Bioscience Reports 3, 921-926 (1983)
Printed in Great Britain
921
Binding oI mitochondrial ATPase from ox heart
to
its naturally occurring inhibitor protein:
Localization by antibody binding
Philip J. 3ACKSON and David A. HARRIS
Department of Biochemistry, University of Leeds,
Leeds LS2 9JT, U.K.
(Received 23 August 1983)
The naturally occurring ATPase inhibitor protein from
ox heart mitochondria was cross-linked to its binding
s i t e on the m i t o c h o n d r i a l ATPase using 1-ethyl-3(dimethylamino)propyl carbodiimide.
The cross-linked
p r o d u c t , when t r a n s f e r r e d e l e c t r o p h o r e t i c a l l y to a
n i t r o c e l l u l o s e sheet~ reacted with antibodies directed
against the inhibitor protein and the 8-subunit of the
ATPase. It was concluded that the binding site for the
inhibitor protein lies on the 8-subunit.
A naturally occurring inhibitor protein can be isolated from ox
heart mitochondria. This protein inhibits both ATP hydrolysis and ATP
synthesis by the mitochondrial ATPase (FI-ATPase) ( l ) .
Only one
mol of inhibitor protein is needed for complete inhibition of ATP
hydrolysis by one mol of F t (2), even though the F l molecule contains
three copies of each of its largest subunits (a and 8) (3).
It was
interesting, therefore,~ to consider whether the inhibitor protein binds
to one of the smaller F t subunits (present in amounts of one mol/mol
FI) , or whether binding to only one of the larger subunits is sufficient
to prevent a c t i v i t y of the whole protein.
Vignais and coworkers have investigated this problem using inhibitor
protein labelled on several amino groups with t~C-labelled thiocyanates
(4) or imidates (_5), and inducing cross-linking either by photochemical
(5) or purely chemical (~) means.
They concluded that the ATPase
i n h i b i t o r protein bound to the 8-subunit of the Ft-ATPase.
The
8-subunit, however, was i d e n t i f i e d on the basis of its relative
r e a c t i v i t i e s w i t h dicyclohexyl carbodiimide and N-ethyl maleimide,
neither of which is a totally specific reagent.
We have recently prepared a radio-iodinated inhibitor in which the
single tyrosine residue is labelled to a very high specific radioactivity
without loss of inhibitory a c t i v i t y (6).
Further, antibodies to the
individual subunits of F 1 and to the inhibitor protein can be prepared.
Thus we were able to re-investigate the site of inhibitor binding using
much more specific and sensitive techniques than were previously
available.
Using l-ethyl-3(dimethylamino)propyl carbodiimide (EDC)
to cross-link Fl_ATPase to its inhibitor protein, and specific antibodies
to i d e n t i f y the composition of protein bands after analytical gel
electrophoresis (7), we were able to confirm that the 8-subunit of F 1
bears the binding site for inhibitor protein, both in the free and the
membrane-bound enzyme.
01983
The Biochemical Society
922
Materials
3ACKSON & HARRIS
and M e t h o d s
FI-ATPase , ATPase inhibitor protein (native and radio-iodinated),
and i n h i b i t o r - d e f i c i e n t submitochondrial particles were prepared as
previously (6), and the oligomycin-sensitive ATPase as described by
Serrano and Racker (8). Antibodies to inhibitor protein were prepared
from rabbit serum, after fortnightly subcutaneous injections in Freund's
complete adjuvant.
To increase its immunogenicity~ the inhibitor
protein was cross-linked to haemocyanin (1:2.5 by mass) using EDC
(9).
After the first three injections, inhibitor protein cross-linked
without haemocyanin was used as immunogen. The haemocyanin was a
kind gift of Dr. E. 3. Wood, Leeds University.
Antibodies to the
individual F 1 subunits were a gift of Dr. 3. Walker, MRC Unit for
M o l e c u l a r Biology, C a m b r i d g e , U.K.
Gel e l e c t r o p h o r e s i s and
electrophoretic transfer of proteins onto nitrocellulose were carried out
as described by Towbin et al. (10).
Results
and D i s c u s s i o n
Cross-linking inhibitor protein to its binding site
The interaction between the F -1A T P a s e and its naturally occurring
inhibitor protein is not normally covalent.
Fig. 1 shows the subunit
pattern observed after gel electrophoresis of the Fl/inhibitor complex
in dodecyl sulphate.
If the complex is prepared in the absence of
cross-linker (track 1A), 6 bands are observed, corresponding to the 5
subunits of F 1 ( ~ - e) and, intermediate in molecular weight between
the ~ and e subunits, the inhibitor protein. This agrees with previous
work (2).
When the complex is prepared, using radiolabelled inhibitor protein,
in the presence of EDC (tracks 1B,1C) additional bands are observed.
A strong band, Y (corresponding to mol.wt. I00 000), is observed near
the top of the gel, and fainter bands appear lower down, at mol. wts.
of about 19 000, 32 000, and 39 000.
