F0F1-ATPase of plant mitochondria: Isolation and polypeptide composition

Vol.

170,

August

No.

3, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

16, 1990

Pages

FoFl-ATPase

AND POLYPEPTIDE

Beston

HAMASUR and Elzbieta

of Biochemistry,

Stockholm Received

June

25,

OF PLANT MITOCHONDRIA:

ISOLATION

Department

University,

1352-1358

COMPOSITION GLASER*

Arrhenius S-106

Laboratories,

91 Stockholm,

Sweden

1990

SUMMARY: A simple and high yield purification procedure for the isolation of F,Fl-ATPase from spinach leaf mitochondria has been developed. This is the first report concerning purification and composition of the plant mitochondrial F,Fl-ATPase. The enzyme is selectively extracted from inner membrane vesicles with the zwitterionic detergent, 3-[(3-cholamidopropyl) dimethyl arnnoniol-l-propane sulfonate (CHAPS). The purified enzyme exhibits a high oligomycin-sensitive ATPase activity (3,6 pmol. min-l . mg-1). SDS-PAGE of the purified F,Fl-ATPase complex reveals protein bands of molecular masses of 54 kDa (Fla,B), 33 kDa (Fly), 28 kDa, 23 kDa, 21 kDa (F16), 18.5 kDa, 15 kDa, 10.5 kDa, 9.5 kDa (Flc) and 8.5 kDa. All polypeptides migrate as one complex in a polyacrylamide gradient gel under non-denaturing conditions in the presence of 0.1% Triton X-100. Five polypeptides could be identified as subunits of Fl. Polypeptides of molecular masses 28 kDa, 23 kDa, 18.5 kDa, 15 kDa, 10.5 kDa, 9.5 kDa and 8.5 kDa constitute the F, part of the complex. Our results show that polypeptide composition of the plant mitochondrial F, differs from other eukaryotic F, of yeast, marnnals and chloroplasts. ': 1990 Academic Press, 1°C. The membrane-bound talyze is

the

terminal

highly

F1 part

activity

(1) the

species

examined. the

which

membrane

subunit

of mitochondria,

of the

and can be easily

and the

through

rast,

reaction

conserved

rophilic

F,Fl-ATPases

contains hydrophobic (2).

the

oxidativedissociated

F1 consists

of five

composition

of the membrane

*To whom correspondence

should

into

two distinct

nucleotide-binding

of the subunits

and bacteria

and photophosphorylation.

membrane-integral

The F1 part

chloroplasts

site part,

enzyme

has similar

designated sector

Fo,

The enzyme parts,

and catalyzes

F. which

a,B,y,G varies

ca-

conducts structure

the

hyd-

ATPase protons in all

and E. In contamong different

be addressed.

Abbreviations: F. and F1, proton-translocating and catalytic moieties of the mitochondrial Al 'P synthase, respectively; OSCP, oligomycin sensitivity conferring protein; DCCD, N,N'-dicyclohexylcarbodiimide; CHAPS, 3-[(3-cholamidopropy?)dimethylamnoni o]-1-propanesulfonate; DTT, dithiothreitol; MOPS, 3-(N-morpholine)-propane sulfonic acid; PMSF, phenylmethylsulfonylfluoride; EDTA, ethylenediaminotetraacetic disodium salt; Tricine, N-[2-hydroxy-l,l-bis-(hydroxymethyl)+ ethyl glycine; PVDF, polyvinylidine difluoride. 0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, inc. All rights of reproduction in any form reserved.

1352

Vol.

170,

species.

No.

3,

1990

A composition

roplast

CF,

(5)

protein

ding

bacterial

F,FI-ATPases

have

membranes

purification

present

complete

stable

and functionally

ATPases

rat

(12)

isolated

from

other

associated

been purified

from

bovine (13).

of the

active. spinach

We discuss mitochondrial

leaf

has been

from

procedure mitochondria.

correspondence F,FI-ATPase

in the

a variety

of

and yeast there

plant

is

chlo-

reported

endogenous

(9,lO)

However,

enzyme

a simple

spinach

heart

COMMUNICATIONS

4 subunits

a loosely

we describe from

(3,4), enzyme

(8),

composition

F,FI-ATPase

the

liver

F.

RESEARCH

mitochondrial

and chloroplasts

report,

of the

of

of

and subunit

In the

nits

in F, of the contains

the mitochondria

BIOPHYSICAL

in E. coli

enzyme also (7).

AND

of 3 subunits

and 5-8

The mitochondrial bitor

BIOCHEMICAL

(6).

ATPase

inhi-

sources

inclu-

(ll),

no report

of the

mitochondria.

for

the The

of the to the

first

isolation

enzyme is

isolated individual

subunits

subu-

of the

FoFI-

sources.

