Light-dependent proton and rubidium translocation in membrane vesicles from Halobacterium halobium

Vol. 64, No. 3, 1975

BIOCHEMICAL

LIGHT-DEPENDENT

PROTON AND RUBIDIUM TRANSLOCATION

IN MEMBRANE VESICLES

Baruch

Section

April

I.

FROM HALOBACTERIUM HALOBIUM

Kanner

and Efraim

of Biochemistry,

Cornell Received

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Molecular

University,

Ithaca,

Racker

& Cell

New York

Biology 14853

23,1975

SUMMARY A procedure for the isolation of membrane vesicles after sonication of Halobacterium halobium is described. Upon illumination these vesicles took up rubidium. This process was stimulated 3 to 7 fold by valinomycin, and inhibited by uncouplers of oxidative phosphorylation or by nigericin. In of low the light, these vesicles extruded protons. However, on addition concentrations of uncoupler the direction of proton movement was reversed. All proton movements were abolished by high concentrations of uncoupler or by nigericin. These observations suggest that part of the vesicle population was inverted and less sensitive to uncouplers.

Halobacterium produce

purple

rhodopsin acts

halobium, membrane

(1,2,3).

It

patches

proton

on illumination

of starved

was an increase

in the

in contrast

intact

cells,

intracellular

bacteria

ATPase

light,

of ATP (5,6).

formation

aration

of right

protons

and uptake

side-in

contain

at low a single

from --in vivo

concentrations,

protein,

bacterio-

studies

Moreover,

concomitantly

oxygen

it with

that

efflux

ATP concentration.

complex

vesicles

of rubidium.

took these

up protons

We describe

Purified

which

catalyze

into

(4)

that there

membrane liposomes,

which

In the presence

vesicles

in this

protein

of protons,

(5,6,7).

inside-out

this

was observed

have been incorporated

of the mitochondrial the

the light

pump (3).

of bacteriorhodopsin to the

in

which

was concluded

as a light-driven

preparations

grown

catalyzed

communication

light-dependent

efflux

in

the

the prepof

Vol. 64, No. 3, 1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Methods Halobacterium grown

halobium

batchwise

reciprocal four

in 2 liter

shaking

fluorescent

lamps

10 g.

After

proton

ug of membrane

3 M KCl.

Rubidium

taining

membrane

volume

of 0.6 ml.

tubes

Valinomycin,

were

incubated

was started

by the

carrier-free

a6RbCl.

temperature.

Three

through sheet

(Edmond

tensity were

withdrawn,

NaCl,

dried

free

86 RbCl was purchased

Preparation halobium

was grown,

stationary

phase

were

inoculated

under

( A660

at 2 pg per

for

5 min.

con-

assay.

2 x lo6

waterbath

ergs/cm2sec.

was passed

plastic

conditions

the

washed

filter-

light

Aliquots

filters,

cpm

at room

light

and a yellow

these

The

The reaction

on and the

filter

to Lowry

Results

and Discussion

vesicles

et al. Inc.

ICN (Cleveland,

with

in(0.1

ml)

4 M

(8).

Bacteriological

(Glenwood,

Ill.).

Carrier-

Ohio).

and bacteriorhodopsin--Halobacterium

the conditions

from a slant.

tubes

counter.

diagnostics,

>.

test

and 4 M NaCl in a final

turned

pm millipore

with

the medium was

in glass

in a shaking

Under

from

= 1.7

that

was used

6 x lo5

according

of membrane

1000 ml:

the pH of the

of KC1 and approximately

heat

in a gas flow

from Wilson

per

at room temperature

performed

placed

on 0.45

was determined

was obtained

were

was approximately

and counted

with

g and bacteriological

paper

in the dark

#809).

and filtered

Protein peptone

Filter

0.265

to 800 pg of protein)

waterfilled

Scientific

in the tubes

were

of 200 W each were

a 10 cm pathlength

illuminated

contained

(6) except

of 1.8 pmoles

lamps

medium

when present,

The tubes

were

was measured

at room temperature addition

slow

filter

as described

(400

of medium with

1 liter

NaOH.

experiments

vesicles

with

soft

translocation

uptake

was

g, CaC12.2H20,

through

protein

H. G. Khorana)

The growth

to pH 7.2 with

Light-driven

to us by Dr.

