An Australian halobacterium contains a novel proton pump retinal protein: Archaerhodopsin

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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

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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

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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.

References i. Oesterhelt, D. and Stoeckenius, W. (1971) Nature New Biol., 233, 149-152 2. Mukohata, Y., Matsuno-Yagi, A. and Kaji, Y. (1980) in Saline Environment (Morishita, H. and Masui, M. eds.) pp31-37, Business Center for Academic Societies Japan, Tokyo 3. gogomolni, R.A. and Spudich, J.L. (1982) Proc. Natl. Acad. Sci. US, 79, 6250-6254 4. Hazemoto, N., Kamo, N., Terayama, Y., Kobatake, Y. and Tsuda, M. (1983) Biophys. J., 44, 59-64 5. Takahashi, T., Mochizuki, Y., Kamo, N. and Kobatake, Y.(1985) Biochem. Biophys. Res. Commun., 127, 99-105 6. Matsuno-Yagi, A. and Mukohata, Y. (1980) Arch. Biochem. Biophys., 199, 297-303 7. Matsuno-Yagi, A. and Mukohata, Y. (1977) Biochem. Biophys. Res. Commun., 78, 237-243 8. Mandel, M. and Marmur, J. (1968) Methods Enz., 12B, 195-206 9. Ross, H.N.M., Collins, M.D., Tindall, B.J. and Grant, W.D. (1981) J. Gen. Microbiol., 123, 75-80

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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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