An Australian halobacterium contains a novel proton pump retinal protein: Archaerhodopsin
Descrição do Produto
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 151, No. 3,1988
Pages 1339-1345
March 30,1988
AN AUSTRALIAN
HALOBACTERIUM
CONTAINS
A NOVEL PROTON PD~IP RETINAL
PROTEIN:
ARCHAERHODOPSIN
Yasuo Mukohata,
Yasuo Sugiyama,
Kunio Ihara and Manabu Yoshida
Department of Biology, Faculty of Science, Osaka University, Toyonaka 560 Japan Received February 18, 1988
Summary: A bacterial strain collected from Western Australia carries all the specific features of Halobacteria, an extremely halophilic archeabacteria. This strain, Halobacterium sp. aus-l, contains a retinal protein which does differ from bacteriorhodopsin but still pumps out protons in the light. This novel proton pump is named "archearhodopsin". © 1988 Academic Press, Inc.
Bacteriorhodopsin (3-5)
were
found
in
(bR;l), the
halorhodopsin
strains
of
(hR;2)
Halobacterium
distributed among laboratories in the world.
and
sensory
halobium,
rhodopsins
which
have
been
Halobacteria in nature may carry
novel light-energy transducers other than those found in H. halobium. Several salt
lakes
orange,
strains in
of extreme
Western
halophiles
Australia.
were
Colonies
collected
of
pink and red and were either translucent
these
strains
or opaque.
colony was seen to be orange red and extremely halophilic all the respects examined
tested.
for its response
This bacterium to light,
(Halobacterium
because
from clay pans were
and
yellow,
One translucent
archaebacteria from sp.
aus-l)
was
first
it looked like the "red strain",
H. halobium RImR (6), in which we discovered hR (7). H. sp. aus-I contains a retinal protein (now named archaerhodopsin) which pumps
out protons
in the light
and is distinct
from hR.
Here we sketch the
bacteria and the proton pump. Materials
and Methods
The bacterial strain described here was collected from a nameless clay pan near Leonora, Western Australia. Halophiles formed colonies on agar plates containing 2% agar in the culture medium (25% NaCl, 0.2% KCI, i% MgSO. 7H^O, 0.02% CaClp, 0.3% sodium citrate, and 0.33% polypeptone (Daigo-Eiyo Co,40sa~a) at pH 7.4 in tap water (7)). An orange red translucent colony was isolated and
Abbreviations: bR, bacteriorhodopsin; CCCP, carnonylcyanide m-chlorophenylyhydrazone; CItE , nonaethyleneglycol dodecylether; hR, halorhodopsin$ PAGE, 2 9 polyacrylamid-e gel electrophoresis; SDS, sodium dodecylsulfate; TPMP , triphenylmet hylpho sphonium cation.
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0006-291X/88 $1.50 Copyright © 1988 by Academic Press, lnc, All rights of reproduction in any form reserved.
Vol. 151, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
inoculated in the culture medium. The process was repeated and the strain was stabilized. Growth of cells (Halobacterium sp. aus-l) in the culture medium at 40°C with vigorous aeration was followed by optical density (680 nm) at various concentrations of NaCI with or without penicillin G. The shape of the bacterium was examined under an optical microscope. The GC content of its major DNA was estimated from the bouyant density by CsCI density gradient centrifugation and from Tm by uv hyperchromicity (8). Membrane lipids were extracted with chloroform/methanol and analyzed by a methanolysis/silica-gel chromatography method (9). Cell envelope vesicles (right-side-out) were prepared by sonication and for ATP synthesis, vesicles were sonicated again in the presence of substrates as described (i0). ATP synthesis was assayed at 30°C with intact cells and the substrate-stuffed vesicles in an air-tight glass vessel (under air or N2) by pH jump (outside acidic (ii)) as well as by actinic illumination. ATP was determined by a luciferin-luciferase method. Western blotting with the anti= Halo-ATPase antibody was carried out on the membrane proteins solubilized from the vesicles with 1% SDS. This polyclonal anti-Halo-ATPase antibody ~12) had been raised in rabbit against the isolated ATPase (the major part of H -translocating ATP synthase (13)) of H. halobium (14). The light-induced pH change of suspensions of cells or vesicles, and the concomitant change in membrane potential were recorded at neutral pH at 30°C by routine methods as in (15). Actinic light (about 105 lux) was provided through a yellow glass filter (Toshiba Y48), except for action spectra experiments where interference filters were used at a calibrated light energy. A claret-colored membrane was isolated from the vesicle membrane by a method similar to that for purple membrane isolation (16) including sucrose density gradient centrifugatiOn.o The NH20H (i M~ treatment was tested on the claret membrane at 20 C in the light (about 5xlO lux), which would result in cleavage of Schiff's base (17), if any, in pigment protein(s) in the claret membrane. Laser flash photolysis was examined on the claret membrane by an ordinary assembly which will be described elsewhere. The pigment protein (archaerhodopsin) was isolated from the claret membrane by an analogous method applied for hR isolation (18) by using ClpE q. The pigment protein in 0.3% SDS was digested by lysyl endopeptidas~- 6f Achromobacter lyticus (Wako Chemicals, Osaka) at various substrate/enzyme ratio in 50 mM Tris-HCl (pH = 8.5) at 37°C for 12hrs. The N-terminal amino acid and partial amino acid sequences of the digested peptides were obtained by an amino acid sequencer. Protein was determined by the Lowry method (19) using BSA as a standard. PAGE in the presence of SDS was the same as that of Laemmli (20). Reagents used were the highest grade available. Results and Discussion
Characteristics
of the bacterial
gation are summarized in Table I.
