Vol. 170, No. 3, 1990 August 16, 1990
BIOCHEMICAL
Solubilitation DCCD-Sensitive
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1339-1345
and Functional Na+/H+-Antiporter halobium
Reconstitution of The from Helobacterium
Tetsuya KONISHI* and Naoyuki MURAKAMI Department of Radiochemistry-Biophysics, Niigata College of Pharmacy, Kamishin’ei 5-13-2, Niigata, 950-21 Japan Received
July
10,
1990
Nat/H+ exchange activity was solubilized from Halobacterium halo6ium with octyl-p-Dglucoside (OG) and was reconstituted into the bacteria rhodopsin incorporated liposomes (BR-liposomes) by the detergent-dialysis method. Light illumination stimulated uphill =Na+ uptake into the reconstituted conjugate proteoliposomes. The zNa+ uptake was FCCPsensitive and was dependent on the amounts of OG-extract applied. On the other hand, the proteoliposomes reconstituted with the membrane fraction pretreated with N,N’dicyclohexylcarbodiimide (DCCD) did not exhibit the lightdependent pNa+ uptake, thus, DCCD-sensitiie. When the reconstituted proteoliposome was incubated with [%]DCCD, radio-labels appeared slightly on 50K but mainly on 1 lK-Dalton component, which are the same components labeled in the intact membrane vesicles. It is concluded that halobacterial DCCD-sensitive Nat/H+-antiporter was solubilized and reconstituted in the conjugate BR-liposomes with preserved functional unit. a 1990Academic me**, Inc. Our resent studies (14) have demonstrated several unique properties of the Nat/H+-antiporter in H.ha/obium that are different from those found in Those are the DCCD sensitivii (1,2), ApH and AY cooperativity (3.4) AYdependent gating (2-4) and unidirectional Nat transport non-halophiles.
from the cytoplasm to the external medium (2,4).
In spite of the extensive
knowledge on the functional properties of the halobacterial antiporter, the precise molecular features are still clouded. To elucidate further the molecular mechanism of activation, we intended to solubilize the Nat/H+ antiporter with octyl-p-Dglucoside from the membrane vesicles of H.ha/obium and to reconstitute it into liposomes. The antiporter activity from the non-halophilic *
To
whom
correspondence
should
be
addressed.
. .
Abbrevlatlons: OG; octyt-fi-Dglucoside, FCCP; carbonyl cyanide gtrifluoroAT, transmethoxypheylhydrazone, APH; transmembrane pHgradient, membrane electrical potential, A& + ; electrochemical potential of proton, Mes; 4-morpholineethanesulfonic DCCD; N,N’dicyclohexylcarbodiimide, acid. 0006-291W90
1339
All
Copyright 0 1990 rights of reproduction
$1.50
by Academic Press, Inc. in any form reserved.
Vol.
170,
No.
