Na+H+ exchange in the cyanobacterium Synechococcus 6311

Vol.

122,

No.

July

18, 1984

1, 1984

BIOCHEMICAL

AND

RESEARCH

COMMUNICATIONS Pages

Na+/H+

EXCHANGE

Eduardo

IN

THE

Blumwaldl*+

CYANOBACTERIUM

J.

Mario

June

5,

SYNECHOCOCCUS

Wolosin*

*Department of Physiology-Anatomy Applied Sciences Division, University of California, Received

BIOPHYSICAL

and

Lester

452-459

6311 Packer*+

and +Membrane Bioenergetics Lawrence Berkeley Laboratory, Berkeley, California 94720

Group,

1984

SUMMARY: The cyanobacterium Synechococcus 6311 adapts to grow in 0.6 M NaCl by developing an efficient system for sodium extrusion. In the present investigation cells loaded with NaCl were subjected to a large dilution. Changes in fluorescence quenching of acridine orange as a function of transmembrane Na+ gradients provide evidence that Na+/H+ exchange activity greatly enhanced in salt-adapted cells.

Mechanisms

of

cyanobacterium NaCl.

process

organic

and

[1,21.

When

tion 0.6

by

to

mM NaCl,

indicating

the

cellular the a Na+/H+

investigate

to

seems

to

NaCl have

adaptation active

Na'

Na+

10n leave from: Hebrew University

0.6

includes

intracellular

high

extrusion located

another

the

TPT

40

in

the

Agricultural P.O.

plasmalemma. in

suspended

these

Box

salicylanilide), (Triphenyltin),

cells

grow

in

0.6

in

0.6

hrs.

sodium

of

before

muscorum leading

suggested nidulans

it and

in

mechanism

events

been

Anacystis Hence,

growth

extrusion

initiation

550

concentraof

Nostoc

has

M

reading

sodium 40

of

mechanism

content:

mM after

it

water

extrusion

cyanobacterium,

cyanobacterium

the

to

intracellular

Furthermore,

salt.

processes

(Tetrachloro

is

fresh

adapt

a Na+

effective

effect

in

Department of of Jerusalem,

an

the

accumulation

sodium

at of

In

in

salt

This

stabilizing

can

and

low

M NaCl.

a specific

transport

TCS Abbreviations: m-chloro-phenylhydrazone), acetic acid).

which

intracellular

presence

in

organism

in

in

[Z].

to

antiporter

grown

increase

exposure

investigated

substances

6311

growth,

Naf

an

osmoregulatory

during

[3,41,

being

adaptation

decreases

adaptation

that

salt

a marked

min.

during

to

are 6311,

Synechococcus is

30

of

inorganic

there

mM after

transport

Synechococcus

The

NaCl,

ion

is

151 is

driven

important

after

Botany, Faculty of Agriculture 12, Rehovot 76100, Israel. ECCP (Fluorocarbonyl EDTA, (ethylene diamine

to

adaptation.

Cyanide, tetra-

M

Vol.

122,

We report movement

No.

1, 1984

now

that

of

H+

BIOCHEMICAL

Na+

gradients

existence,

in

transmembrane

suggesting

the

AND

BIOPHYSICAL

in

RESEARCH

COMMUNICATIONS

Synechococcus

that

organism,

6311 of

induce

a Naf/Hf

exchange

system.