strain isolated in the present investi-
All these features
terium is in the family of extremely halophilic (21).
suggested that the bac-
archaebacteria,
Halobacterium
The strain is thus tentatively classified into H. sp. aus-l.
Table I. Summarized features of a bacterium collected from a clay pan in Western Australia (Halobacterium sp. aus-l) Shape under an optical microscope: NaCI requirement in growth: Nutrient: Antibiotics resistivity: Membrane lipids: Major pigment: GC content of major DNA:
thin rod; av. 5 pm long more than 15% no growth on carbohydrates grow in 300 ug/ml penicillin G non-saponifiable ether lipids bacterioruberin (cf. Fig. 3B) 65%
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Vol. 151, No. 3, 1988
The
intracellular
reaction under
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
level
v e s s e l w i t h N 2. The
actinic
light
(uncoupler).
The
actinic
by
The
anti-Halo-ATPase (86
and
blotting), supports
(Fig.
light
as
anaerobic
i).
This
and/or
(13))
sp.
ATP
ATP
jump
aus-i
as
much
those
is
close
was
as
of
abolished
vesicles H.
the
air
by
also
halobium
of H.
halobium. halobium,
the
increased + 1 m M TPMP
vesicles
sp.
This
in
synthesized
characteristic
proteins
H.
to H.
the
then r e v e r s i b l y
envelope
identified
as
exchanging
formation
cell
(12)
by
level was
in the m e m b r a n e
sensitively H.
pH
antibody
64 kDa
that
decreased
substrate-stuffed
ATP
units
ATP
(II).
paired
aus-i
sub-
(Western
immuno-reaction
particularly
in r e s p e c t s
to this key enzyme. The pH of cell itant
suspension
(in NaCI)
increased
w i t h ATP f o r m a t i o n (Fig. i). The d i r e c t i o n + . The pH of s u s p e n s i o n s of cell envelope
by TPMP ic
light
depending
inside-negative NaCI) CCCP
on
the
membrane
regardless
of
(protonophore)
supporting potential
the
apparent
abolished
~ - ~
~
pH= 7.0
A
on f
off
~
A
TPMP+ on
T
T
Y
direction the
under
or KCI;
Fig.
pH
2),
changes
(data
component
and
actin-
-180 not
the
mV
in
shown).
the m e m b r a n e
on
off
on
V
•
V
4 M NaCI
reversed
whereas
(about
off •
TPMP+
protein
pH= 7.0
1 off
CCCP
T
~J
~5 o
~10 ngH+/mg f .....
efflux
~
5 M KCI
o',2 E
pH = 7.0
30°C
F-<
I
I
0
I
I
2O Time
I
I
TPMP* (or vaL) ~j
/,
under N 2
E c
Q
concom-
was
changed
monotonously of
light
of the pH change
pH d e c r e a s e
20 ngH*
actinic
vesicles
(NaCl
increased
both
/mg
A
salt
under
I
CCCP
40 (rnin)
~\%~'~[ "~.~
Fig. 1 (left). Energetic responses of Halobacterium sp. aus-i to light. The air above the cell suspension (1.6 mg/ml) in the reaction vessel had been purged by N_ 30 min before the first actinic illumination. The aerobic ATP level was s~own by the closed circle. The upward DH signal denotes the increase of pH reading. On and off indicate the start and the end of actinic illumination. TPMP is a liposoluble cation that functions as an uncoupler by cancelling the membrane potential. Fig. 2 (right). pH Changes of the right-side-out vesicles of Halobacterium sp. aus-i in the light. The vesicles were illuminated either in 4 M NaCI o~ in 3 M KCI at the initial (dark) pH of 7.0 and 30°C. CCCP (5 ~M) or TPMP (i mM) or valinomycin (val. 5 FM; broken curves) was added at the time shown.