organisms monitoring
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
in the reconstituted proteoliposomes are usually determined by the intraliposomal pH after a Na+-pulse using pH-sensitiie fluores-
cence probes or by determining zNa+ transport coupled to pH-jump (58). However. these method can not adapt for the halobacterial antiporter, because simple Na+- and/or H+-pulse unable to facilitate the exchange process without the presence of AY and a simultaneous imposition of both ApH and AY are necessitates for the antiporter activation (24). Combined use of the valinomycin-K+ diffusion and the ammonium diffusion are adapted for the simultaneous
impositions
transport
(9.10)
systems
of
AY and ApH to facilitate the A&+-coupled
but it was claimed that valinomycin
caused
artificial
zNa+ movement across the membrane (11) Therefore, in the present studies we examined the possible use of bacteriorhodopsin (BR) as a Ah+ generator; The conjugate liposomes examined for its lightdependent zNa+ solubilized
DCCD-sensitive
containing BR uptake. Results
Na+/H+ antiporter
and antiporter was showed that the OG
was functionally
reconstituted
in
the liposomes. METHODS
AND MATERIALS
e Vesicles Prm n and So/ubi/~7atioa; Membrane vesicles from H.ha/obium R,M, (bR+, hR+) were prepared as described (1). The membrane vesicles were suspended in a medium containing 2.9 M KCI, 100 mM NaCl, 1 mM Mes (pH 6.0) (Buffer A) at the concentration of 5 mg/ml. For preparating the DCCD-pretreated membrane vesicles, vesicles were treated with DCCD for 12 hr (at 200 nmole DCCD/mg of vesicle protein) at 4OC, then collected by centrifugation and washed onoe with Buffer A. For solubilizing the membrane proteins, vesicles was added by OG (protein to OG weight ratio of 0.8) and was incubated for 2 hr at 4OC. After the centrifugation at 150,OOOxg for 60 min, the supernatant was used for reconstitution. From 50 to 60 % of the membrane protein were extracted under the condition. Protein concentration was determined by using a BCA Protein Assay Reagent (Pierce Chemical Co.) using bovine serum albumin as a standard. Pre.patation of BR lbosomes: Large unilamellar liposomes were prepared by the reverse-phase evaporation as described (12) using a mixture of phosphatidylcholine and cholesterol (mole ratio 1O:l) in 2.9 M KCI, 100 mM NaCI. 10 mM Mes (pH 6.0) (Buffer B). Purple membrane prepared according to (13) was mixed with the liposomes (a lipid to protein weight ratio of 60) with shaking. The mixture was freezed at -4oOC and slowly thawed at 4OC, then sonicated for 5 min (50 % duty) in a water bath-type sonicator (HEAT Systems Ultrasonics Model W-225R). This procedure was repeated 3 times, and finally the mixture was centrifuged for 20 min at 15,000 rpm with HfTACHl model CR15B centrifuge to discard aggregated fraction. Lipid content in the proteoliposome was determined by a Phospholipid-test kit (Wake Pure Chemical Industries). ~titution; The m-extract (containing 10 mg protein) was mixed with the BR-liposomes. The mixture was incubated for 30 min at room temperature, then dialyzed for 48-72 hr against Buffer B at 4%. Initial concentration of OG was less than 0.5 %(w/v) to avoid randomization of BR orientation in the membrane. Reconstituted proteoliposomes were collected by centrifugation at 1340
Vol.
170,
No.
3, 1990
BIOCHEMICAL
AND
100,OOOxg for 30 min and washed fraction.
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
twice with the buffer B to remove
soluble
ment of Ne+&+erter acti& Light-dependent zNa+ uptake was determined by the centriige-column method. The reconstituted proteoliposome was incubated in Buffer A containing “NaCI under illumination with a slide projector lamp (560 W). An aliquot of the reaction mixture was taken into a mini-column at the desired incubation time. The column was packed with Shephadex G-50 (equilibrated in 3 M KCI, 1 mM Mes (pH 6.0)) and was centrifuged for 30 set at 1,000 rpm in a Hitachi model CR15B centrifuge before use. Proteoliposomes passed through the column by centrifuging at the same condition were collected in scintillation vials. The radioactivity was determined in a Packard Model 2200CA liquid scintillation counter. HPLC-Analysis of PClDCCD bindina wmwnents in liwsom Reconstituted proteoliposomes were incubated with [“JC)DCEt for 12 hr at a concentration of 100 nmole/mg of protein. After passing through a Shephadex LH-20 column equilibrated with 3M KCI and a deionized water equilibrated Shephadex G-25 column, the liposomal proteins were precipitated by 10 % trichloroacetic acid. The precipitate was solubilized in 4 % sodium dodecylsulfate (SDS) and subjected to gel-permeation HPLC using Toyo-Soda Model HLC603A equipped with a G3000 SW column. Materiak~ Phosphatidylcholine was obtained from Nippon Fine Chemical Co, Ltd., ZNaCI (carrier free) from New England Nuclear, FCCP from Tokyo Kasei Co., Ltd. Gctylglucoside, cholesterol and all other reagents used were purchased from Wako Pure Chemical Co. Ltd.