METHODS Synechococcus 6311 cells were grown as described previously [I] in either After 96 hours, low salt (11 mM Na+) or high salt (0.6 M NaCl) growth media. cells were harvested by centrifugation (lO,OOOxg, LO min) at 25' C and resusPermeability to NaCl was measured by following pended in growth medium (pH 7.8). osmotic volume changes after NaCl-induced stress using an ESR method for internal cell volume described in 121. In this method an ESR signal, representing the concentration of rapidly permeable spin probe TEMPONE, is selectively measured inside the cells by quenching the external signal with impermeable paramagnetic ions. Kinetic resolution was achieved using a rapid mixing apparatus [2]. Development of pH gradients in response to outwardly directed Na+ gradients was followed by the fluorescence quenching of acridine orange [6]. To 6 ~1 of NaCl loaded Synechococcus generate outwardly directed Na+ gradients, 6311 cells were diluted in 333-fold solutions containing 1 uM acridine orange 0.6 M mannitol and 25 mM N-methylglucamine-gluconate (Eastman Kodak, Co.), (prepared by titrating solutions of D-gluconic acid lactone with N-methylglucamine) pH 7.8 and concentrations of Na+ of Kf salts as indicated. For salt loading cells suspended in their growth medium were sedimented in an Eppendorf centrifuge, resuspended with one volume of growth medium, supplemented with NaCl to the final concentration indicated in the figures, and kept at room temperature in the dark. Final protein concentration was 8.5-9.5 mg/ml (approx. 2 mg chlorophyll/ml). Changes in the fluorescence were monitored with a PerkinElmer MPF 44A spectrofluorimeter (excitation, 493 nm; emission, 530 nm), at room The excitation slit was set at 1 nm to minimize induction of photemperature. tosynthetic activity.

RESULTS Comparison

of

Control ferred

1.0

Thereafter, within

entry

of

suggests will

incubation

reaching

and and

upon

storage

M NaCl

0.6

volume

grown

shown)

in

cells

of dark

0.6

or

with of

2 hours

acridine were

cells within

swell

cells for 1.0

the

to

NaCl

either

tested,

453

of of

after

original the

volume

is

salt-induced NaCl

M or

1 M NaCl

equilibration

fluorescence but

no

significant

in (Fig.

due

stress

solutions.

0.6 orange

1).

their

recovery

trans-

(Fig.

analysis

minutes,

M NaCl

when

ms

reaching

that

a few

shrink 200

Elemental

confirms

the

either

to

grown

respectively.

incubated

up

and

permeability

quenching of

cells

M NaCl

not

cells

adapted

a minimum

seconds

with

in times

60

This

of

resulted

0.6

(results

significant

Dilution medium

M NaCl,

NaCl.

that, be

mM Na+)

15

cells

salt

(11

control

re-swolen to

and

grown

into

volume

control

Na+-free 2).

Different

difference

Vol.

122,

No.

1, 1984

BIOCHEMICAL

AND

Control

1.0 M N&l

Salt

grown

was

observed.

addition was

1.

54

obtained % Triton

failed

to

induce

a Na + dependent

for

of

the

the

reversible

salt-loading

was of

of

the

of

either

cells

of

the

level

the

specific

(control

and

of

salt-grown),

conditions:

0.6

M NaCl

NH4C1,

due

extent,

or

with

or

detergent

These

results

to

the

formation

achieved

concentration but

t

same

NH4Cl

fluorescence NaCl

upon

the

with

was

Tab t Loading

about

signal.

quenching

6311.

restored

Na2SO4,

fluorescence

fluorescence

independent

to

pH gradient

the

Because

by Synechococcus

was

fluorescence,

of

COMMUNICATIONS

~

fluorescence

addition

pH gradient. Na2S04

quenched

increase

M N&I)

response to NaCl mixing with NaCl.

transmembrane

A second

further the

osmotic time of

Recovery

collapsing

X-100.

that

of

mM Na2S04.

by

0.04

addition

of the indicate

A fraction of

indicate

Kinetics Arrows

RESEARCH

cells 10 s

cells (0.6 10 s

1.0 M NaCl

Fig.

BIOPHYSICAL

had

after used

a linear

Loading conditions: 1 .O M NaCl

Triton x-100

Fig.

2.

Quenching

of

acridine

Synechococcus

experiment indicated

fluorescence a Na+-free loaded cells were diluted NH4C1 or Triton X-100

6311 NaCl NazS04,

orange

cells

into

454

of

by dilution of NaCl-loaded To start the medium. into test medium; where 0.04% were added.

rela-

Vol.

122,

No.

BIOCHEMICAL

1, 1984

Cells to Na, diluting

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

added or K,SO, medium Final

I

[N, + 1 ImM)

1oc

75

2.0 5 1.5 8

20

5a

\b;6s;M s

I

*

“y

4

36 mM Na,SO,

0.1

I

I

I

0.5

1.0

1.5

el

3.

