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withdrawn,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.
1061
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.
1061