The flasks

37°C.

g, KC1 2.0

filtration

medium was adjusted

50-150

filled

from above.

9.75

given

Erleneyers

at approximately

NaCl 250 g, MgS04, peptone

S9 (kindly

described

under

This

took

about

The cells

were

collected

1055

Methods,

7 days when

to the

early

the flasks

by centrifugation

for

Vol. 64, No. 3,1975

BIOCHEMICAL

20 min at LO,000 medium without about

x g and were peptone),

equal

were

volume

washed

once

and finally

5% of the volume

the cells

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of the

stored

suspended

culture

at 4'C.

15 min,

the supernatant

was centrifuged

45 min.

The precipitate

was washed

of the volume

of the washed

lowing

preparative

stable

for

over

experiments intact absence

performed

8 months

with

respect

these

or presence

lation

vesicles

did

water

For this

and exposed

centrifugation

tate

three

times

at a protein

aration

is

cribed

concentration

essentially

electrophoresis

pure

and is

previously

extruded

protons

were

of pH on the Fig.

2.

of proton atoms

This

a single

reconstitutively

major

active

in

at 4" were All

In contrast either

used were

to

in the

for

the iso-

resuspended

(neutralized

with

the purple

and resuspended

of 2 to 4 mglml.

fol-

efflux.

acid

x g, 15 min)

water

exhibiting

stored

the vesicles

(133,000 distilled

and all

precipi-

in distilled

bacteriorhodopsin band

prep-

in acrylamide

the sonication

system

gel des-

(6).

Light-dependent cles

with

in 4 M NaCl at 25%

uptake

to 1% deoxycholic

an

x g for

was also

purpose

twice

After

water

oxygen

with

at 133,000

preparation.

catalyze

immediately

x g for

proton

The preparation

of glycerol.

NaOH, pH 8.0). was washed

not

this

at

at 13,000

The vesicles

to light-induced with

medium

2 min in a 10 KH3

Harvesting

at 4".

performed

of bacteriorhodopsin.

in distilled

once and resuspended

were

were

for

centrifugation

suspension.

salts

growth

was diluted

in a 50 Ti rotor

bacterial

(the

processed

30 ml batches

After

steps

on vesicles

bacteria

output.

the Basal

suspension

in

Sonifier

medium

When not

The bacterial

Ratheon

salts

in

medium.

of 4 M NaCl and sonicated at maximal

in Basal

proton

protons taken

into up.

This

light-dependent

At pH 3.5 extrusion

the medium cycle

varied

1).

considerably

from Proton

1056

numerous

by these

&fold

the membrane

vesi-

was turned

off,

When the light

be repeated

extrusion

was about

at pH 5.8.

illumination

(Fig.

could

proton

the extent

H+ per mg protein

translocation--Upon

higher batch

extrusion

times.

vesicles than

to batch

is

The effect shown in

at pH 6.0. between

was virtually

The extent 4 to 16 ng

abolished

by

Vol. 64, No. 3, 1975

Figure

1.

BIOCHEMICAL

Light-driven

proton

pH was adjusted

to 5.75

of the pH meter

was standardized

(2 pg/ml),

of the uncoupler

by high with

effect

was also

concentrations

that

there

inside-out.

the

proton inside-out

slower

kinetics

tion.

The ratio

activity

the data in

uptake.

the right

these

side-out

varied

A similar

the two types

of vesicles.

was high which vesicles

in preparations

to actively

*Abbreviations used: DCCD, N,N'-dicyclohexylcarbodiimide; acetonyl)-acetone; FCCP, p-trifluoromethoxycarbonylcyanide

transport take

phenome-

side-in

and one

If

of opposite this

is

cor-

and exhibit

support

this

extrusion/light-dependent

had low Rb'

1057

this

the resultant

to uncouplers

as

was observed for

one right

of evidence

was reversed 1) as well

reveral

1 are

proton

In the presence

(Fig.

shown in Fig.

of HCl.

