Isolation of a major hydrophobic protein of the mitochondrial inner membrane

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol.55,No.3,1973

ISOLATION OF A MAJOR HYDROPHOBIC PROTEIN OF THE MITOCHONDRIAL INNER MEMBRANE Roderick

A. Capaldif,

Hirochika

Komai and Douglas R. Hunter

Institute for Enzyme Research University of Wisconsin Madison, Wisconsin 53706 Received

October

5,1973

SUMMARY: A protein of molecular weight 29,000 has been isolated from the mltochondrial inner membrane. It is a major component of Racker's hydrophobic protein mixture and is also rather selectively released from the inner membrane by lysolecithin treatment. Data indicate that the 29,000 component may be as much as 10% of the total protein of the inner membrane.

INTRODUCTION The mitochondrial ponents. four

inner

These are for

electron

oligomycin

complexes sensitive

Complexes I (5),

the most part isolated

II

(5,6),

to almost

five

complexes:

(5,7)

The polypeptide

and IV (8-10)

ATPase (11,12)

all

into

number of protein

by Green and his collaborators

III

sensitive

a large

organized

ATPase complex (3,4).

chain and oligomycin are referrable

membrane contains

of the electron

have been identified.

These

the major bands seen in sodium dodecyl

found in significant

component.

29,000 is not however We have succeeded

Its method of preparation

in this

sulfate

One major com-

in isolating

is detailed

of

transfer

ponent of molecular

complexes.

and the

compositions

(ETP) (5).

in any of these

the

(1,2)

polyacyl amide gels of inner membrane preparation weight

com-

amounts

and purifying

this

paper.

MATERIALS AND METHODS ETP were prepared

They were treated

'Present

address:

Eugene, Oregon

according

with lysolecithin

Institute

to the method of Linnane and Ziegler as described

of Molecular

97403.

Copyright 0 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.

655

Biology,

by Komai --et al.

University

(13).

(14).

of Oregon,

The

BIOCHEMICAL

Vol.55,No.3,1973

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Mobility

Figure-l. Densitometric traces of various fractions derived from the mitochondnal inner membrane, run on 10% polyacylamide gels in the presence of 1% SDS and 5 mM B-mercaptothanol. Trace a, ETP; Trace b, Racker's hydrophobic mixture; Trace c, the supernatant S2 from lysolecithin treated ETP; Trace d, pure 29,000 component after chromatography through Biogel A-5 M in 1% SDS and 0.5 mM dithiothreitol. A number of bands are identified by size as follows: 65000, NADH dehydrogenase; 55000 and 52000, the major subunits of Fl; 50000, core protein of Complex III; 48000, one of the b cytochromes of Complex III. supernatant fraction

S2 was rich was prepared

The 29,000 lysolecithin the above

as described

molecular

treatment fractions

in the 29,000

weight or from

(5-10

mg/ml)

component

(15).

The hydrophobic

by Kagawa and Racker protein

the

was further

hydrophobic

were

fraction

made 5 mM with

656

protein

(16).

purified

from

as follows: respect

S2 of the 5 ml of

to dithiothreitol

BIOCHEMICAL

Vol.55,No.3,1973

and then

were

reagent.

This

at 105,000 sulphate for

brought solution

x g for

respect

was incubated

120 min.

to urea

on ice

by addition

for

was resuspended

(SDS) and 5 mM B-mercaptoethanol

and dissolved

This

Laboratories,

solution

Inc.)

monitored

for

by dialysis

in 1% SDS and 0.5

absorbance against

48 hours.

was chromatographed

concentrated

at 280 nm.

a large

Ethanol

volume

was removed

and the gels

--et al.

were

(17).

fixed

in

Protein

concentration

Polyacrylamide

and stained

Biogel-A-5

gel

to 1OO'C M (Bio-rad

from

were

the purified

the cold

room

and the

protein

protein (4'C)

for

was

was estimated

by the

electrophoresis

as described

dodecyl

Fractions

SDS was removed

evaporation

centrifuged

by heating

dithiothreitol.

of 90% ethanol

solid

in 3% sodium

through

by rotary

by freeze-drying.

of Lowry

ti

of the

30 min and then

The pellet

1 min.

method

to 8 M with

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

was performed

by Fairbanks

--et al.

(18).

RESULTS AND DISCUSSION A typical sulphate

and run

Band number component gels

densitometric

14 is

the

(trace mixture,

It

dalton

Figure

is

inner also

1 (trace

precipitated

threitol,

Figure

the yield

of highly

shown

a). This

by its

bovine

serum albumin,

ovalbumin,

and cytochrome

5.

the major

component

preparations,

component

1 (trace

as estimated

ribonuclease

membrane

in Figure

dodecyl

in the preparation.

proteins

lysolecithin

contaminating chromatography

in 1% sodium

in supernatant

as shown

of Racker's

migration

S2 of

in Figure

hydrophobic

on

1 protein

b).