RESULTS
AND
DISCUSSION
Fig. 1 shows a typical profile of light-dependent pNa+ uptake into the reconstituted proteoliposomes. Over the time-course of 12-min incubation, no light-stimulated
zNa+
uptake
was
observed
whereas
a rapid ENa+ uptake was occurred
prepared
from the OGextract
in the control
in the conjugate
of the membrane
BR-liposomes, proteoliposomes
and the BR-liposomes
by the
detergentdailysis procedure. Since there is no transmembrane Na+ gradient before illumination start, =Na+ is considered to be activefy transported against its concentration gradient. When the conjugate proteoliposomes were incubated
with
FCCP
prior to illumination,
the active
zNa+
uptake
was
eliminated, indicating that the Aj&+ created by BR is the driving force for the zNa+ uptake. To make it certain that the observed =Na+ uptake is really due to the
CG
containing stimulated uptake
solubilized
Na+/H+
antiporter,
the
conjugate
various amounts of OG-extract were prepared and the lightnNa+ uptake were examined. As shown in TABLE I, the aNa+
activity was totally dependent
on the amounts
of OG-extract
the reconstitution. In order to determine whether the observed =Na+ by the DCCD-sensitive Na+/H+ antiporter characterized halobacterial
proteoliposomes
membrane
(l-4),
the effect of DCCD 1341
used for
uptake is mediated previously in the
on the nNa+
uptake
was
Vol.
170,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
6 G‘ P
0
5
15
10
TIME
(min)
zNa+-uptake into the conjugate Fia. 1. Time course of lightdependent proteoliposomes. zNa+-uptake into the conjugate proteoliposome was determined under illumination ( 0 ) or in the dark ( 0 ). ( A ); FCCP (2pM) was added prior to BR-liposome under illumination. ( 0 ); zNa+-uptake in the control illumination.
studied.
Since DCCD addition inhibits BR’s H+ pump in the reconstituted
proteoliposomes (not shown), the OG-extract
was obtained
from
the
membrane vesicles pretreated by DCCD and was reconstituted into BRliposomes. In the DCCD-proteoliposomes thus prepared, the light-stimulated zNa+ uptake was completely inhibited (Fig. 2). Since the H+ pumping activity of BR and the membrane leakiness of the DCCD-proteoliposomes were the TABLE I of zNa+ uptake activity in the reconstituted on the amount of octylglucoside-soluble extract
Dependence proteoliposomes
Cctylgulcoside-Extract (mg protein)
aNa+-uptake activity (nmol Na+/mg PC# at 4min)
0 3.0 6.0 10.0
1.5 1.9 2.7 5.2
t f 2 +
0.3 0.4 0.3 0.3
Octylglucoside-extract (3mg protein/ml) in the buffer containing 2.9 M KCI, 1OOmM NaCI. lOmM Mes (pH=6.0), 12% OG was mixed with BR-liposomes. Then the mixtures were dialyzed for 72 hr at 4%. against Buffer B. The extent of zNa+-uptake was determined at 4 min of illumination. #: PC; phosphatidylcholine. 1342
Vol.
BIOCHEMICAL
170, No. 3, 1990
E
c
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
4
0
0
I
I
1
5
10
15
TIME
(min)
w.
Effect of DCCD on light-dependent zNa+-uptake. The OGextract from the DCCD-pretreated vesicle membrane was Monensin (0.2pM) was added at the reconstituted into the BR-liposome. arrow indicated. ( 0 ); zNa+-uptake under illumination, ( 0 ); dark control.
same as that of the control BR-liposomes, the lack of =Na+ uptake is resulted from the DCCD effect on the antiporter. This conclusion was also supported by the evidence that the addition of monensin, a Na+/H+ exchange ionophore,
300 0 Membrane n Conjugate
1200
Vesicle proteoliposomd
200
4
800 +
ii P
100 400
0
0 5
10
20
15 Elution
Time
25
30
(min)
HPLC analysis of DCCD-binding components. Reconstituted proteoliposomes and intact membrane labeled with [l%]DCCD and the labeled peptide components by gel-permiation HPLC using Toyo-Soda G3OOB SW column.