1341
5 I
rr, in
Vol. 151, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS +
potential
increase
pH decrease results
(Fig.
component,
suggest
2).
system
(e.g.,
The action spectrum
and valinomycin
possibly by depressing
that
a
light-driven
functions on the membrane, uptake
TPMP
the membrane
electrogenic
if a Na+-dependent,
Na+/H + antiport
intensified
potential.
proton-extruding
membrane-potential
(22,23))
is
assumed
for the rate of proton pumping
bR-like pigment is functioning.
(in KCI)
the
These pump
sensitive H +
as in H. halobium.
(Fig. 3C) suggests that a
The spectrum may be distorted due to shadowing
by bacterioruberin as in the case of hR (7). The claret membrane could be separated by density gradient centrifugation at 35 - 38 w/w% sucrose, whereas purple membrane at 40% (16).
The proteolipo-
somes prepared with the claret membrane and asolectin liposomes by sonication, showed
CCCP-sensitive
membrane. ment
(17).
proton
uptake
The claret membrane was
in the
light
as well
partly bleached
The bleaching left a rough absorption
was
spectroscopically
all-trans retinal,
converted
the chromophore
0.2
.1~ 0
O.
by the NH20H-light
the
Because the bleached
original
of the proton pump pigment
|
I
I
!
one
by
adding
is suggested to
I
Cloret mernbrone
_
0.1
0
>~
to
treat-
=
0.3
~
|
back
of purple
spectrum of bacterioruberin
and also gave a difference spectrum of a bR-like pigment. pigment
as those
....
I
..
I
I
0.5
~
_~q)
',,
,6
n.
Q,-, Oo
,o 0
i
500
I
400
I
I
500
600
Wavelength
.
700
(nrn)
Fig. 3. Spectra related to archaerhodopsin. A) the absorption spectrum of the purified archaerhodopsin (60 ~g/ml) in 2 M NaCl containing 0.5% C.12.9.E B) Absorption spectra of the claret membrane (50 ~g protein/ml) before Lso±la line) and after (broken line) bleaching by the NH_0H treatment. C) Action spectrum for the rate of light-driven pH decrease of Zcell envelope vesicles in 3 M KCI at the initial (dark) pH=7.0.
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Vol. 151, No. 3, 1988
be
retinal.
about
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The
i:i mole
the pigment,
claret
ratio.
membrane
Laser
which was
flash
followed
intermediate
maximum
410 nm similar
The
isolated
spectrum
with
to
of
that
bK
its molecular Although similar
membrane 0.3%
not was
visible
(16).
above
cleaved cut
protein
(in
transient within
about
showed
the absorption
SDS-PAGE
time
two
C12E9 )
showed
3A),
of the
pigment
an
which
protein
each
acid sequence
ogies
upto
(proton
Therefore, light-driven
pump the
proteins
patterns
4).
(Fig.
sp.
4)
including (i))
and
retinal
aus-i
where
proton pump,
are very
were
bR in purple hydrolysed
sp.
2
3
II) shows homol-
aus-i
pump (27)) is clearly
and we now name it to be
4
5
6
kDa 29--
Q ~
14.4
Z
a.2-+
Fig. 4. SDS-PAGE patterns of the hydrolysates of archaerhodopsin and bacteriorhodopsin. Archaerhodopsin (lanes i-3) and bacteriorhodopsin (lanes 4-6) were solubilized in 0.3% SDS and digested with lysyl endopeptidase at enzyme/substrate ratios of nil (lanes i and 4), 1:400 (2 and 5), 1:200 (3 and 6), for 2 hr at pH 8.5 and 37°C. The hydrolysates were applied on SDS-PAGE and stained by Coomassie brilliant 51ue. Markers with their M are also shown. r
1343
was
The primary
"archaerhodopsin".
1
in
hydrolysates,
the N-terminus
30% with hR (chloride in H.
suggested
in the claret mem-
that in Table
protein
similar
26 kDa).
of these
Furthermore,
absorption
is very
of bR (25) or Ala of hR (26).
(so far resolved,
50% with bR both.
of pyro-Glu
Both
SDS-PAGE
(Fig.
in H. protein
the conditions
(25).
The
other
protein
the pigment under
peptides
endopeptidase. from
amino
the distinguished
of the
of
i0 msec.
of bR (24).
nm (Fig.
