-depleted ASU particles, this organization is somewhat
perturbed, as if the removal of F
results in the inward
collapse of the b subunits toward each other, also bringing
F
close to d. As a consequence, in ASU particles,
but not in SMP, b + b and d +
F
cross-linked dimers can form (the latter is marked by an arrowhead in Fig. 5A, lane 11). When
MBP was used for cross-linking of ASU particles, b +
F
was formed as in MBP-treated SMP, but the yield of b + OSCP dimer was greatly diminished (Fig. 4, lane
6). Instead, there was the appearance of a band qualifying as a b + b dimer, which was not seen in MBP-treated
SMP. Interestingly, the addition of F
to ASU particles
prior to MBP treatment restored the formation of the b +
OSCP product and diminished the yield of the b + b dimer (Fig. 5, lanes 3, 4, 7,
and 8). These results suggest that (i) F
removal
diminishes the interaction of b and OSCP, which is consistent
with the fact that extraction of F
also results in
labilization of OSCP, and (ii) the readdition of F
to ASU
particles reestablishes the close association of b and OSCP,
which is also consistent with the fact that function can be restored to
ASU particles by the addition of F
and OSCP (Matsuno-Yagi
and Hatefi, 1984). Recent data of Abrahams et al.(1994) on the
crystal structure of bovine F
indicate that
protrudes
30 Å from the core of F
, which the authors believe
forms part of the stalk. Fig. 6A shows a possible
location for the tail of
surrounded by b, d,
and OSCP. This possible location inside the stalk is consistent with
our inability to find cross-linked products of
with any of the
stalk subunits. It also agrees with the presumed inward collapse of the
stalk to allow the formation of b + b and d + F
cross-linked products when
together
with the remainder of F
is removed from SMP. As was shown
in Fig. 2, both
and
form multiple cross-linked
products when SMP or isolated F
preparations were treated
with DST (or EDC, not shown). However, like
,
also failed to
cross-link to any stalk subunit. The data of Abrahams et
al.(1994) on the crystal structure of bovine F
do not
show the location of
. It may be of interest to add in this regard
that E. coli
and
cross-link via a disulfide bond
in the presence of CuCl
(Mendel-Hartvig and Capaldi, 1991),
a G29D mutation of the E. coli
renders the F
-deficient (Maggio et al., 1988), and tryptic
removal of 15 NH
-terminal residues of the
subunits of E. coli F
destroys the ability of the
reconstituted
to bind
(Dunn et al., 1980). As mentioned earlier, E. coli
is considered to be analogous to the bovine OSCP (Walker et al., 1982; Ovchinnikov et al., 1984; see also Dunn
and Heppel, 1981). However, the results just described make one wonder
about the location of
in relation to the distal end of E.
coli F
, where by analogy to the bovine F
the NH
termini of the
subunits would be located
(see Abrahams et al., 1994).
Figure 6:
Schematic representation of the near
neighbor relationships of subunits b, OSCP, d,
F, and A6L in bovine submitochondrial particles.
Cross-linked dimers of subunits were identified where circles representing the subunits overlap. Because knowledge of the
structures of these subunits is limited, the diameters of the circles were deliberately made the same to avoid any inference
regarding the relative sizes and shapes of these subunits. A,
end-on view of a cut parallel to the membrane through the bottom
(proximal end) of F
. B, end-on view of a cut
parallel to the membrane at the membrane-matrix
interface.
Finally, it should be
mentioned that Joshi and Burrows (1990; see also Archinard et
al., 1986) have previously detected in a preparation of bovine ATP
synthase treated with dithiobis(succinimidylpropionate), which
cross-links amino groups at a distance of 12 Å, many of the
cross-linked products we have characterized here. They employed
antibodies to whole F, OSCP, F
, A6L, a, and c, but not to the individual subunits of
F
and subunit d. Therefore, some of the
cross-linked products were identified in part on the basis of their
mobility on two-dimensional SDS gels. The cross-linked products they
report, which we have not seen in SMP with our three cross-linking
reagents, are those of F
with
,
, and A6L. They
also claim the formation of F
+ d dimer (they
refer to d as the 20-kDa subunit), which we only find in
DST-treated ASU particles, but not in intact SMP.