Quenching of acridine orange fluorescence by dilution of NaCl loaded Synechococcus 6311 cells into a Na+ or Kf containing medium. Conditions as in Fig. 2 except salt-adapted cells were used and or @ wss initially present or added later to the reaction Na+ mixture. Inset: double log plot of relation between fluorescence and Na+ concentration in the test medium.

to

the

amount

the

irreversible

that optical

Table

quenching These the

1).

differences

pH

probe.

However,

larger

quenching

decreased probe

Na-+ studies. concentration

was

used

specificity. The

Only

Na+-dependence in

the

diluting

cells.

was

scaled

in

salt-grown,

loading

case,

acridine

salt

the

medium

cells

quenching

resulted lev-

in

of

fluorescence

control

cells.

even

larger

when

1 M. 1 nM 9-amino to

the

orange

con-

fluorescence

extent

were

due

while

was

fluorescence

the

using

acridine

the

than

was

it

quenching

100%

M NaCl

obtained

cell

Na+

acridine

as

absorbance

was

gradient-sensitive

appeared

to

be

employed

in

the

a more

sen-

experiments.

salt-adapted of

to

procedure

subsequent

medium,

Hence,

grown

orange

Thus, in

the

larger and

uptake fluorescence

0.6

this

lo-fold.

the

with

were

with

the

loaded

shown) in

than

and

the

(not

of of

control in

results

in

Na2SOb)

5-fold

between

three-fold

sitive

of cells

concentration

Similar the

For was

included

absorbance

addition

attained

NaCl

cells

component

and

(after

3,

of

opacity

achieved

(Fig.

EF a

0.5 : D

Fig.

tionship

from

t

3_

[Na + 1 out

Log

cluded

1.0

\

cells extent is

of shown 455

were

fluorescence in

Figure

quenching 3.

The

level

subsequent on of

the

Vol.

122,

No.

1, 1984

BIOCHEMICAL

AND

BIOPHYSICAL

TABLE Effect

of

Ionophores

and Medium

Addition

n.d.

Acidification Extent

of

on Fluorescence Fluorescence Percent

the

diluting

of

The

greater

of

permeable

cation

resulting

in In

0.6

ent

After

dependent Na+/$

tration

the

turn,

in

showed

with

with

external will

suspended

1 hour

of

quenching

remaining a linear

of

K+

is

present

Na+

by

K+

(72

mM)

in

fluorescence,

Recovery expected, release

the

signal

present was

the

but

significant

Na+

to

- Q)

for

in at

the

least quenched

as

introduction

by

diluting 6-fold

of

fluorescence of

counter

specificity,

medium cells

Figures

fluorescence A double

test

these

(Methods,

relationship

the

fluorescence

a growth

incubation

(100

of

of

K+.

initially

a

flow,

pH gradient.

in

described

K+

facilitate

the

was

Moreover,

to

option

and

most

K+.

quenched compared

of

of

high

as

Na2S04

Replacement

with

experimental

as

and

slower

medium loss

stoichiometry. (Q)

recovery

of

Na+

whether

afterwards. in

recovery of

in

procedure

quenched

of

of

achieved

pH gradient)

washed

NaCl.

lution

be

a different

were

added

6-fold

addition

loss

independent

or

still rate

upon

(i.e.,

38
resulted

was

could

medium.

cells

also

recovery

quenching

was

medium

medium

rate

Recovery

added co Na-free medium at pH 7.8 as in Figure 1. After maximum quenching, ionophores or acid (H2SO4) were added. This was min later by 50 mil Na2SO4 to induce complete recovery of fluoresper cent). The values given are the fraction of fluorescence min after the indicated additions.

attained

diluting

Recovery

= non-detectable

fluorescence in

COMMUNICATIONS

I

TCS (5uM) FCCP (HUM) EDTA (0.5uM) TCS + EDTA FCCP + EDTA TPT (1 uM) pH to 7.3 pH to 6.6 pH to 5.3 50 mM Na2S04 Cells were fluorescence followed 1 cence (100 regained 1

RESEARCH

was

in

which

were

0.6

M KC1

subjected

l-2).

to

No

significant

of

the

replaced the

same K+ gradi-

observed.

logarithmic

plot

fluorescence

vs

the

with

of

1 (Fig.

a slope 456

salt-grown

ratio

external 3,

between Na+

inset).

concenA 1:l

di-

Vol.

