Localisation of the hydrophilic C terminal part of the ATP synthase subunit 8 of Saccharomyces cerevisiae

Vol.

138,

No.

July

16,

1986

1, 1986

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

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Pages 78-86

LOCALISATION OF THE HYDROPHILIC C TERMINAL SYNTHASE SUBUNIT 8 OF Sccharomvcesmm Jean

VELOURS and Bernard

Institut de Biochimie Cellulaire 1, rue Camille St Saens 33077

PART

OF

THE ATP

GUERIN

et de Neurochimie BORDEAUX Cedex

du CNRS FRANCE

Received May 20, 1986 The hydrophobic subunit 8 of the yeast ATP synthese W8S modified using the non-penetrating amino reactive specific reagent :isethionylacetimidate. The polypeptide w8s modified when using the isolated ATP sYnth8Se and sodium bromide-treated submitochondrial particules. It is shown that the only lysine of the protein was modified by the reagent. It is concluded that the hydrophilic C terminal part of the protein containing lysine 47 is located on the inner side of the inner mitochondrial membrane. 0 1986

Academic

The

Press,

membrane-embedded

three

hydrophobic

yeast.

The

(subunit third ATP

Inc.

and

product

of

structure

of

molecular

(85%)

8s

Prediction

is

hydrophobic ABBRE-VIATIPNS pOrtiOnS of iA1 * PMSF ; : PITC PTC : t1c :

this

code

encoded is

is

measured

circular

have

shown which

by

respectively

which

88~1

gene

isolation

and

118 amino

the

protein

Inc. reserved.

78

to the

the acids

This In

long(?). content

spectroscopy subunit may traverse

a

primary

ahelical

:Fl and Fo : periqheral and integral the H+tr8nslOC8tipg ATP SynthaSf?, isethionyl8cetimid8te, phenylmethylsulfonyl fluoride, phenylisothiocyanater phenylthiocerbamyl, thin layer chromatography,

$1.50 0 1986 by Academic Press, of reprodurrion in any ,form

The

(6).

solvents(h).

dichroism that

belongs

in organic

is

in

of 8000 Mr (1.2.3).

which

the

the

the

contains DNA

proteins

5855 Da, and has 8 high

by

with

for

6)

soluble

reported

ATP synthase

ribosomes

proteolipid

weight

domain

loci

the

by the mitochondrial

Mr (subunit

8).

we

methods

of

the mitochondrial

(4.5)

psper

part encoded

0112

21500

(subunit

preceeding

0006-291X/86 Copyright All rights

and

9)

polypeptide,

Its

subunits

Olil

synthase

Fo

(8).

8 contains the membrane

inner

a

Vol.

138,

No.

1, 1986

mitochondrial of

the

outside

membrane protein the

terminus

BIOCHEMICAL

is

mitochondrial

once

localized

(7).

BIOPHYSICAL

RESEARCH

Furthermore

the

a hydrophilic

contains membrane.

AND

In

this on

domain

paper, the

we inner

COMMUNICATIONS

C terminal

which

may protrude

propose side

Part

of

that

the C

the

inner

membrane.

Isefhionyl(lacetimidate 2, (0.9-1.5 were obtained TBq/mg) Centre,Amersham. IAI, Lysyl-lysine Sigma. PITC was from Pierce.

(2.1 GBq/'mmolf and 330t from the Radiochemical and PMSF were purchased from

PreDarations

Cells of th; diploid yeast strain "yeast foam" were grown aerobica:Lly with 2% galactose as carbon source(g). Mitochondria were prepared by the manual shaking method in the Presence of 1 mM PMSF(10). In vivo labelled mitochondria were obtained from small scale experiments using 35SO 2 as label, in the presence of cycloheximide(l1). The ATP synthase was immunoprecipitated by Fl antiserum as in (12). 3sS-.Labelled mitochondria were suspended in the isolation buffer;O.iiM mannitol, 10mM sodium phosphate,lmM PMSF pH 7.0, One half of the sample was centrifuged and the mitochondrial pellet was suspended in the following incubation buffer:0.6M mannitol,O.lM triethanolamine, 1mM PMSF pH a.0 at a protein concentration of 2.7 mg/ml. The other half of the sample was at 80 volts (Hanemass sonicator). Sodium sonicated for 3 min. was added at a final concentration of 4 M and the mixture bromide for 1 hour at O"C.The submitochondrial particules was incubated were recovered by centrifugation in a Beckmam airfuge at 1OOOOOg 10 min. was washed with the isolation for The floating layer buffer, and the pellet obtained after centrifugation was finally suspended in the incubation buffer at a protein concentration of 2.7 mg/ml. IWlif’ication

