A yeast mitochondrial inner membrane 30K hydrophobic protein: comparison with subunit 32K of the cytochrome bc 1 complex

Vol. 110, No. 3, 1983 February 10, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages

945-950

A YEAST MITOCHONDRIAL INNER MEMBRANE 30K HYDROPHOBIC PROTEIN : COMPARISON WITH SUBUNIT 32K OF THE CYTOCHROME BC 1 COMPLEX :: M. ALKOUTAYNI and A.J.C. STAHL Laboratoire

Louis

Pasteur,

SUMMARY: A yeast mitochondrial inner membrane hydrophobic protein been isolated and compared to subunit 32K of the yeast cytochrome complex. Both proteins are translated on mitochondrial ribosomes, nearly the same molecular weight and similar aminoacid compositions. parison was carried out by immunological techniques with specific dies, and by studyingjyeast strains having mutations in the COB of the mitochondrial DNA. Results show that the two proteins are tical.

30K has bc 1 have Comantiboregion not iden-

Received

de Biochimie, B.P.

December

INTRODUCTION the yeast

14,

Facultd de Pharmacie, Universite 10, 67048 STRASBOURG, France

1982

: A 30 kilodalton

mitochondrial

(30K(3 hydrophobic

inner

membrane.

protein

Aminoacid

was extracted

composition,

from

apparent

molar ratio of acidic to basic aminoacids, and polarity molecular weight, were previously determined (1). Interaction between this protein and the other

inner

membrane

No information Among the sized (3,

constituents,

is available

other

known

4) has similar

compare

the

me-linked

concerning

yeast

on mitoribosomes,

subunit

properties

two proteins

assay),

tation

of protein-antibody

complexes

other

strains

the mitochondrial the

DNA are

30K hydrophobic

MATERIALS

3 yeast protein

was studied

of this

membrane cytochrome

30K hydrophobic

by Staphylococcus --------------------mutations

bearing

used to study

the

influence

(2).

protein.

proteins

synthe-

bc 1 complex

protein.

three immunological procedures Ouchterlony gel immunodiffusion

On the

hand,

function inner

32K of the

to the

using

phospholipids

the

mitochondrial

only

immunosorbent

especially

Therefore

we

: ELISA (Enzyand precipi-

aureus. in the COB region of the mutation

of on

synthesis.

AND METHODS

in (5). Saccharomyces cerevisiae strain p+ IL 8-8C is grown as described P~eparaEi~n-oEmIEo~~ionaria is performed according to (6) except that cyto92390 VILLENEUVE LA GARENNE) is used (7). Mitochondria helicase (I.B.F., are further purified on a sucrose gradient (8). Separation, delipidation and purification of the inner membrane 30K hydrophobic protein is performed as described in (1). Antibodies against the hydrophobic proteins are produced in rabbits by means of four consecutive injections of the hydrophobic protein in a 8 week period : the first injection in the muscle comprised 0.6 mg of protein in 1% (w/v) SDS solution supplemented with complete Freund's adjuvant ; the :; To whom all

correspondence

should

be addressed. 0006-291X/83/030945-06$01.50/0

945

Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 110, No. 3, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

