Binding of dicyclohexylcarbodiimide to a native F1-ATPase - inhibitor protein complex isolated from bovine heart mitochondria

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 867-873

Vo1.152, No. 2,1988 April 29, 1988

BINDING

OP DICYCLOHEXYLCARBODIIMIDE TO A NATIVE PI-ATPase - INHIBITOR PROTEIN COMPLEX ISOLATED PROM BOVINE HEART MITOCHONDRIA

Carmen Beltr~n, A. G6mez-Puyou and M. Tuena de G6mez-Puyou Instituto

de

Fisiologfa Celular, Universidad Nacional Aut6noma Apartado Postal 70-600, 04510 M~xico, D.F. M@xico

de

M~xico,

Received March 15, 1988

SUMMARY: The effect and the binding of dicyclohexylcarbodiimide (DCCD) to a soluble native Fi-ATPase-inhibitor protein complex (FI-iP) isolated from heart mitochondria was studied. About one mol DCCD bound per mol FI-IP complex; this inhibited its ATPase activity by more than 95%, ever under conditions that led to maximal hydrolysis. Bound DCCD localized to B-subunits of the FI-IP complex. Cross-linking of the DCCD labeled complex with N-(ethoxy-carbonyl)-2ethoxydihydroquinoline yielded a protein with a Mr 65,000-67,000 that contained IP as evidenced by its reaction with IP antibodies. No ~-subunits were detected in this cross-linked product. The Mr 65,000-67,000 protein corresponds to Bsubunits cross-linked with IP (Klein et al, Biochemistry 1980; 19, 2919-2925). However, no DCCD was found in the cross-linked B-subunit-IP product of labeled native FI-IP. Thus the ~-subunit in contact with IP is distinct from the other two ~-subunits of the enzyme. © 1988 AcademlcPress,Inc.

INTRODUCTION: The H+-ATP synthases of energy-transducing membranes catalyze the synthesis electron

of ATP with the energy of electrochemical H + gradients derived transport.

catalytic portion,

They F I.

are

composed

of a membrane

factor

The latter has five different subunits

(F0), ~3,

from and

a

B3, Y , ~,

and g in the indicated stoichiometry (i) with Mr 54,000, 50,000, 33,000, 17,500 and 5,700 respectively low

molecular

weight

(2).

In addition mitochondrial F I contains a detachable

protein (IP) that inhibits ATP hydrolysis

(3)

and

essential for maximal accumulation of ATP during oxidative phosphorylation By

cross-linking studies of soluble and particulate F I reconstituted with

Klein

et al (5) and Jackson and Harris (6) showed that IP is in close

is (4). IP,

contact

with one of the three B-subunits of F I. In F 1 from E. coli, the g-subunit exerts a role analogous to that of IP in mitochondria

(7,8),

and

it was shown (9,10) that the ~-subunit of

interacts with g does not react with dicyclohexylcarbodiimide

867

(DCCD).

EF 1 Thus

that it

0006-291X/88 $1.50 Copyr~ht © 1988byAcademic Press, ~c. Allrigh~ofreproduction in anyform reserved.

Vol. 152, No. 2, 1988

was

proposed

reactivity

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

that the three ~-subunits of EF 1 were not equivalent.

Here

to DCCD of the ~-subunits of a soluble FI-IP complex isolated

the from

submitochondrial particles which intrinsically have their ATPases controlled by IP

was explored.

The results show that the B-subunit that interacts

with

IP

fails to react with DCCD.