F r o m the corresponding autoradiograph (tracks 2A,B,C), we see
that band Y does not contain inhibitor protein, while the other 3 bands
all do.
In agreement with Klein et al. (4), we conclude that these
bands represent polymers of the inhibitor protein (I2, I3, and I~), since
their intensities are increased if the inhibitor:F 1 ratio is increased
(tracks B).
In addition, they bind only antibody to the inhibitor
protein, not antibody to the other subunits (Fig. 2).
The trimer 13
seems to run anomalously slowly in this gel system.
In addition, the autoradiograph reveals that one band (X), containing the r a d i o a c t i v e inhibitor protein appears in the presence of
c r o s s - l i n k e r at about 65 000 d a l t o n s (track 2C).
This band is
abolished if the c o m p l e x is prepared with the same amount of
radiolabelled inhibitor but with a 20-fold excess of unlabelled inhibitor
protein (track 2B).
This is consistent with the view that Band X
represents a complex of inhibitor protein with its binding site on Fl,
since the specific activity of the complex is reduced in the presence
of unlabelled inhibitor protein. It is not simply a polymer of inhibitor
p r o t e i n alone, since p o l y m e r i z a t i o n i n c r e a s e s at higher protein
c o n c e n t r a t i o n s (tracks B).
The use of substoichiometric amounts
MITOCHONDRIAL ATPase-INHIBITOR BINDING
923
Fig. i.
Cross-linking of inhibitor protein to
FI-ATPase.
Purified F 1 (50 ~g) was incubated in 50
~I of i0 mM sodium phosphate~ i mM ATP (pH 6.7 with
NaOH) containing i ~g of radio-iodinated inhibitor
protein (2 x I0 ~ c.p.m./~g). Further additions were
as follows:
Track A~ 100 nmol of MgATP.
Track B, 16 ~g of unlabelled inhibitor protein~ 100
nmol of EDC~ and i00 nmol of MgATP.
Track C, i00 nmol of EDC and i00 nmol of MgATP.
After 60 min at 20~
the reactions were stopped by
addition of 3 ~mol of Tris, and the samples analysed
by NaDodSO4-gel electrophoresis using a 5-15% linear
gradient of acrylamide, in the discontinuous buffer
system of Laemmli (12).
The gel was then stained
for protein with Coomassie Brilliant Blue R250
(tracks IAmBiC), dried~ and autoradiographed (tracks
2A~B~C) for 2 weeks at -70OC~ using Fuji RX X-ray
film.
inhibitor protein in these studies (cf. references # and 5) is likely to
prevent significant cross-linking to non-specific binding sites on F I.
Analysis of the cross-linked product
To determine the composition of Band X~ the protein bands from a
s i m i l a r gel ( p r e p a r e d using u n l a b e l l e d i n h i b i t o r p r o t e i n ) w e r e
e l e c t r o p h o r e t i c a l l y transferred to a nitrocellulose sheet, and analysed
by t r e a t m e n t , first with antibodies to the individual subunits, and
s u b s e q u e n t l y with r a d i o - i o d i n a t e d p r o t e i n A.
P r o t e i n A (from
924
JACKSON & HARRIS
Staphylococcus aureus) binds t i g h t l y to F c region of IgG.
Thus when
the nitrocellulose sheet is incubated with antibody and free antibody is
washed away, subsequent incubation with radio-iodinated protein A will
specifically radiolabe] any antibody/antigen complex on the sheet (7).
The results are shown in Fig. 2. Tracks B represent the subunits
of an F l - i n h i b i t o r complex prepared in the absence of cross-linker.
With the exception of anti-y antibody (which reacts to some extent
w i t h the ~ or B subunit of Ft) the antibodies bind well to the
expected subunit and hardly at all to the others. Non-immune serum
(tracks 6A,B) does not bind. Separate gels, not shown here, confirm
Fig. 2.
Antibody labelling of the protein bands
after Fl-inhibitor cross-linking.
Cross-linking and electrophoresis were carried out
as in Fig. i, except that a ratio of 50 ~g of F 1 to
i ~g of unlabelled inhibitor protein was used in
each case.
The tracks contain: A, complete system;
B, EDC omitted; C, inhibitor protein omitted.
The far left tracks were stained for protein as in
Fig. i. The remaining tracks were transferred to a
nitrocellulose sheet (See Materials and Methods),
and further treated with (a) i mg/ml haemoglobin / 1
mg/ml bovine serum albumin to block unsaturated
b i n d i n g Sites on the nitrocellulose
(I h), (b)
50-500 pg of antibody (2 h) followed by (c) 0.2 pg
of radio-iodinated protein A (15 x I06 c.p.m/Ng).
All additions were made in 3 ml of buffer containing
170 mM NaCI, i0 mM Na2HP04, 1.8 mM KH2P04~ 3.3 ~M
KCI, 0.2% Triton X-100 pH 7.2, plus haemoglobin and
albumin as in a.