second is 1 mg in 1 %0 SDS intravenously, the third is 0.6 mg in 1 %. SDS subcutaneously supplemented with complete Freund's adjuvant and the last is 1 mg in 1 $0 SDS intravenously. Antibodies against the yeast apocytochrome b were kindly provided by G. SCBATZ and are described in (9). The immunological analysis of the hydrophobic protein is performed as in (10) for ELISA and as in (11, 12) for the Ouchterlony immunodiffusion. Precipitation of antigens complexed with antibodies by the ------r-------. Staphylococcus aureus protein A (13) is realized as follows : the 30K hydrophobic protein T----is mixed with antibodies against the cytochrome b ; then S. aureus bearing ------T-protein A is added to precipitate the complex ; the antigen-antibodies complexes are eluted with a tris-buffer 10 mmol/l, 2-mercaptoethanol 0.15 mol/l, EDTA 1 mmol/l, glycerol 10 % (v/v) containing 1 % SDS. Eluted products are deposited on 9 per cent polyacrylamide gel. The same procedure is realized with a total inner membrane proteins solution containing the as a positif control and with the 30K hydrophobic cytochrome bc 1 complex, protein mixed with non immunized rabbit serum as a negatif control. Gel electrophoresis is carried out in presence of 1 8 (w/v) SDS according to (14) with 4 mol/l urea added. Protein is estimated by the method of Lowry (15) with bovine serum albumin as a standard. The mutant yeasts used are : mit- cyt b-226, a pin point mutation in COB region (16) ; Gl71, a box (4-2) mutation leading to synthesis of 14 K cytochrome b (17), instead of normal 32 K cytochrome b as in the wild strain; G 625 a large deletion, with no cytochrome b synthesis at all (18). They are grown anaerobically as described in (5), except that the concentration of the galactose in the growth medium is increased to 10 % and glucose is omitted. Preparation of promitochondria is performed as in (19). RESULTS 1. The 30K hydrophobic phobic

protein

liberated) does not against

protein

antibodies

tested

by ELISA

shows a maximal

against

colour

at 400 nm when the antigen-antibody improve

with

cytochrome A

higher

amounts

b antibodies,

of the

specific

intensity ratio

the hydrophobic

(p. nitroaniline

(w/v)

antibodies

30K hydro-

is

l/10

(Figure

1).

protein

does not

; it Tested react.

401 1.0

(La

0.6

0.3

1

1

10 20

I

I

1

1

50

100 anti

150

200

pg

Figure

protein

1 : Titration curve of 3OK hydrophobic yeast mitochondrial inner membrane protein (10 ).lg) with specific antibodies, by ELISA . Secondary antibodies are labeled with E. coli alcaline phosphatase. 946

Vol. 110, No. 3, 1983

BIOCHEMICAL

AND BIOPHYSICAL

2

RESEARCH COMMUNICATIONS

4

Densitometer

tract

0.5 E c it

0.4

m

0.2

;i;

0.1

0.3

8

0

5 0 2

0.5 0.4

9

0.3 0.2 0.1 0 0.5 0.4 0.3

0.2 0.1 0

Direction

Figure

Total mitochondrial cytochrome b). 3OK hydrophobic 3OK hydrophobic (negatif control).

b) c)

2. Complex

precipitation

densitometer

trace

and

with

inner

and 30K hydrophobic

shown

the

to be precipitated

3. Ouchterlony cytochrome protein diffusion obtained

total

immunodiffusion

membrane

fraction,

specific

antibodies

with

the or

: The center

precipitation But on the

and staining. either

b antibodies well

the peripheral-well

; no detectable

cytochrome

with

(Results

the

electrophoresis b antibodies,

serum

; peaks

32K of cytochrome protein

Subunit

proteins (Fig. contains

b. is

32K of solution

is

2a). 20 ~1 of the

10 ~1 of 30K hydrophobic

lines could be obtained after other hand precipitation lines are b antibodies

hydrophobic

not

2b).

membrane

subunit.

30K hydrophobic

when the 30K hydrophobic (Fig.

32~

b

rabbit

4 to subunit

inner

serum

cytochrome

non immunized

by cytochrome

b antibodies,

with

against

b antibodies

in the

rabbit

2 shows gel

obtained

proteins

against

cytochrome

b present

immunized

with

(containing

; 4 : cytochrome

complexes

be obtained

non

A : Figure

to immunoglobulines, could

with

fraction

membrane

protein

1, 2, 3 correspond treated

with

protein

proteins

membrane

protein. protein

of the three

total

No precipitation

inner

: immunoglobuline

1,2,3

cytochrome

b

2 : 9 % polyacrylamfde gel electrophoresis of immune complexes obtained with yeast cytochrome b antibodies, precipitated A and eluted with tris-buffer. -S.- - aureus - - - - - - protein a)

protein

of migration

shown). 941

protein

tested tested

against against

the

inner its

Vol. 110, No. 3, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

1

A400

1

2

3

4 pg

Figure

for

the

The hydrophobic of protein

is

Assayed can be found

from p+ yeast the three

strain.