MATERIALS

AND METHODS

Spinach (Spinacia oleracea L.) was grown under artifical light Plant material: at 180C with a light period of 12 hrs in nutrient solutions as described in (14). Spinach leaf mitochondria and submiPreparation of spinach leaf mitochondria: tochondrial oarticles were prepared according to the Procedure described earlier in our laboratory (15) with the exception that 3 uM leupeptin and pepstatin were included in the medium before sonication during preparation of the particles. The spinach leaf submitochondriPurification of oligomycin-sensitive FoFI-ATPase: al particles were suspended at 20 mg/ml-in0,25 sucrose and 10 mM MOPS, pH 8.0 and diluted to 2 mg/ml in a medium containing 0,15 M K2HP04, 1 mM ATP, 1 mM MgC12, 25 mM EDTA, 0,5 mM DTT and 5% ethylene glycol, pH 8.0 and centrifuged for 45 min at 105000 g at 3°C. The pellet was washed in the same medium twice. The final pellet 1 mM ATP, 1 mM MgC12, 25 mM EDTA, 0,5 mM OTT and was resuspended in 50 mM Tricine, 5% ethylene glycol pH 8.0 (TA buffer) at a protein concentration of 4 mg/ml. The suspension was stirred on ice for 20 min with 0.75% (w/v) CHAPS in TA buffer and centrifuged for 1 h at 105000 g at 3°C. The supernatant containing solubilized crude F,FI was layered on a discontinous sucrose gradient. Layers of 1 ml 50% sucrose, 2 ml 25% sucrose and 9 ml 20% sucrose in TA buffer containing 0,2 % CHAPS were placed in a 13 ml Beckman tube. The crude FoFI (1 ml, 1,5 mg) was layered on top of the gradient. After centrifugation for 14 hrs at 27,000 rpm in a SW 40 rotor at 3"C, fractions of 0,7 ml were collected by bottom-puncture. Isolation of FI-ATPase: The FI-ATPase of spinach leaf mitochondria was isolated by a modified chloroform extraction procedure (16,17). Labelling with [14C]-DCCD: [I4C]-DCCD dissolved in toluene was dried under vacuum and dissolved in ethanol. Purified FoFI was incubated with [14C]DCCD/ethanol at a concentration of 5 nmol [I4C]-DCCD/mg protein for 3 h at 4°C (18). PAGE electrophoresis: SDS-PAGE was conducted in the buffer system of Laemnli (19) using a lo-30% linear acrylamide gradient in the presence of 4 M urea. In the PAGE system without SDS, 3-15x linear acrylamide gradient gels were used as in (20) except that 0.01 % (v/v) Triton X-100 was used instead of nonaethyleneglycoldodecyl. Imnunological analysis: Im'nunodecoration of Westernblots (21) was used for the quantification of the FI-ATPase during the purification procedure. Determination of proteins: Protein was determined with the Bio-Rad protein assay reagent of Bradford (22). Assay for ATPase activity: ATPase activity was determined spectrophotometrically at 37'C as described previously (23). 1353

Vol.

BIOCHEMICALAND

170, No. 3, 1990

BIOPHYSICALRESEARCH

COMMUNICATIONS

RESULTS AND DISCUSSION Purification

of FoFl-ATPase --

The F,Fl

ATPase

complex

of spinach

Washing

of the

step

procedure:

(1)

dium

containing

25 mM EDTA.

with

0.75%

(w/v)

a discontinous Fig.

CHAPS. (3) sucrose

1 shows

Spinach

leaf

EDTA (lane

fied

FoFl

(lane

Y subunit

with

using ticles

with

shown

isolated

purification

antibodies

EDTA removes

tion

(93%).

of the

of the

in a three

particles

of the membrane

fractions are are

with

bound

solubilized

crude

of the

enzyme.

the

a me-

FoF1-ATPase FoF1-ATPase

by the

kDa

gradient

F1 (lane

spinach

leaf

of the

content

in the

The removed

particles F,Fl-ATPase recovery

with

of the F1

mitochondrial

submitochondrial

all

puri-

e).

cross-reactivity

of almost

a high

ab

sucrose

was-

protein

Fl-ATPase CHAPS results

as shown

F1 pardoes

in a spe-

by the

of Fl-ATPase

(91%).

cde

96 KS-3020.114.b 8.24

1.

SDS-PAGE of different fractions during purification of the spinach leaf mitochondrial F,Fl-ATPase. Subtnitochondrial particles before (lane a) and after washing with EDTA (lane b), crude F,Fl (lane c), sucrose gradient purified F,Fl (lane d), isolated spinach leaf mitochondrial F1 (lane e).