of

the

subunit

fl with

IAL

:

35S-ATP synthase immunoprecipitates obtained from 2 mg of 35S-labe:Lled mitochondrial protein were suspended in 0.1 ml of 10 centrifugation the Pellets were mM sodium phosphate pH 7.0. After for 30 min. at O'C in 0.05 ml of 0.1 M triethanolamine incubated or not 4 M of sodium bromide. IA1 PH a . 0 some of which contained was added at a final concentration of 50mM and incubation was 24°C. The reaction was stopped by carried out for 90 min. at addition of 0.01 ml of 1 M tris-HCl pH 7.0, and the suspension was extracted with 20 volumes of chloroform:methanol (2:l) for 2.5 hours at room temperature. 4 volumes of water were added for washing and the organic phase was concentrated under nitrogen. The dried residue was dissolved in 0.02 ml of chloroform:methanol (2:l) and analyzed by chromatography as in (7). SsS-labelled mitochondria and submitochondrial particules (0.5 in 0.18 ml of incubation buffer) were incubated for mg protein 45min. at 24°C with 27 mM and 55 mM of IA1 (10 umoles IA1 and 20 moles IAI/ mg of protein respectively). The reaction was steppe 8 with tris-HCl as above . Mitochondria and submitochondrial were particules centrifuged. The pellet was suspended in 0.02 ml of incubation buffer and the proteolipids were extracted upon 79

Vol.

138,

No.

1, 1986

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

chloroform:methanol (2:l). The soluble addition of 1 ml of the aqueous phase was fraction was washed with 0.25 ml of water. organic phase containing the proteolipids was discarded and the The dried residue was solubilized in 0.02 dried under nitrogen. ml of chloroform:methanol (2:l) and 0.08 ml of pre-cooled diethyl ether was added. The mixture was centrifuged and the pellet was dissolved in 0.02 ml of chloroform:methanol (2:l). The labelling of mitochondria and submitochondrial particules by S'C] -IA1 was performed as above, but the proteolipids were precipitated twice with diethyl ether in order to remove most of of ["CC]-IA1 and labelled lipids. the degradation products 14 C -IAI ng of the subunit 8 by Unlabelled ATP synthase immunoprecipitates obtained from 1 mg of mitochondrial protein were dissociated with sodfvm bromide as above. They were incubated with 8.8 u moles of L c]-IAI (18.5 MBq)(total volume:0.35 mli for 90 min. at 2ll'C. The proteolipids were extracted with 7 ml of chloroform:methanol (2:l). The insoluble residue was then eliminated by centrifugation . After mixing with 1.5 ml of water, the aqueous phase was discarded and the organic phase was dried under nitrogen. Next, 80~s of unlabelled subunit 8 were added as carrier. The proteolipids were precipitated upon diethyl ether addition as above. The proteic pellet was solubilized in 0.1 ml of chloroform:methanol(2:1) and chromatographed as in (7). The area between Rf 0.6 and 0.7 was scraped and extracted with 10 ml of chloroform:methanol (2:l) for one hour at room temperature. The insoluble residue was eliminated by centrifugation. 11 volumes of water were added and the organic phase was concentrated to dryness. The dried residue was hydrolyzed with 6N HCl and the amino acids were coupled to PITC as in (13). bgparation of PTC.N6-acetwo luti of lysyl-lysine in 0.2 M triethanolamine PH 9.0 was 1 1-I mole IA1 for 2 hours at 2&'C. After 10 incubated with 5 ,,moles of min. of incubation the pH was adjusted to 9 by addition of of the reaction was 0.3 ml). triethanolamine (the total volume The mixture was dried and hydrolyzed with 0.1 ml of 6N HCl for 211 hours at 105'C. The amino acids were coupled with PITC as in Analysis of PTC derivatives was performed by tic on reverse (13). KC 18F) using methanol:water(l:l), 0.05 M phase plates (Whatman ammonium acetate pH 6.8 as solvent.