MATERIALS AND METHODS: Biological Material: Mitochondria and Mg-ATP submitochondrial from bovine hearts were prepared as in refs. i0 and 3 respectively. IP was purified as in ref. 12. The native soluble FI-IP complex was isolated as described previously (13) following the work of Feinstein and Moudrianakis (14). FI-IP was stored in small aliquots at -70°C until the time of the experiment. Assay of ATPase Activity and Activation of FI-IP. ATPase activity was measured at pH 8.0 and 23°C spectrophotometricall~ in the presence of an ATP regenerating system (15). The soluble FI-IP complex exhibits a low activity that is increased 50 to i00 fold to a value of close to 100 u m o l min-lmg -I by incubation in 250 mM sucrose/50 mM tris-acetate pH 8.0/10 mM ATP/I.5 mM EDTA at 50°C (14). Binding of [14C]DCCD to F]-IP and Cross-linking. FI-IP was filtered by centrifugation through Sephadex G-50 columns (16) equilibrated with 50 mM MOPStris pH 7.0, 4 mM ATP, 2 mM EDTA, and incubated with [14C]DCCD (Amersham International) (4 x 104 cpm/nmol) as described under Results. At the desired times the mixture was filtered through centrifuge columns. In the filtrate protein and radioactivity was determined, and the amount of [14C]DCCD bound per FI-IP calculated, using a molecular weight of 357,000, (347,000 for F I ref 17, and i0,000 for IP). Cross-linking of the [14C]DCCD labeled FI-IP was carried out with N(ethoxy-carbonyl)-2-ethoxy dihydroquinoline (EEDQ) (5); after incubation with [14C]DCCD, the mixture was centrifuged through columns equilibrated with 250 mM sucrose/ i0 mM MOPS-HCI pH 6.6/2 mM Mg-ATP; 2 mM EEDQ was added and incubated for 20 min at 30°C. At this time an equal volume of 125 mM Tris-HCl pH 6.8/20% glycerol/4% sodium dodecylsulfate (SDS) /10% B-mercaptoethanol/0.001% bromophenol blue, was added, and subsequently analyzed by SDSpolyacrylamide gel electrophoresis (PAGE). When an acid-PAGE was run, to the filtered labeled enzyme an equal volume of 200 mM KH2PO 4 pH 4.0/0.28% Bmercaptoethanol; 70 mM tetradecyltrimethylammonium bromide/17% sucrose/O.001% Coomassie R-250 was added prior to its application in the gel. In some cases FI-IP was cross-linked with EEDQ and subsequently labeled with [14C]N ethylmaleimide (NEM) exactly as Klein et al (5); the products were analyzed by SDS-PAGE. Gel Electrophoresis. SDS-PAGE was run as described by L~mmli (18) with a 5% polyacrylamide stacking gel and a 13.5% polyacrylamide separating gel. AcidPAGE was carried out as in 19. The gels were stained with 0.05% Coomassie R-250 in 20% methanol / 7% acetic acid. Destaining was carried out with the same solution without Coomassie. The gels were scanned in a Beckman DU-50 Spectrophotometer at 560 nm; this was followed by slicing of the gel into 2 mm fractions. The slices were dissolved in 0.4 ml 30% H202 at 60°C for 6 hrs. The radioactivity of the fractions was determined by liquid scintillation. Immunological Detection of Inhibitor Protein. Electrophoretically resolved proteins by SDS-PAGE were transferred to millipore nitrocellulose sheets (20). The nitrocellulose filter was saturated with 0.5% bovine serum albumin (BSA) in i0 mM phosphate pH 7.4/150 mM NaCI (PBS) for 1 hr. at 23°C. The sheet was then incubated with 0.5% serum anti-IP (raised in rabbits) in 0.5% BSA in PBS for 2 h. Subsequently it was washed with 0.5% BSA in PBS for 1 hr with 3 changes. Immune complexes on nitrocellulose filters were detected by incubation with protein A gold for 3 hrs at room temperature, followed by two washes of five min each with PBS, one wash with 1% Triton X-100 in PBS, and finally two washes

868

Vol. 152, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

with PBS alone for i0 min. The reaction with antibody was evidenced as a pink spot. Protein was measured by the method of Lowry (21), using BSA as standard.

RESULTS:

The FI-IP complex isolated from submitochondrial particles

rich

in

inhibitor protein exhibited an ATPase activity of 1 to 2 pmol min -I mg -I. Under conditions

that

abolished

the inhibitory action of

IP

(14),

the

activity

increased to nearly i00 pmol min -I mg -I (Fig. IA). It is known that the binding of

one mol DCCD per mol F 1 free of IP inhibits its ATPase activity

(5,22).

To

explore if FI-IP interacted with DCCD, FI-IP was incubated with DCCD, and after elimination

of free DCCD,

its ATPase activity was assayed after exposing

complex to activating conditions. its

ATPase activity,

The incubation of FI-IP with DCCD diminished

as well as the extent of ATPase activity

incubation in the activation mixture with the binding of DCCD to FI-IP. the

activity

(Fig.

i,

reached

(Fig.

reached

after

IA). This effect of DCCD correlated

With approx, one [14C]DCCD bound per FI-IP,

after activation was about 95% lower than

the

control

A and B). Thus the reactivity of the enzyme to DCCD was not affected

by the inhibitor protein. increased,

the

In the absence of DCCD, the activity of the complex

but to a value about 80% lower than that attained after full activa-

B

A 100

>------------I--

-------I

Q2

E

E

N

E i o

50

0.!