122,

No.

stoichiometry

of

tionship

obtained

external

Na+

with

H'

is

base

monoamine

is

permeable,

and

(Alout

such

are

free

this

quantity

free

has

100 the

[Na+]

out in

among

other

needs

to

on

signal

to

observed

were

of

present

dye

to

medium in

the

only

inside molecules

orange

[12,13]. where

= (l+n)xVinx[Hf]in/ of

Q /(LOO

a slope

of

1,

fraction

of

for

[Na+lout

might

low reach

The 1)

due, H+

more of

that

compartment

provided

be

resilience

suggests

- Q)

preloaded

equilibrium,

extruded.

cellular

for

fluorescence

plot

(Table

the

[A]out~Vout,

with

to

[Alin and

a small

since be

form

acridine of

Q)

line

in

has the

bound

logarithmic

only

[Na+]

Na ' of

still

-

Deviation

depletion more

Q/(100

i.e.,

a weak

H+ gradient

straight

constant,

amine

dye,

a double

For

where

total

percent

transmembrane

Na+

intracellular

For

unaccumulated

(I),

and

the

significant

relevant

for

exchange. of

ionophores.

Na+/H+

exchange

activity,

on

recovery

of

the

~JM FCCP

the

quenched

gain

further

increases

in

recovery

centrations.

The to

be

the

localized

ionophore,

medium. at

The the

of

insight

EDTA extracellular

was induced

rate

effect

and

studied gradual

were

however,

into

ionophores

fluorescence ionophores,

Hf-

further

effect

Hf

added

or

To

5 VM TCS,

may

a

of

acidification

concentrations

Effect

No

to

of

the

of

[ll,lZ], the

relaand

unprotoned

in

the

The

(I).

volume. with

H+ accumulation.

accumulated

(Fig.3).

equilibration

whose

number

COMMUNICATIONS

fluorescence

The

Therefore

the

to

follows:

lo),

the

correlate

amount

nearly

reasons, be

quenched

yield

remains

dissipated

to

relationship

will

as

intracellular

volume.

Given

remaining

concentrations

n is

the

the

to

respectively.

response

extracellular

[Na+]

Na+/H+

in

(pK

dye

is

data

= LH+I~~/[H+I~~~

where

observed

V outx~H+lout’

is

orange

shown

the

RESEARCH

= [Na+]in/[Na+]out

compartments

been

by

explained

(unbound)

- Q represents

is

be

[Alin/[Alout

Vin

BIOPHYSICAL

quenched

[H+]~~/]H+~,,,

that

and

AND

indicated

of

acridine

holds

molecule

Q,

versus

ratio

[A]inx(n+l)xVin,

quenching,

Na+

as

the

is

can

aqueous

each

Na+

the

when

it

equals

that

exchange

for

achieved

cell

V out

Na+/H+

concentration

extracellular

Thus,

BIOCHEMICAL

1, 1984

became was

immediate,

surface.

457

(Table

using more

TPT,

properties

changes

and

obtaind

the

in 1).

limited higher

effective

of

the

external

pH

Addition signal

of recovery.

ionophore when

suggesting

that

a Cl-/OH

exchange

con-

EDTA

was

its

action iono-

6

Vol.

122,

No.

phore,

induced

to

(from

7.3

tion

to

1, 1984

almost 7.8)

pH 6.6

required

BIOCHEMICAL

complete

did

not

induced

change

to

BIOPHYSICAL

fluorescence

moderate

acidification

AND

RESEARCH

recovery.

the

fluorescence

signal

recovery.

COMMUNICATIONS

Changing signal.

the

medium

Further

Full

pH

acidifica-

fluorescence

recovery

pH 5.3.

DISCUSSION Ion

transport

$-gradient 151

demonstrated

Raboy

and

Padan

regards

the

active

tion

with

Na+

Using

6311

in was

the

The

will

on

be dependent

higher

permeability

a significant lized

NaCl

crease

in

ance.