1354

not

in the was-

67-

Fig.

on

procedure.

a) and after

The F1 protein

protein.

of the with

c),

mitochondrial

Washing

of the washed

% protein

(lane

isolated

recovery

purification

to (lane

immunological

original

solubilization 18.8

the

the

prior

F,Fl leaf

technique.

28% of the

Treatment

shown

crude

from

blot

during

spinach

against

as indicated

and quantitative

solubilization

Also

Western

Fl-ATPase

hed particles cific

the

submitochondrial

lization

particles

was estimated

quantitative

contain

b).

1 summarizes fractions

was purified

gradient.

d) and the

different

Solubi

Centrifuga

submitochondrial

with

mitochondria

iso lated

SDS-PAGE of different

hing

Table

(2)

leaf

The

Vol.

170, No. 3, 1990

BIOCHEMICALAND

TABLE 1. Purlffcatlon

of

Fractions

Submitochondrial

Crude

l

high

the

of the

3.1

1.0

100

13

72

0.52

4.0

1.3

93

18.8

0.51

15.0

5.0

91

4.8

0.48

54.0

17.4

85

it

in

the

lue

to the

is

is

close

purity

total

of the

TABLE 2. ATPase

0.48

f

in the

ATPase

activity

of submitochondrial

actfvity

and

of the particles

oligomycin

SenSltiVlty

speclflc ()h~l~mg-~

particles

Washed-submitochondrial

particles

FoF1-ATPase

Sucrose gradlent F,FT-ATPase

purified

oligomydn/mg

wfth

antf-

gradients

purified

is

as

FoF1-ATPase,

and from

the

5.9

0.57

purified

% of the

of total

mass of F1

mg of Fl.

FoF1,

as

recovery

the molecular

contains

the

(21).

which

This

va-

indicates

enzyme.

ATPase

Submitochondrial

on sucrose

value

FoFl

mg, found

F1 +' subunit technique

constitutes

different with

of dtfferent

protein

were

used

fractions

EDTA results

purification

is

shown

activity

of

steps

Increase total

. mln-l)

@aol.min-l)

0.4

7.2

1.3

16.9

the spec. activity -fold

the

spinach

of

Oligomycin* sensftlvity %

1.0

80

3.25

75

1.8

6.12

4.5

75

3.6

3.17

9.0

50

In the

assays.

1355

in

in an increase

F,Fl-ATPase

Fractions

* 2 nmol

recovery

of the blot

Assuming

mg purified

mitochondrial

Crude

F,Fl-ATPase

-fold

in the

F,Fl-ATPase

mitochondria.

0.88

F1 content

times

From this

that

leaf

F,Fl,

Oligomycin-sensitive 2. Washing

17.4

particles.

spinach

Western

FoF1-ATPase

enriched

can be calculated

protein

cross-reactivity FT using

oligomycin-sensitive

Fl-ATPase

to be 65% of

Table

0.56

0.88

submitochondrial

enzyme,

Purification

purlfied

as 85%. The

membrane

high

100

3.4

recovery

to

F,Fl-ATPase

18

F,Fl-ATPase

compared

mltochondrial

Fl-ATPase* liq x

FL-ATPase content was determlned from bmnunologlcal bodies against the isolated spinach leaf mitochondrlal

final

leaf

x

particles

Sucrose gradlent F,FL-ATPase

spinach

COMMUNICATIONS

Protein mg

particles

Washed-submitochondrial

the

BIOPHYSICALRESEARCH

leaf

of

Vol.

total

170, No. 3, 1990

ATPase

activity

due to the ally

release

however

procedure

the

specific

were

intrinsic

aches

were

used;

phoresis

of

this

me polypeptide of the

A

stained

mine1

which

is

The enzyme

of the

total

during

the purification

min-l

probably is

ATPase

parti-

activi-

mg-1.

l

purified

as that

shown

complex

protein

bands

bands

21 kDa,

18.5

In order

to examine

F,FI-ATPase

complex

enzyme

was analyzed

conditions

one single band

ten protein

23 kDa,

d).

of the

protein

F,FI-ATPase

blue

enzyme

pm01 *

revealed

28 kDa,

1 lane

of SDS, only single

l

protein.

decrease

of the

to 3,6

pmol

inhibitor

by the

non-denaturating

pattern

purified

Coorrmasie

Fig.

1) When the

absence

0,4

to 16.9

ATPase

activity

enzyme

components

under

7.2

RESEARCH COMMUNICATIONS

FoFl-ATPase --

33 kDa,

kDa (cf

bands

electrophoresis

from

purified

of 54 kDa,

kDa and 8.5

the

ATPase

of the

of the

masses

and in

from

endogenous

g-fold

compositon

SDS-PAGE

9.5

enzyme

by CHAPS as indicated

increases

Subunit

cular

of the of the

inactivated

ty,

BIOCHEMICALANDBIOPHYSICAL

in the

with kDa,

15 kDa,

whether

all

presence

in Fig.

d).

by laser

on the SDS-PAGE.