mutants

same mobility

extracted

7

6

However

strain.

expected even

it

yeast.

contains

only

wild

gel

proteins

DNA mutants

the

log

a very

phase small

protein

cell amount

extracted

electrophoresis

of

show a 30K band with

protein

extracted

and the

two mitochondrial

strain

of the

30K hydrophobic

from

the + the p strain. mutants

30K hydrophobic

protein

and for

protein cytochrome

b. Results in Figure 3 show that 30K hydrophobic protein when compared to the

does not does react

14 K instead

contain with

any cytochrome the

cytochrome

32 K cytochrome

of

b, as b antibodies

(9),

b, as in the

DISCUSSION Yeast

mitochondrial

mitochondrial

origin.

synthetized another

inner

known

inner

membrane

proteins

The hydrophobic

on mitochondrial membrane

:

cytochrome

contain mutant

only

the hydrophobic

the presence

against

The G625 mutant ; the Gl71

wild

membrane

for

against still

with

of the hydrophobic

antibodies

the two mutants

of Lowry,

SDS-polyacrylamide

inner

of the yeast

antibodies

in 3 mitochondrial

in each case from

by the method

and G625 are analyzed

by ELISA with

protein

as compared

total

as that

membranes

b, with wild

30K hydrophobic

protein

promitochondria.

Gl71

6

protein

3 : Immune reactivi+ty measured by ELISA mitochondrial inner membrane proteins from p yeast (O,O),from.mutants Gl71 with cytochrome b 14 000 (A,A) and ~625 without cytochrome b (0,m) against 30K hydrophobic protein antibodies (0,A.o) or against cytochrome b antibodies (O,A,W.

4. Search

Inner

5

of

nuclear

protein

studied

in this

(1).

We compare

this

ribosomes protein,

may be of either

taking 948

into

account

work

or is

protein the

following

with

Vol. 110, No. 3, 1983 properties

BIOCHEMICAL

: aminoacid

aminoacids,

composition,

polarity

can be noticed cytochrome

AND BIOPHYSICAL

molar ratio of acidic to basic molecular weight. A satisfactory

and apparent

between

the hydrophobic

bc 1 complex

RESEARCH COMMUNICATIONS

described

protein

by Katan

and subunit

and Groot

(3, 4) but

obtained There is

here, show antigenic differences between both. no cross reaction between the 3OK hydrophobic protein

from

inner

the

cytochrome

membrane

immunochemical

involves

might poor

in

figure

toward

mutations

in the COB region

are

able

still

in the

p

+

strain,

Mutation

respiratory

is apparently react well

hand yeast protein

in smaller

in the COB region but

amounts.

with

It

must

does not

hinder

respire

of the

synthetize

the

3OK hydrophobic

results the

cytochrome

suggest

32K subunit

of yeast

protein

that

with

the hydrophobic

(G625),

the 30K hydrophobic b, derived

our results. protein

is

are

still

of the mitochondrial

of the cytochrome

cytochrome

accordance

b at all

protein

bc 1 complex.

impair

3OK hydrophobic

is

The precise

a does

to synthetize

membrane.

not

Our

identical

with

The amino

acid

composition

analysis

(21)

is

from DNA sequence

now under

able

inner

the

mitochon-

protein. Our results show that a mitochondrial mutant synthetizing 14 K cytochrome b (G171) and an other mitochondrial mutant which not

one found

that

DNA does

synthesis

but

as the of entire

of the mitochondrial

localization

investigation

case,

having

be noticed

in place

the the

identical

properties

(20),

protein,

not with

strains

DNA do not

and immunological promitochondria

antibodies

30K hydrophobic

This antibodies

3OK hydrophobic

to the native

and the

extracted

specific

only

function,

membrane,

protein.

though

synthetize

: in gel,

as compared

of the mitochondrial

to synthetize

of the

The different

us the same results

protein

On the other

electrophoretical

mutants dria.