F&J.
1343
vesicles were were analyzed
i P
Vol.
170,
No.
BIOCHEMICAL
3, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
caused a large uptake of aNa+ into the DCCD-proteolipcsomes illumination (Fig. 2). Since the silver-stained peptide patterns
by light on SDS-gel
after PAGE were same in the control and DCCD
(not shown),
the possibility
that intrinsic
reconstitution
procedure
DCCD-sensitive
Na+/H+
antiporter
might
can be excluded. antiporter
DCCD-binding
component(s),
have lost through Therefore
is functionally
conjugate BR-liposomes. To rationalize the relation between uptake activity were
the steps
it is concluded reconstituted
of
that the into
the
the Na+/H+ antiport activity and the
the conjugate
labeled
proteoliposomes
with [%]DCCD
proteoliposomes
with
and the radio-labeled
nNa+ proteins
were analyzed by HPLC. Of the two DCCD-binding components with 50K and llK-Dalton found in the native vesicle membrane (14), 11K components were mainly labeled but 50K labeled
in the conjugate proteoliposomes in parallel with =Na+ uptake activity (Fig. 3). Therefore the 11 K peptide is likely the component related to the Na+/H+ antiporter as is previously suggested (1,14),
but further
reconstitution
functional
method reported
slightly
studies
are required
here provided
for the conclusion.
a promising
The
way to identify
the
functional unit of the halobacterial Na+/H+ antiporter for the next step. It is also true that the present conjugate reconstitution method using BR liposome is quite
useful
requires
for investigating
other
[email protected]+ coupled
both ApH and AY? simultaneously
transport
system
which
for its operation.
ACKNOWLEDGMENTS This work was supported by a Grant-in-Aid for Scientific Research in priority areas of “Bioenergetics” to TK. and also in part by a Grant-in-Aid (01708243) to NM. from the Ministry of Education, Science and Culture, Japan. REFERENCES 1. a) Murakami, b) Murakami,
N., Konishi, T. (1985) J. Biochem. (Tokyo) 98, 897907 N., Konishi, T. (1988) J. Biochem.(Tokyo) U&231-236
2. Konishi, T., Murakami, 3. Murakami, 4. Murakami,
,
N. (1988) FEBS Let-t. 226, 270-274
N., Konishi, T (1989) Arch. Biochem., Biophys. 271, 515523 N., Konishi, T (1999) Arch. Biochem. Biophys. in press
5. LeBelle, E.F (1984) B&him.
Biophys. Acta. 770, 79-92
6. Seto-Young. D., Garcia, M.L., Krulwich, TA. (1985) J. Biol. Chem. 260, 11393-l 1395 7. Weinman, E.J., Shenolikar, S., Cragoe, E.J.Jr., Dubinsky,W.F? (1988) J. Membr. Biol. m, l-9 8. Padan, E.. Maisler. N.. Taglicht. D., Karpel, R., Schuldiner. S. (1989) J. Biol. Chem. 264, 20297-20302 1344
Vol.
170,
No.
3, 1990
9. Bassilana, 1022 10. Nakamura, 11. Eisenman, in “Progress
BIOCHEMICAL
M., Damiano,
AND
E., Leblanc,
BIOPHYSICAL
RESEARCH
G. (1984) Biochemistry
COMMUNICATIONS
a,
1015
T, Hsu, C., Rosen, B.F? (1986) J. Biol. Chem. 261, 678-683 G., Szabo,G.,
G., Ciani, S., McLaughlin,
in Surface and Membrane
pp. 139-241, Academic 12. Konishi, T. Murakami,
Press,
Science”
(Danielli, J.F, ed.),
New York
N., Hatano. Y. Nakazato,
Biophys. Acta.=, 278-282 13. Oesterhelt, D., Stoeckenius, W. (1974) Methods 14. Konishi, T , Murakami,
S., Krasne, S. (1972)
K. (1986) Biochim. Enzymol. 31, 667678
N. (1984) FEBS Lett. 169, 283286
1345