DCCD*(~OOUM)

extrusion

explanation

of vesicles,

The

the response

with

greatly.

(2 ug/ml)

A possible

Two lines

for

indicated.

be observed

proton

are more resistant

of uncoupler

and low for

also

(5 x 10e4 M).

movements

assayed

of 2.5 nmoles

added where

could

by nigericin

of light-dependent

port

were

effects

x 10m6 M).

case,

of proton

in the presence

only

(lo-5M)

light-dependent

abolished

vesicles

take

expect

by addition

may be two populations

In this

light-dependent

M) the

FCCP*(2

were

and at the end of the experiments

these

of 1799

the uncoupler

non is

rect,

1799*(10W5

from g.

in 1 ml of 3 M KC1 (6).

up to 2-fold

but

isolated

pg of protein)

translocation

(2 pg) and 1799

Stimulations

or valinomycin

and this

(120

proton

nigericin.

by vesicles

vesicles

light-dependent

Nigericin

2 pg/ml

translocation

Membrane

halobium.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

explanaproton

with

a high

activity.

Rb+

uptrans-

One would

up Rb+ in exchange 1799, bis-(hexafluorophenylhydrazone.

for

Vol. 64, No. 3,1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

60.

2Oa IO.

3

4

6

7

6

PH

Figure

2.

The effect

of pH on light-driven

from E. halobium. for

Membrane

light-dependent

the response pulses

H+.

Moreover

which

isolated

Addition

of uncoupler

was apparently

riched

in inside-out

vesicles.

Light-dependent

Ginsburg These

--et al. authors

and that bacteria,

sodium

were

of the

After

capacity

assayed

external

pH.

each measurement

was measured

by sequential

populations

with

addition

(9)

that

suggested

still

of

known

of small

the cation Rb+ uptake

is

bound is

energy

potassium

but

cation is

inside

a permeant the cell. and that

1058

effect.

there

This en-

in an environment It

the bacteria ion

in the absence

significantly

low potassium.

somehow within dependent

this

grow usually

the predominant

centrifugation,

uptake

enhanced

heterogenous

(4 M) and relatively

that

proton

dramatically

Rb+ uptake--Halobacteria present

by differential

nigericin-sensitive

population

high

0.1 N HCl.

the buffering

vesicle

vesicle

with

pg of protein)

as a function

The pH was increased

light-dependent,

of uncoupler.

in vesicles

of 25 mM KOH.

we have

displayed

with

translocation (140

extrusion

to 3.44

to light,

of HCl.

amounts

vesicles

proton

The pH was adjusted

proton

was shown by is

potassium.

in the Halobacteria,

We observed is

a rapid

that efflux

in intact in

the

Vol. 64, No. 3,1975

BIOCHEMICAL

AND BIOPHYSICAL

(0) - valinomycin

(b)+

in

time

Figure

3.

Light-dependent

rubidium

Experiments absence

were

(1.2

incubation

in

rubidium

light

dependent

2 ug/ml

inhibited

about

KC1 was replaced to be required valinomycin bium.

for

from

was sensitive transport

rubidium

markedly

The extent

ranging

In

3-7

activity

have

vesicles

In the dark,efflux

thus

far

1799 were

obtained

rubidium

uptake

in membrane

again

varied

3).

cells

was recovered

Attempts

to isolate

Nigericin

valinomycin

appears

3 shows that

vesicles

of II. halo-

of vesicles,

or absence

of valinomycin

30 to 50% of the Rb+

in the vesicles an ionophore

for

when tested Rbf from

been unsuccessful.

of the rubidium

occurred

1059

from

at

when the unlabeled

Fig.