the

we extracted

A, immunoprecipitation

toward

protein.

results

Purification of the 3OK hydrophobic solvents. The procedure might induce inner

the native

3 shows that

30K hydrophobic

solubility,

gaves

of the hydrophobic

have good specificity

the

and the 32~ subunit

same membrane.

of protein assay.

the mitochondrial

affinity

because native

aid

the use of organic changes

present

raised but

the

immunosorbent

conformational protein

in the

of the problem

with

or enzyme-linked

mitochondria

present

approaches

immunoprecipitation protein

of yeast

bc 1 complex

analogy

32K of the

and the in our

in

function

of

laboratory.

ACKNOWLEDGMENTS We thank

J.

Strasbourg) H. Fukuhara (Gif-sur-Yvette) (Basel) by the

and M. Acker

Malgras

(Centre

de Transfusion

Sanguine

for help in preparation of the 3OK protein antibodies, (Orsay) for the gift of mutant mit- cyt b- 226, P.P. for

the

gift

of mutants

for the gift of antiapocytochrome Centre National de la Recherche 949

G171 and ~625, b serum. Scientifique.

This

de SJonimski,

and G. schatz work

was supported

Vol. 110, No. 3, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNlCATlONS

REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

19 20 21

- Felter, S. and Stahl, A.J.C. (1978) Biochimie, 60, 1175-1179. - Alkoutayni, M. and Stahl, A.J.C. (1982) submitted for publication. - Katan, M-B., Pool, L. and Groot, G.S.P. (1976) Eur. J. Biochem., 65, 95-105. - Katan, M.B. and Groot, G.S.P. (1976) Genetics and Biogenesis of chloroplasts and Mitochondria, Bticher, Th. Neupert, W., Sebald, W., Werner, S., North-Holland Publishing Company, Amsterdam, 273-280. - Schneller, J.M., Schneller, C., Martin, R. and Stahl, A.J.C. (1976) Nucleic Acids Res., 3, 1151-1165. - Faye, G., Kujawa, C. and Fukuhara, H. (1974) J. Mol. Biol., 88, 185-203. - Diatewa, M., Viard, I. and Stahl, A.J.C. (1980) Biochimie, 6x67-69. - Accoceberry, B. and Stahl, A.J.C. (1972) C.R. Acad. Sci. Pazs, 274, 3135-3138. - Solioz., M. and Schatz, G. (1979) J. Biol. Chem., 254, 9331-9334. - Engvall, E. and Perlmann, P. (1972) J. Immunol., 109, 129-135. - Ouchterlony, 0. (1953) Acta. Pathol. Microbial. Stand., 2, 231-240. - Poyton, R.O. and Schatz, G. (1975) J. Biol. Chem., 250, 762-766. - Kessler, S.W. (1975) J. Immunol., 115, 1617-1624. - Weber, K. and Osborn, M. (1969) J. Biol. Chem., 244, 4406-4412. - Lowry, O.H., Rosebrough, M.J., Farr, A.L. and Randall R.J. (1951) J. Biol. Chem., 193, 265-275. - Wesolowski, M. and Fukuhara, H. (1979) Mol. Gen. Genet., 170, 35-45. - Lazowska, J., Jacq, C. and Slonimski, P.P. (1980) Cell, 22, 333-348. - Slonimski, P.P., Pajot, P., Jacq, C., Foucher, M., Porrzn, G., Kochko, A. and Lamouroux, A. (1978) in Biochemistry and Genetics of Yeasts (Bacila, M., Horecker, B.L. and Stappani, A.O.M., eds) 339-368, Academic Press, New-York. - Accoceberry, B. and Stahl, A.J.C. (1970) Bull. Sot. Chim. Biol., 52, 113-123. - Schatz, G. and Saltzgaber, J. (1969) Biochim. Biophys. Res. Commun., 37, 996-1001. - gbrega, F.G. and Tzagoloff, A. (1980) J. Biol. Chem., 255, 9828-9837.

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