2A). 2B)

2) Different

kDa,

the protein approgel

of Triton

(Fig.

of SDS (Fig.

examined

10.5

on polyacrylamide

in the presence

were

mole-

two experimental

band was observed

1 (lane

apparent

X-100

Reelectroshows

the

sa-

preparations

scanning

of the

A constant

ratio

A

6

between

the

6

kDa

02

C

-front Fig.

2.

PAGE of the isolated spinach leaf mitochondrial F,Fl-ATPase denaturating (A) and denaturing conditions (6). The protein Fig. 2A was reelectrophorized in the presence of SOS (Fig.

Fig.

3.

Identification of the subunits of the isolated spinach leaf mitochondrial F,Fl-ATPase (B) by autoradiography of the [14C]-OCC0 labelled F,Fl (A) comigration with the purified potato ATPase inhibitor protein (C) and by Western blot of the isolated F,Fl with antibodies against the PVP protein (26) of beef heart mitochondrial F,Fl (D). 1356

under nonband shown in 29).

D

Vol.

No. 3, 1990

170,

content

of different

These

two experimental

intrinsic

BIOCHEMICALANDBIOPHYSICALRESEARCH components approaches

components

Five basis

of the

of

fied

FI-ATPase

with

antibodies

of the

relative of

mobility

against

10.5

kDa,

protein

9.5

was

[14]-DCCD

identified

and mamnalian (Fig.

F.

the

subunit

conferral

of FI spinach

(not

shown),

are

leaf

mitochondrial

with

the

suggest

that

23 kDa protein with

tochondrial beef

heart

shown

15 kDa and 8.5 identifing subunits.

her

subunits

differs

eukaryotic

our

results

F, moieties

kDa protein

protein

(24).

to oligomycin, (25)

(cf.

correspond

of the

coli.

mamnals

amino

FoFI-ATPase

from

the

previously

heart

PVP protein

in the acid

of

e) we

of about

Polypeptides

we are

J-terminal the

to the

20 kDa miof

of 28 kDa, process

sequences

spinach

in

23 kDa

1, lane

of bovine

dep-

protein

of the

Fig.

as

between

particles

comigration

PVP protein

b of k.

as well

of an OSCP-like

FI

kDa

c of the E. coli

submitochondrial

isolated

15 kDa,

with

The 10.5

existence

puri-

shown).

The 9.5

experiments

Currently,

composition

of yeast,

subunit

inhibitor

purified

show that

in polypeptide

kDa,

to OSCP. The polypeptide

identity.

by analysis

to the

Due to the

to subunit

of unknown

(not

complex.

interaction

heart

and may thus

to be homologous

In sumnary,

tochondria

30)

the

18.5

by its

reconstitution

of the

in the

against

23 kDa,

on the

interaction

FI-ATPase

FoFI-ATPase

may correspond

(Fig.

kDa are

these

indicative

are

to the

1) and by their

CF,.

ATPase

in

proteins

as compared

of the

chloroplast

and beef

present

antibodies

F,FI-ATPase

thus

FoFI-ATPase.

polypeptide

Fig.

F, part

mitochondrial

FI-ATPase

protein

cross-reacts

of the

sensitivity

mitochondrial

gel

protein

endogenous

leaf

of oligomycin

leaf

leted

to the

spinach

the

shown).

as FI subunits

mitochondrial

corresponds

III

found

identified

of 28 kDa,

DCCD-binding

and subunit

of the

spinach

the

This

3C) may correspond

Sensitivity

kDa constitute

as the

3A).

(cf.

masses

all

(not

complex. were

leaf

preparations

that

polyacrylamide

spinach

of molecular

kDa and 8.5

(Fig.

F,FI-ATPase

mitochondria

purified

Seven polypeptides

indicate

on the

leaf

examined

F,Fl-ATPase

of the

spinach

in the

strongly

purified

components

their

was found

COMMUNICATIONS

leaf

of of the mi-

characterized

ot-

and chloroplasts.

Acknowledgments Antibodies against PVP protein Professor F. Guerrieri. We would Norling for valuable discussions.

from beef heart mitochondria were a kind gift like to thank Professor B. Andersson and docent The research was supported by grants from the 1357

of 6.

Vol.

170, No. 3, 1990

Swedish Natural Science Johanssons Foundations.

BIOCHEMICALAND

Research

Council

BIOPHYSICALRESEARCH

and from

Carl

Tryggers

COMMUNICATIONS

and O.E.

and Edla

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