Figure could

1

shows

cross The

35.

the

contains

residue

is

a

free

amino

to

be

part

an

group.

provides of

the

protein

which

only

lysine

residue Thus, target

information (matricial

domain

presumably

domain the

blocked,

ideal

hydrophobic

membrane,

hydrophilic

membrane

could

the

&7 is

from

probably

the

a protein on the side 80

which

residue

the

only group

Since

amino

outside

localisation or intermembrane

the

the

N terminal

acid

containing

of lysine

labelling

Protein

15 to residue

protrudes

residue.

the s amino for

with

&7 appears

reagent, of the

which

C terminal

side).

Vol.

138,

No.

1, 1986

BIOCHEMICAL

1

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

10

f-MPQLVPFYFMNQLTYGFLLMITLLILFSOFFLPMIL

hydrophobic

hydrophilic

domain

domain

\

iSU2X-l:

Amino acid sequence of the subunit 8. The sequence was described in (7). The amino acid residues are represented in the one-letter code as defined in (18). + basic residue: * The star indicates the only lysine residue of the subunit 8.

To

this

end, this

chosen; with

shows

2

modification

that

of

bromide

when

addition.

of

70%

sample

subunit

and

only

when

whole

as in

subunit

8.

of the

the

in

the

The intense

subunit

to

significant plates of

(0.67

subunit

8

upon

autoradiography

showed that

the

sodium

sodium

bromide-treated This

was not

8

Fl

or

depleted

8 (Fig.X-D).A

spot

an

was dissociated

sample.

2, and represented

Figure

tic amount

(Fig.3A-B)

subunit

a

to

result

could

due to Fl.

mitochondria IA1

IA1

on

untreated

hindrance

of

in

(la).

to

A larger

ATP synthase

that

modified

Rf:0.67 of

34%

Addition

particules

8).

8 was modified

shows

3

using

shown).

the

led

was

conditions

unlabelled

proteolipids

subunit

by a steric

Figure

of

physiological

of

synthase

Densitometry

the

be explained

Rf

reagent

and alcohols

addition

for

0.6

modified

was

the

under

amidines

ATP

of

non-penetrating

reacts

to form

amines

35S-immunoprecipitated

instead

insoluble

was IA1 which

primary

Figure

lipid

a polar,

modified

by IAI

mitoplast

(not

submitochondrial new spot

about

at Rf 0.4

34%

appeared

of the

corresponded

at

quantity

to subunit

9 (7). We

investigated

membranes. contaminating

In

the

reaction

spite lipids

of and

of the diethyl

degradation 81

labelled ether products

IA1 to unlabelled precipitation

steps

of IA1 were still

Vol.

138,

No.

1, 1986

BIOCHEMICAL

Rf ABC

AND

BIOPHYSICAL

-Front

RESEARCH

Rf ABC

0.67 0.6

COMMUNICATIONS

0 E -Front

0.67

iB

0.4

-Origin

02

1 -Origin

03

Fiaure:

P

1

Modification of the proteolipids of ~%)ATP synthase by unlabelled IAI. 35 S-immunoprecipitates were incubated in the absence (A) or presence (B) of IAI. The proteolipids were extracted and chromatographed on tic plates. (C): UM sodium bromide pretreated ATP synthase immunoprecipitate incubated with IAI. Finure:

Modification of the proteolipids at the mitochondrial and levels. The proteolipids were submitochondrial particule purified (see method section) and extracted, partially chromatographed as in Figure 2. extracted from whole 3!S-labelled A and B : Proteolipids of IAI/ mg of mitochondria incubated with 10 u and 20 p moles protein respectively. from 35S-labelled sodium C and D : Proteolipids extracted bromide-treated submitochondrial particules incubated with 10 and 20 pmoles of IAI/ mg of protein respectively. E: control: proteolipids extracted from 3%-labelled mitochondria incubated without IAI.

present.