E

(2_ I.C 10

20

30

40

50

60

Minutes

I

I

i

I

I

I

t0

20

30

40

50

60

Minutes

Figure I. Effect of DCCD on the ATPase Activity of FI-IP and Binding of [14C]DCCD to FI-IP. In A. FI-IP was incubated in 50 mM MOP-tris pH 7.0, 4 mM ATP, 2 mM EDTA with (A,O) and without ( O , 1 ) 200 ~M DCCD at 24°C. In O a n d A , at the indicated times ATPase activity was assayed. In the traces depicted by .andOaliquots of the mixture were filtered through centrifuge columns equilibrated with activating buffer, incubated at 50°C for 70 min., and ATPase activity measured. B. FI-IP (1.7 mg/ml) was incubated as in A with 240 ~ M [14C]DCCD. At the indicated times aliquots were withdrawn and filtered twice in centrifuge columns. In the filtrate radioactivity and protein were determined.

869

Vol. 152, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

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Figure 2. Electrophoretic Analysis of [14C]DCCD Labeled FI-IP and Crosslinked FI-IP. FI-IP was incubated with [14C]DCCD for 4 hours to a value of 2.1 mol DCCD per mol FI-IP. After filtration the enzyme was analyzed by A. SDS-PAGE B. Acid PAGE. C shows the SDS-PAGE analysis of cross-linked FI-IP, and the radioactivity profile of the sliced gel (cross-hatched). The inset in C shows the molecular weight of the complex $-IP; a mixture of seven different proteins of 66.45, 36, 29, 24, 20 and 14 KDa (Dalton Mark VII-L, Sigma) were used as standards. The drawings at the top in A and C show the reactivity to antisera against IP.

tion (Fig. IA); this indicated that throughout the time of incubation with DCCD more than 80% of the total enzyme population remained controlled by IP. FI-IP

labeled

exhibited the ~, g-subunits. Most

of

B, %

[14C]DCCD

was

, 6, and ~-subunits,

analyzed

in

the

radioactivity was found in the B -subunit, the

% subunit.

with

a

small

In gels ran under acid conditions,

label was in ~-subunits.

interacted

SDS-PAGE.

Thus,

the B-subunits of FI-IP.

The

enzyme

and IP which migrated between ~ and

clearer separation of ~ and $-subunits was achieved that

by

The radioactivity of slices of the gel was determined

the

detected

with

2A).

amount

was

in

which

a

(Fig. 2B), it was confirmed

as in F 1 free of FI-IP labeled

cross-linked with EEDQ, and analyzed in SDS gels

(Fig.

IP

(5,22),

with

DCCD

[14C]DCCD

was

(Fig. 2C). Close to 50% of the

radioactivity was found at the origin of the gel; a substantial amount of label was in the region that corresponded to B-subunits that had not undergone crosslinking. Klein et al (5) showed that F 1 reconstituted with excess IP, labeled 65,000

with

[14C]DCCD

- 67,000

that

and cross-linked with EEDQ yielded a contained radioactive ~-subunits and

870

subsequently

product IP.

of

Jackson

Mr and

Vol. 152, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

3600

•

~

w 3200. ¢.)

2800 ~ 2,~,o~r z

2000

'~

lSOO

E

~4

T..-, 120C

o

40C 0 0

4 cm

2

6

Figure 3. SDS-PAGE of [14C]NEM Labeled Cross-linked FI-IP. FI-IP was cross-linked with 2 mM EEDQ as in Fig. 2. After filtration, cross-linked FI-IP was incubated with 3 mM [14C]NEM as in (5). The [14C] radioactivity profile (cross-hatched area) and the densitometric trace are shown.

Harris

(6)

also

detected

reconstituted

FI-IP complex.

products

our

weight,

of

this

band

in

The existence

native FI-IP preparations

and the presence

possibility

corresponded

that

did

the

The

of

a

of this band among the cross-linked was

evidenced (Fig.

by

its

2C). The latter was

directed against IP (Fig. complex

the

molecular

Mr

2C, inset).

65,000-67,000

observa-

FI-IP labeled in the ~, y , and E-subunits with in the Mr 65,000-67,000

protein band

band

[14C]NEM

(Fig.

3).

and in agreement with Klein et al (5) and Jackson and Harris

(6), it is concluded cross-linked

native

products

cross-linked with IP was ruled out by the

not exhibit radioactivity

On these grounds,

its

in

to ~-subunits

tion that cross-linked

cross-linked

of IP in this protein band

detected by its reaction with antibodies The

the

that the Mr 65,000-67,000

band,

corresponded

to B-subunits

with IP.

insets of Figure 2,

A and C are drawings of the reaction of IP

antibodies which illustrate

that after cross-linking

FI-IP, all IP localized to the Mr 65,000-67,000 all IP was cross-linked the Mr 65,000-67,000

to ~-subunits,

protein.

of

with

[14C]DCCD-labeled

band. Thus notwithstanding

that

hardly any radioactivity was detected in

This indicates

IP did not react with DCCD.