pattern
bleaching
in
caused
life
0.5%
at 560
by chymotrypsin
into
differed
20% with
longest
bacterioruberin
recovery
to M intermediate
maximum
features
found to be Thr instead
and
of the
of bR in H. halobium,
SDS by lysyl
however,
The
photolysis
and
size to be very close to those of bR and hR (about
to those
brane was
pigment
the
retinal
by spontaneous
The photochemical at around
contained
Vol. 151, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table II. Partial amino acid sequences of the N-terminal region of archaerhodopsin, bacteriorhodopsin and halorhodopsin Archaerhodopsin Thr Ala Ala Val Gly Ala Asp Leu Leu Gly Asp Gly Arg Pro Glu Bacteriorhodopsin (29) pyro-Glu Ala Gln Ile Thr Gly Arg Pro Glu Halorhodopsin (26) Ala Val Arg Glu Asn Ala Leu Leu aR) bR) hR)
Thr Leu ??? Leu Gly lie Gly Thr Leu Leu Met Leu lie Gly Thr Phe Tyr Phe Trp Ile Trp Leu Ala Leu Gly Thr Ala Leu Met Gly Leu Gly Thr Leu Tyr Phe Ser Ser Ser Leu Trp Val Asn Val Ala Leu Ala Gly Ile Ala Ile Leu Val Phe
HR-like pigment has not been detected in this bacterium. dopsin-like pigment exists in a small quantity,
A sensory rho-
as judged from the behavior of
the bacteria to near uv light.
Concluding Remarks In 1977, by finding hR (2) we showed that bR is not the sole light-energy transducer in (laboratory kept) halobacteria (7). The finding from the present investigation suggests that the light-driven proton pump is not always bR. One will find the third proton pump and/or pumps for other ions from other halobacteria in natural habitat. Actually, we identified the third proton pumping rhodopsin in another Australian strain, H. sp. aus-2. Physicochemical nature of these bacterial rhodopsin pumps would be compared with their primary structures to find a clue to learn the molecular architecture/mechanism of pumps. The strategy would be cooperative with site-directed mutagenesis of bR (28). The results may also be correlated with evolutionary aspects of retinal pumps and/or halobacteria.
Acknowledgments It should be acknowledged here that the survey for halophilic microorganisms in Australia in 1983 was fully supported by the Japan Society for Promotion of Science. Y. M. is grateful to other staffs of the survey team, Drs. R. Chujo (Tokyo Institute of Technology), F. Tokunaga (Tohoku University), T. Konishi (Niigata College of Pharmacy), K. Tsujimoto (University of Electro = Communications) and Y. Takeuchi (Jichi Medical School). We are indebted to Prof. H. Kagamiyama and Dr. S. Kuramitsu of Osaka Medical College for the amino acid sequence analysis. This work was supported partly by a Grant-in-Aid for Scientific Research in Priority Areas of "Bioenergetics" to Y. M. (#62617003) from the Ministry of Education, Science and Culture of Japan.
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i0. Mukohata, Y. and Yoshida, M. (1987) J. Biochem., 101, 311-318 ii. Mukohata, Y., Isoyama, M. and Fuke, A. (1986) J. Biochem., 99, 1-8 12. Mukohata, Y., lhara, K., Yoshida, M., Konishi, J., Sugiyama, Y. and Yoshida, M. (1987) Arch. Biochem. Biophys., 259, 650-653 13. Nanba, T. and Mukohata, Y. (1987) J. Biochem., 102, 591-598 14. Mukohata, Y. and Yoshida, M. (1987) J. Biochem., 102, 797-802 15. Mukohata, Y. and Kaji, Y. (1981) Arch. Biochem. Biophys. 206, 72-76 16. Oesterhelt, D. and Stoeckenius, W. (1974) Methods Enz., 31, 661-678 17. Oesterhelt, D., Schuhmann, L. and Gruber, H. (1974) FEBS Lett., 44, 257-261 18. Sugiyama, Y. and Mukohata, Y. (1984) J. Biochem., 96, 413-420 19. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem., 193, 265-275 20. Laemmli, U.K., (1970) Nature, 227, 680-685 21. Kushner, D.J. (1985) in The Bacteria VIII (Woese, C.R. and Wolfe, R.S. eds.) pp171-214 , Academic Press, Orlando 22. Lanyi, J.K. and Silverman, M.P. (1976) J. Biol. Chem., 254, 4750-4755 23. Konishi, T. and Murakami, N. (1988) FEBS Lett., 226, 270-274 24. Oesterhelt, D. and Hess, B. (1973) Eur. J. Biochem., 37, 316-326 25. Gerber, G.E., Anderegg, R.J., Herlihy, W.C., Gray, C.P., Biemann, K. and Khorana, H.G. (1979) Proc. Natl. Acad. Sci. US, 76, 227-231 26. Blanck, A. and Oesterhelt, D. (1987) EMBO J., 6, 265-273 27. Schobert, B. and Lanyi, J.K. (1982) J. Biol. Chem., 257, 10306-10313 28. Hackett, N.R., Stern, L.J., Chao, B.H., Kronis, K.A. and Khorana, H.G. (1987) J. Biol. Chem., 262, 9277-9284 29. Ovchinnikov, Yu. A., Abdulaev, N.G., Feigina, M.Yu., Kiselev, A.V. and Lobanov, N.A. (1979) FEBS Lett. iOO, 219-224
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