8 M urea,

column

is

the major

of the

of other

standard

protein

Treatment with

of 29,000

chymotrypsinogen,

treated

c).

the

is

polypeptide

weight

with

dehydrogenase,

lysolecithin

gels

predominating

has a molecular

The 29,000

of ETP solubilized

on 10% polyacrylamide

in comparison

lactic

trace

supernatant

the

29,000

proteins. through

1 (trace purified

d).

molecular

Purification Biogel Using protein

or hydrophobic

A-5 m in the

weight

protein

to homogeneity

fraction

fairly

free

was achieved

1% SDS and 0.5 mM dithio-

lysolecithin

method

was 5-8% of the starting

657

protein

of preparation, inner

membrane

by

BIOCHEMICAL

Vol. 55, No. 3,1973

preparation,

even after

is a major

component

The fact

that

a lipoprotein

by the

fraction,

the

while in

mitochondrial

is

then,

obscure portion

are

from

component

that

protein

therefore

membrane.

it

outl'

is

concentration.

distributed

This along

and solubilized

contention with

is

a large

amount

left

after

of this

mg/mg protein,

cf.

we have isolated membrane

with It

0.5-0.6

a major

intrinsic

a molecular

weight

hydrophobic

required

for

electron

transport

however,

protein

reconstitution

Its that

fraction,

ETP).

of the

of 29,000.

may be significant,

is in

protein

of protein

fraction

mg/mg protein

the

function protein

a mixture

of oxidative

of

supported

of the hydrophobic

removal

as

in an area

in the preparation

of Racker's

which

This

"scooped

is solubilized

at present.

proteins the

lipid

protein

(0.3

inner

a major

suggests

the precipitate

lipid

inner

selectively

1.5 mg/mg protein)

In sumnary

remains

rather

a high

that

(about

depleted

is

RESEARCH COMMUNICATIONS

described.

of the mitochondrial it

with

fact

lipid

the manipulations

by lysolecithin,

the membrane

AND BIOPHYSICAL

of

phosphorylation

and ATP synthesizing

complexes

(19).

ACKNOWLEDGMENTS We are grateful ment of this work. National Institute

to Dr. David E. Green for his interest This work was funded in part by grant of General Medical Science (USPHS).

in and encourageGM-12847 from the

REFERENCES 1.

Hatefi, Y., Haavik, A.G., Res. Cotnnun. 4, 441.

2.

Green, D.E., and Fleischer, S. (1962) in Horizons in Biochemistry, M. Kasha and B. Pullman, eds. AcademicPress, New York, p. 381.

3.

Tzagoloff, 243, 2405.

4.

Kagawa,

5.

Capaldi, R.A. in Mammalian Cell D.M. Robinson,Tds. Butterworth

A.,

Y.,

Y.

Byington,

K.H.,

and Racker,

6.

Hatefi,

7.

Baum, H., Silman, Chem. 242, 4876.

Ann.

8.

Capaldi,

9.

Kuboyama,

R.A., M.,

and Griffiths,

N.Y.

Acad.

F.C.,

J. Biol. Membranes, and Co.,

Sci.,

Rieske,

and Hayashi, Fong,

and MacLennan,

E. (1966)

H.I.,

D.E.

(1961)

D.H.

Biochem.

(1968)

Chem. 241,

Biophys.

J. Biol.

Chem.

2467.

Vol. II, G.A. Jamieson London, in press.

and

in press. J.S.,

and Lipton,

H. (1972) and King,

FEBS Letters T.E.

658

(1972)

S.H. 2&, J. Biol.

(1967)

J. Biol.

261. Chem. 247,

6375.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 55, No. 3,1973

10.

Mason, T., Ebner, E., Poyton, R., Salsgaber, J., Wharton, D.C., Mennucci, L., and Schatz, G. (1972) in Mitochondria/Biomembranes, Vol. 28, S.G. Van der Bergh, P. Borst, L.L.M.van Deenen, J,.C. Riemersma! E.C. Slater, and J.M. Tager, eds. North Holland/American Elsevier, p. 53.

11.

Capaldi,

12.

Tzagoloff, A., Rubin, Acta 301, 71.

13.

Linnane,

14.

Komai, H., Hunter, Cotmnun. 53, 82.

15.

Komai, H. (1974) Ann. N.Y. Acad. Sci. 277, in press.

16.

Kagawa, Y., and Racker,

17.

Lowry, O.H., Rosenbrough, J. Biol. Chem. 193, 265.

18.

Fairbanks, 2602.

19.

Racker,

R.A.

(1973) Biochem. Biophys. M.S.,

A.W., and Ziegler, D.R.,

G., Steck,

and Sierra,

M.E. (1973) Biochim.

D.M. (1957) and Takahashi,

E. (7977)

L.T.,

E., and Kandrach,

Res. Conznun. 53, 1331.

N.J.,

Biochim. Biophys. Acta &, 120. Y. (1973) Biochem. Biophys. Res.

J. Biol. Fart-,

Chem. 246, 5477.

A.L.,

and Wallach, A. (1971)

and Randall,

D.F.H.

J. Biol.

659

Biophys.

(1971)

R.J.

(1951)

Biochemistry

Chem. 246, 7069.

lo,