Figure

products

.

contains in

a

the

fraction whole a with

4 shows

The

submitochondrial

shoulder

at

sodium

4

mitochondrial

diethyl

particule

Rf 0.67.

C).No

This

product, ether

(not

since

shoulder

was

(Figure it

shown),

82

observed n A).The

disappeared

the

labelled

fraction appeared

submitochondrial

spot

fraction

of

analysis

bromide-depleted

(Figure

non-proteic

a densitometric

(Fig.UB) as a peak particule

at Rf 0.67 peak

in

at Rf 0.6

upon precipitation

the was

Vol.

138,

No. 1, 1986

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Finure:

Labelling of proteolipids by [J1'C -1AI. Unlabelled mitochondria submitochondrial particules (B) and sodium brqmide-treated (A), submitochondrial particules analyzed (EL w;;z incubflted withp'C]-IAI. The proteolipids were as In Figure 2,and the autoradiography was scanned. densitometric analysis of Figure 3D. (D) : Control: su.9: subunit 9 ; su.9 IAI: subunit 9 modified by IA1 su.az subunit a ; Su.8 IAI: subunit 8 modified by IAI.

We verified lysine

target

the

This was possible

117.

during

stable

iS

that

of

IA1

to the

because of

hydrolysis

amidine

The

(15).

subunit

the

8 was

formation,

experiments

which were

as

follows: i)

Unlabelled

and IAI,

lysyl-lysine

ii)

N6-acetamidino

Unlabelled

extracted

ATP

and

-acetamidino

as described

subunit

bromide-treated

8

synthase

hydrolyzed, lysine.

PTC

N6 -acetamidino

&sine

(Rf:0.26)

lysine

Figure lysine

was due to

was prepared

from the peptide

in the method section.

was modified and the thereby

by

c'k 1-IA1

14C-labelled

generating

from sodium subunit

the

8 was

I: 1 14

c

-N6

5B shows the migration

of unlabelled

(Rf:0.6).

amount of PTC

a loss 83

The of the

small

acetamidino

group

under

Vol. 138, No. 1, 1986

0.67.

BIOCHEMICAL

Assuming

our

C

upon

when

using

used.

hydrophilic the

to

note

that

of

the

three

is to

a

backbone

N

widely

of

with

8 is (Fl

membrane.

This

location

the

side).

subunit

other

amino

since the

starting of

is

the side

interesting

to a localisation side

negative may

the

matricial

It

8 led

the

of the

location

on

the

obtained

rejected,

the

of subunit

with

decrease

whatever

results.

on

it

on

the

which

only

and is

C

be

of

related

the

of other

44)

(16)~

that

the yeast

that

location

the membrane,

certain

crossing

the

to the

predicted

was

groups

around

(due

to

located,

be

N

findings from

the 14:

by unpaired residues

proline

in

the

position

generated

of non-integration

could

of

was predicted

was presumably

favor

termini

of

one

carbonyl

in

the

membrane.

one on each

side,

2 So,

of

the

membrane.

chemical

asymmetry

side

domain

mitochondrial

This

outer

be concluded

(7) ; a 6 turn

amide

and

cannot

fact:

hydrophilic

the

particules

labelled

residues

sequence

6),

agreement

was

membrane

outside

in

an eventual

basic

this

primary

and

these

orientation

although

terminus

of

9) was

part

is

ATP

potential.

Thus,

inner

(-subunit From

the

mitochondrial

point

mitochondria

under

protruding

due to competition

47

mitochondrial

:Lnner

membrane

of

8 reacted

bromide-treated

protein

value

whole

C terminal

of

and

lysine

proteolipid

material

the

possibility

on

other

of

submitochondrial

amount

groups

subunit

sodium

This

48%.

son:Lcation.The

label

part

about

inverted

Of

(Cf.

terminal

represent

amount

accessible

conditions

synthase),the

9.