About

upon illumi

was completely

M).

the batches

in the presence (Fig.

process

of E. -- coli,

with

the pre-

figure.

3 shows that

(8 x low5

(10,ll).

of the intact

these

results

this

uptake

and uncoupler

of valinomycin.

and that

or potassium

The Rb+ uptake

in the absence

Fig.

(2 up),

during

in the

vesicles

of stimulation

to nigericin

included

membrane

stimulated

fold.

were

or

Valinomycin

Methods.

and nigericin.

Similar

from 11. halobium.

in the presence

indicated

by the uncoupler

60%.

by RbCl.

under

(8 x 10q5M)

up by the vesicles

and abolished

vesicles

470 ug protein

experiments

of an uncoupler

was taken

minutes

as described

in the

volinomycin

in membrane

with

pg) and 1799

period

the presence

nation

performed

of valinomycin

nigericin

dark

uptake

RESEARCH COMMUNlCATlONS

the membrane

vesicles,

Vol. 64, No. 3, 1975

BIOCHEMICAL

.ac t h m E b +n lY 4 0 E =

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

'Osom40-

3020-

toI 6

I IO time

Figure

4.

Efflux

of rubidium

Membrane

both

in

rapidly

the presence

or absence

point

efflux

was started

times

0.1 ml samples

the presence in its

Finally, g.

halobium

not

These out vesicle translocation

1 30

in minutes vesicles

ug of protein)

were

of valinomycin by turning

were

of g. halobium

(2 ng) off

filtered,

incubated

in the light

for

20 min.

the light.

washed,

in the dark.

At the

in

At this indicated

dried

and counted

(Fig.

4),

as

Methods.

or in

MacDonald according

the absence

under in the

and our

and Lanyi to the

uptake

because

be detected

1 23

of valinomycin

but much more

presence.

sodium-dependent interest,

(470

under

a 20

from membrane

vesicles

described

I I6

of solutes

observed

procedure

described

of 14 C-leucine. these intact

This

experimental bacteria

experiments

preparation

(12)

described

that

vesicles

here,

catalyze

observation

conditions,

prepared

is

the usefulness

in this

in the absence

communication of respiratory

1060

a light-

and

of particular

leucine

transport

(R.MacDonald,personal

illustrate

from

could

communication). of the for

the

energy.

right

side-

study

of

Vol. 64, No. 3, 1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Acknowledgement We wish and Dr. This

to thank

P. Heytler work

Dr.

R. J. Hosley

of DuPont

was supported

for

of Eli

Lilly

for

the

gift

of nigericin

1799.

by NSF Grant

#BMS 71-01311.

References 1. Oesterhelt, D. and Stoeckenius, W. (1971) Nature New Biol., 233, 149-152. 2. Blaurock, A.E. and Stoeckenius, W. (1971) Nature New Biol., 233, 152-155. 3. Oesterhelt, D. and Stoeckenius, W. (1973) Proc. Natl. Acad. Sci. USA, -70. 2853-2857. 4. Danon, A. and Stoeckenius, W. (1974) Proc. Natl. Acad. Sci. USA, 2, 1234-1238. 5. Racker, E. and Stoeckenius, W. (1974) J. Biol. Chem., 249, 662-663. 6. Racker, E. (1973) Biochem. Biophys. Res. Commun., 55, 224-230. 7. Racker, E. and Hinkle, P.C. (1974) J. Membr. Biol. II, 181-188. 8. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem., 193, 265-275. 9. Ginsburg, M., Sachs, L. and Ginsburg, B.Z. (1971) J. Membr. Biol., 2, 78-101. 10. Bhattacharyya, P., Epstein, W. and Silver, S. (1971) Proc. Natl. Acad. Sci. USA, 68, 1488-1492. 11. Lombardi, F.J., Reeves, J.P. and Kaback, H.R. (1973) J. Biol. Chem. 248, 3551-3565. 12. MacDonald, R.E., and Lanyi, J.K., Biochemistry, submitted for publication.

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