The sheets were washed well with
this buffer between each addition and were autoradiographed as in Fig. I.
The antisera used were: I~ anti-~ subunit; 2~ anti-~
subunit; 3, anti-y subunit; 4, anti-~ subunit; 5,
anti-inhibitor protein; 6~ pre-immune serum.
M I T O C H O N D R I A L A T P a s e - I N H I B I T O R BINDING
925
t h a t t h e s e a n t i b o d i e s to the cz and [3 subunits do not cross r e a c t , as
can also be deduced f r o m t r a c k s 1A,2A.
( S o m e p r e p a r a t i o n s of a n t i - ~
and anti-[3 a n t i b o d i e s do in f a c t cross r e a c t , owing to s i m i l a r i t i e s in
s e q u e n c e b e t w e e n t h e s e subunits ( l l ) .
T r a c k s C a r e p r e p a r e d f r o m F l t r e a t e d with EDC in the a b s e n c e of
inhibitor protein.
The h i g h - m o l e c u l a r - w e i g h t band, Y, is seen to
r e p r e s e n t a c o m p l e x of c r o s s - l i n k e d products, c o n t a i n i n g the ~ and [3
subunits of F p
As e x p e c t e d , no band c o r r e s p o n d i n g to X is o b s e r v e d .
In c o n t r a s t to r e f e r e n c e 4, we find no o b s e r v a b l e c h a n g e in band
p a t t e r n w h e t h e r or not cross-linking o c c u r s in the p r e s e n c e or a b s e n c e
of i n h i b i t o r p r o t e i n .
This m a y r e p r e s e n t a lower p e r c e n t a g e of
cross-linking in our studies.
T r a c k s A a r e p r e p a r e d f r o m F, t r e a t e d with EDC in the p r e s e n c e
of inhibitor protein.
Band X (al~out 65 000 d a l t o n s ) is o b s e r v e d to
bind only anti-B and a n t i - i n h i b i t o r antibodies ( t r a c k s 2A,SA).
We
conclud% t h e r e f o r e , t h a t the inhibitor b e c o m e s linked to the [3-subunit
of the A T P a s e .
Since F I binds m a x i m a l l y one mol of inhibitor protein
per tool of FI (2,6), the c o m p l e x p r e s u m a b l y contains one inhibitor
m o l e c u l e and one 6-subun[t.
Since EDC cross-linking o c c u r s d i r e c t l y
b e t w e e n a c a r b o x y l group of one p r o t e i n and an amino group of the
o t h e r , we conclude t h a t the inhibitor-binding s i t e lies on the [3-subunit
of the F I - A T P a s e , in a g r e e m e n t with Klein et al. (~,5).
Fig. 3. Cross-linking of inhibitor protein to other
A T P a s e preparations.
Cross-linking~
electrophoresis~ and autoradiography were carried out as in
Fig. i~ except that F I (50 Hg), in tracks IA,B,C,
was replaced by inhibitor-deficient submitochondrial
particles
(I00 Hg)
(tracks
2A,B,C),
oligomycin-sensitive ATPase (75 Hg) (tracks 3A~B~C), or
omitted entirely (track 4D). Tracks A and C contain
the complete system, and tracks B, the system
lacking EDC.
EDC is present in track D.
Tracks
IB~IC, stained for protein~ are shown on the left.
926
3ACKSON & HARRIS
Binding of i n h i b i t o r p r o t e i n to a single 8-subunit thus seems
sufficient to completely inhibit F! activities, despite there being 3
subunits per molecule of F 1. It is interesting that the active site for
ATP hydrolysis and synthesis is also believed to lie on the B-subunit
(13), althouth it is not yet established whether the inhibitor protein
binds at the active site itself.
The inhibitor-binding site on membrane-bound F[
Fig. 3 shows the results of linking radiolabeIled inhibitor protein to
F! in b o t h its f r e e and m e m b r a n e - b o u n d f o r m s , and to t he
detergent-solubilized oligomycin-sensitive ATPase complex ( F I . F o ) . In
all cases, a radioactive band indistinguishable from Band X is observed.
We t h e r e f o r e c o n c l u d e , in agr e e m ent with r e f e r e n c e 5, that tile
inhibitor-binding site is the same in free and membrane-bound F t . The
greater intensity of Band X observed when mernbrane-bound F L is used
( t r a c k s 2A,C) p r o b a b l y r e f l e c t s the 1 0 - f o l d - h i g h e r a f f i n i t y of
membrane-bound F I for inhibitor protein as compared to free F L (2).
A number of minor, additional radioactive bands are observed when
i n h i b i t o r p r o t e i n is cross-linked to submitochondrial particles, but
because of the larger number of proteins present in these particles,
the composition of these e xt r a bands and their role in specific binding
of the inhibitor protein is not yet established.
Acknowledgements
This work was supported by SERC Grant no. GR/A88460.
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