871

that the ~-subunit

in contact with

Vol. 152, No. 2, 1988

DISCUSSION: that

The

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

present findings,

and those published before (5,6)

in the reconstituted and native FI-IP complex,

contact

with IP.

~-subunit either

a ~-subunit of F 1

~-subunit

alternatively

is

in

In addition it was found here that in the native complex the

in contact with IP has a much lower reactivity

this

indicate

is

intrinsically different from

to

[14C]DCCD.

the

other

Thus

two,

the interaction of one of the $-subunits with IP confers to

or the

subunit a low sensitivity to DCCD. There is an apparent discrepancy between the data

of

Klein et al (5) and the present findings with

respect

labeling of the protein-band that results after cross-linking. analysis

of

to

[14C]DCCD

However a close

the data of the authors shows that only a small fraction

of

the

total

radioactivity in the ~-subunits appeared in the Mr 65,000-67,000 protein

band.

Thus

even

though this group worked with F I in presence of

excess

IP,

there seems to be a higher selectivity toward DCCD of B-subunits that were

not

in contact with IP. of does

ATP

Accordingly, it would appear that synthesis and hydrolysis

as catalyzed by particulate FI-IP (4) would be through an enzyme

not

accordance

have with

three

equivalent B-subunits.

This

conclusion

would

the findings of Capaldi et al (9,10) on EF 1 in which

that be

it

in was

shown that the ~-subunit that interacts with g fails to react with DCCD. As the ~-subunit of EF 1 acts similarly to IP of mitochondria (7,8 but see ref. 23), it is likely that H+-ATP synthases may function with nonequivalent B-subunits.

ACKNOWLEDGEMENTS: This work was supported by grants from the Consejo de Ciencia y Tecnologla (CONACyT), M~xico and the Organization of States.

Nacional American

REFERENCES: i. Hatefi, Y. (1985) Ann. Rev. Biochem. 54, 1015-1069 2. Penefsky, H. S. (1979) Adv. in Enzymol. and Rel. Areas of Mol. Biol. 49: 223-280. 3. Pullman, M. E. and Monroy, G. C. (1963) J. Biol. Chem. 238, 3762-3769. 4. Beltr~n, C., Tuena de G6mez-Puyou, M., Darszon, A. and G6mez-Puyou, A. (1986) Eur. J. Biochemo 160, 163-168. 5. Klein, G., Satre, M., Dianoux, A. C. and Vignais, P. V. (1980) Biochemistry 19, 2919-2925. 6. Jackson, P. J. and Harris, D. A. (1983) Biosc. Reports 3, 921-926. 7. Sternweis, P. C. and Smith, J. B. (1980) Biochemistry 19, 526-531. 8. Dreyfus, G. and Satre, M. (1984) Arch. Biochem. Biophys. 229, 212-219. 9. Tommasino, M. and Capaldi, R. A. (1985) Biochemistry 24, 3972-3976.

872

Vol. 152, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

i0. L~tscher, H. R. and Capaldi, R. A. (1984) Biochem. Biophys. Res. Commun. 121, 331-339. ii. LSw, H. and Vallin, I. (1963) Biochim. Biophys. Acta 69, 361-374. 12. Klein, G., Satre, M., Zaccai, G. and Vignais, P. V. (1982) Biochim. Biophys. Acta 681, 226-232. 13. G6mez-Puyou, A., Tuena de G6mez-Puyou, M., and de Meis, L. (1986) Eur. J. Biochem. 159, 133-140. 14. Feinstein, D. L. and Moudrianakis, E. N. (1984) J. Biol. Chem. 259, 42304236. 15. Pullman, M. E., Penefsky, H. S., Datta, A. and Racker, E. (1960) J. Biol. Chem. 235, 3322-3329. 16. Penefsky, H. S. (1977) J. Biol. Chem. 252, 2891-2899. 17. Knowles, A. F. and Penefsky, H. S. (1972) J. Biol. Chem. 247, 6617-6623. 18. L~mmli, M. K. (1970) Nature 227, 680-685. 19. Fellous, G., Godinot, C., Baubichon, H., DiPietro, A. and Gautheron, D.C. (1984) Biochemistry 23, 5294-5299. 20. Towbin, H., Slachelin, T. and Gordon, J. (1979) Prec. Natl. Acad. Sci. USA 76, 4350-4354. 21. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Rondall, R. J. (1951) J. Biol. Chem. 193, 265-275. 22. Pougeois, R., Satre, M. and Vignais, P. V. (1979) Biochemistry 18, 14081413. 23. Bragg, P. D. and Hou, C. (1987) Biochim. Biophys. Acta 894, 127-137.

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