Although

highly

dependent

of

found

and

in

intracellular

upon

salt-adapted

not

of

cells

by

titration

show

any

are

of significant

cell

that conjunc-

are

and

be

cells

the is the

be

that

of large

of

intracellular

homogenates

from

control

458

in

after

efflux. upon it

NaCl an

the

can

pH range

The dilution, be

uti-

permeability adaptive of

in-

salt

toler-

orange

is

differences

measurements

difference

NaCl

acridine

explained

it

quenching)

development

quenching

to

observed

before lower

driven.

Synechococcus

that,

lost

explanation

likely

ATP

technique,

in

implies

will

the

been

(fluorescence

cells

binding,

a)

plasmalemma.

in

quenching

changes

control

fluorescence

since:

the

suggested

had

demonstrated

accompanies

not

efflux

exchange

salt-adapted

intracellular

synthesis. Plectonema

in [5]

accumulation

Na+

activity

binding made

in

An alternative

extent

be

Na+/H+

intracellular In

exchange the

Hf

between

NaCl

loss.

Na+/H+

trol

did

ratio

H+ accumulation. this

capacity,

to fraction

for

reduces

cells

the

the

quenching the

ATP

was

N-N'-dicyclohexylcarbodiimide,

can

of

which

system

fluorescence

activity

are

antiporter

that

fluorescence

pumps

Paschinger

with

suggesting

magnitude

cyanobacterium

ti

Paschinger

plasmalemma,

another

a Na+/H+

orange

the

nidulans,

presumably

movement, by

acridine

larger

salt-adaptation.

Na+

a electrochemical

energy,

driven

treated

exchange

Much

in

redox

driven

observed,

Na+/@

cells.

studies

cells

sensitive

that

from

of

was

Kf-ATPases;

respiratory

upon

Anacystis

across

and

and

mechanism

extrusion

accumulation

clear

ATP-driven

depend In

H+-extrusion

concluded

the

to

plasmalemma.

ATP-driven

[7] both

appears

the

photosynthetic

that As

across an

upon

boryanum

Na+

cyanobacteria

produced

dependent

is

in

between

only

by

con-

differences buffer

and

salt-adapted

between

4 and

8

Vol.

(data

122,

No.

not

shown);

in

salt

20

percent In

the

and

adapted

Na+/$

of

the

of

this

of

various

of whole

BIOCHEMICAL

1, 1984

cells the

the

[LO,

total

cells,

it

of

suggestion

a are

increases

in

Blumwald

and

protein

content

is

difficult

The

exchange. presence

b)

true the

AND

very

protein

the to

activity

antiporter

in

known do

to

not

occur

exceed

cell. determine

the might

and

exact be

mechanism the

specificity

of

expression

Evidence

plasmalemma.

stoichiometry

COMMUNICATIONS

preparation]

observed the

RESEARCH

concentration in

Packer, of

exchange

Na+/H+

BIOPHYSICAL

in and

support the

effects

ionphores.

ACKNOWLEDGEMENTS This research was supported Office of Basic Energy Sciences, Research Department of the Gas of Soil Science of the University Spath is gratefully acknowledged.

by the Division U.S. Department Research Institute of California.

of Biological Energy Research, of Energy, by the Basic and by the Kearny Foundation Collaboration with Susan

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Blumwald, E., Mehlhorn, R.J. and Packer, L. (1983). Proc. Nat. Acad. Sci. USA 80,2599-2602. Blumwald, E., Mehlhorn, R.J. and Packer, L. (1983). Plant Physiol. 73:377-380. Blumwald, E. and Tel-Or, E. (1982). Arch. Microbial. 132,163-167. Blumwald, E. and Tel-Or, E. (1982). Arch. Microbial. 132,168-172, Paschinger, H. (1977). Arch. Microbial. 113,285-291. Reenstra, W.W., Warnock, D.G., Yee, V.J. and Forte, J.G. (1981). J. Biol. Chem. 256,11663-11666. Raboy, B. and Padan, E. (1978). J. Biol. Chem. 253,3287-3291. Schuldiner, S., Rottemberg, H. and Avron, M. (1971). Eur. J. Biochem. 25,64-70. Lee, H.C. and Forte, J.G. (1978). Biochim. Biophys. Acta 508,339-356. Wolosin, J.M. and Forte, J.G. (1983). J. Membrane Biol. 71,195-207.

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