70% of the

that

experimental

could

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

is

facilitated

towards

the

approach

the

targets

proteolipid,

From prediction E. & mitochondria,

subunit

contains methods,

could

reactive 2 lysine

Hoppe

c, which

is

traverse 85

when

the

is

products. residues

and Sebald

analogous

there

to the membrane

Subunit (1~s

(17)

an

8 and

proposed subunit twice

9 in

a

Vol.

138,

No. 1, 1986

hairpin

like

structure.

and lysine

side

experimental subunit treated

de les

BIOCHEMICAL

Thus,

411 on the

other

(Figure

3)

data g

AND

when

submitochondrial

using

whole

lysine side

RESEARCH

8 would of the

show

COMMUNICATIONS

be located

membrane.

as starting

and

on one

Indeed,

a modification

mitochondria

particules

This work was supported Bordeaux II and the membranes biologiques).

BIOPHYSICAL

of the

our Rf of

sodium-bromide

materials.

by research grants from the Universite CNRS (ATP: Conversion de l'energie dans

l.HENSGENS, L.A.M., GRIVELL, L.A., BORST, P. AND BOS, J.L. (1979) Proc. Nati. Acad. Sci. USA, 76 ,1663-1667. 2.MACIN0, G. and TZAGOLOLOFF, A.(lg7g) J. Biol. Chem., 254, 0617-U623. 3.MACIN0, G. and TZAGOLOFF, A. (1980) Cell, 20, 507-517. U..ESPARZA, M., VELOURS, J. and GUERIN,B. (1981) FEBS Lett., 134, 63-66. 5.MACREADIE. I.G., CHOO, W.M., NOVITSKI, C.E., MARZUKI, S., NAGLEY, P., LINNANE, A.W. and LUKINS, H.B. (1982) Biochim. Int., 5, 129-136. 6.MACREADIE. I.G., NOVITSKI, C.E., MAXWELL, R.J., JOHN, U.. 001, B.G.. MC MULLEN, G.L., LUKINS, H.B., LINNANE, A.W. AND NAGLEY, P. (1983) Nucleic Acids Res., 11, 4435-4451. 7. VELOURS, J., ESPARZA, M., HOPPE, J., SEBALD, W. and GUERIN, B. (1984) The EMBO J. 3. 207-212. 8.DAUMAS, P., HEITZ. F.. VELOURS, J. and GUERIN, B. (198k) Proceedings of the International Forum on Peptides. Cap d'Agde {France). 9.ARSELIN de CHATEAUBODEAU, G., GUERIN, M. AND GUERIN, B. (1976) BIOCHIMIE, 58, 601-601. lO.LANG, B, BURGER, G., DOXIADIS, T., THOMAS, D.Y., BANDLOW, W. and KAUDEWITZ. F. (1977) Anal. Biochem., 77, 110-121. ll.VELOURS, J., GUERIN, M., and GUERIN, B. (1980) Arch. Biochem. Biophys. 201. 615-628. 12.TODD. R.D., MC ADA, P.C., and DOUGLAS, M.G. (1979) J. Biol. Chem. 254, 11134-11141. 13.HEINRIKSON. R.L. and MEREDITH, S.C. (198U) Anal. Biochem., 136, 65-74. lh.;;;fI;;;Y, N.M. and BERG, H.C. (1974) J. Mol. Biol., 87, . 15.REPKE. D.W. and ZULL, J.E. (1972) J. Biol. Chem.. 247, 218g-21911. 16. SEBALD. W., HOPPE, J., and WACHTER, E. (1979) In Quagliariello, E. et al. (Eds), Function and Molecular Aspects of Biomembrane Transport, Elsevier/North-Holland, Amsterdam, pp. 63-74. 17.HOPPE. J. and SEBALD, W. (1984) Biochim. Biophys. Acta., 768, l-27. lS.IUPAC-IUB Commission on Biochemical Nomemclature (1968). J. Biol. Chem., 2L13, 3557-3559.

86