263 - Proteolytic Studies of Chain Cleavage and Proton Pump Activity of Bacteriorhodopsin in Purple Membranes

Bzoelecfroc~er,zisfryy

azd

Bioetzevgetics

5,

723-740

(1975)

_

263 - Proteolytic Studies of Chain Cleavage and-Proion Pump Activity of Bacteriorhodopsin in Purple Membranes-* by KINKO TWJI and KURT ROSEXHECK Department

of Membrane

Research,

The LVelzmann Institute

of Science, Rehovot.

Israel

Manuscript

received

June 26th

197s

Cleavage of bacteriorhodopsin in purple membranes of Halobacterby proteolytic digestion led to fragments, the molecular weights of which depended on the enzyme used. On SDS-poly-acrylamide gels trypsin gave bands corresponding to 33,000 and zz,ooo daltpns, pronase - Z,OOO and 17,400 daltons, and papain - zr,ooo and 16,600 daltons, when the enzyme treatment was carried out in the dark. When the purple membranes were illuminated during the pronase digestion, an additional small fragment (M.\V. - Soo), of primarily hydrophobic amino acid composition, was split off. The kinetics of the light-induced pH changes in suspensions of reconstituted proteoliposomes incorporating enzymatically modified bacteriorhodopsin could be fitted by two first-order processes, one being about twenty times faster than the other. After trypsin treatment this kinetics was not significantly changed. Pronase treatment drastically reduced the light-induced pH changes, acting mainly on the amplitude of the slower process. The apparent rate constants of both processes were markedly increased_ Similar, but less drastic effects were found in papain-treated membranes. The increases in the rate constants of the slow phase could be accounted for by assuming that the proton leak through the proteoliposome membrane was increased by the proteolytic cleavage of the % bacteriorhodopsin chain. ~ZCWL hnlobi~r~t

,

Introduction

The purple membrane is one of the cell membrane fragments of the bacterium Halobacterium hat?obi~rnt.L Bacteriorhodopsin, the only protein of the purple membrane, consists of a single polypeptide chain halophilic

* Discussed at the 4th International \\-oods Hole (Mass.). 2-S October 1977.

Symposium

on Bloelectrochemistry.

i*4

Tsuji

and

Rosenheck

with a molecular weight of -Z~,OOO da1tons.l It has a broad absorption band bound by a protonated SCHIFF base linkage to one of the lysme residues with a ma,ximum at 570 nm, which is attributed to a retinal molecule constitutes about 75 weight in the protein cha.in.lDe Bacteriorhodopsin percent of the purple membrane, the remainder being lipids l Each segments oriented nearly protein molecule comprises seven a-helical to the membrane plane and almost totally nnmersed perpendicularl> xithin the lipid bilayer 3-5 Bacteriorhodopsin exists in a stable dark-adapted form with an absorbance ma_ximum around 560 nm and a metastable light-adapted form with an absorbance ma_ximum around 570 nm 6 Retinal extracted from dark-adapted bacteriorhodopsin is reported to be a mixture of while the light-adapted form 1s in the nil13-cis and all-tram forms,’ tlaws configuration * Bactenorhodopsin which contams a&tram retmal responds to a hght flash with a transient shift of its absorption peak from 570 nm to 412 nm ma a series of very short-lived intermediates, accompanied by proton release on one side of the membrane and proton uptake on the opposite srde.zng Subsequently the 570 nm absorption is regenerated, again bl- passmg through a senes of spectrally distinct intermediates. The nature of the coupling between the photochemical reactron and its function as a hght-dnven proton cycle of bactenorhodopsm*.lO pump is a subject of much concern to those investigating energy transduction in biological membranes_ One of the possible approaches to this sublect is the parallel study of the extent to which these two processes are influenced by chemicalrr or structural modifications of the purple Recently, an investigation of the modifications membrane constituents. in the kinetic properties of the photocycle, induced by limited protely-tic cleavage of the bactenorhodopsin chain ttt satzr. was camed out usmg pronase as the proteolytic enzyme-l2 The present work is concerned with the manner in which the kinetic and quantrtative aspects of the proton pump activity are affected by selective, proteolytic cleavage of the protein chain The amino acid composition and molecular weight distribution of chain fragments, after tqpsin, pronase and papain treatment of the purple membrane, were determined and the proton pump activity of the resulting modsed protein was measured by monitoring lightinduced pH changes in suspensrons of reconstituted proteoliposomes. The enzymatic dlgestrons were carried out in both the dark-adapted, as well as the light-adapted, proton-pumping state of bacteriorhodopsm, using steady illumination for the latter case, in order to test whether an>- evidence could be found for a bght-mduced conformatronal change, that might be coupled to the photochermcal cycle.

ExperImental

HaZobncterrwlz Ilalobtzox M-I was groom as described by DANOK and STOECKESIUS.~~ Purple membrane was prepared from the cells of

Proteolysis

of Bacteriorhodopsin

., 7%

Haikbattmizrtn hatobiium according to the methqd of OESTERRRLT and STOECILENIUS.~~ The amino-acid composition of the bacteriorhc+L dopsin (see Table I) was sinnlar to that reported by OESTERHELT.and The molecular weight of the bacteriorhodopsin was STOECKENIUSl estimated to be 24,000 by sodrum dodecyl sulfate-acrylamide? gel electroBacteriorhodopsin concentrations in the purple membrane phoresis

the

suspension were determined on the basis of a molar extinction of 63,000 M-km-r at 570 nm a Table

Ammo-acid composition I amino-acid/m01 bacteriorhodopsin termined for experimental reasons

coeffiuent

of the purple membrane.. Values are in mol Tryptophan and cysteine were not de-

Lysme Hlstxline Argmine Aspartic acid Threonine Senne Glutamlc acid Proline Glycme _Uamne Valine Metfuonine Isoleucine Leucine Tyrosine Phenylalamne

7 I 7 18 16 16 18 I2 24

26 I7 7 12

27 8 II

were prepared accordmg to L?_ACKER~~~~~.from Proteoliposokes purple membrane or enzymatically m,oillsed protein of purple membrane and soya-bean liprd ; the nuxture of 3 cm3 of purple membrane suspe~nsion (about 40 nmole of bacteriorhodopsm) in I MNaCl or’o.5 Afg,SO, and soya-bean lipid was somcated for I hour in a LABORATORIESSUPPLIES sonicator. Trypsin and pronase were purchased from SIGMA, papam suspfnsioh was from WORTHINGTONBIOCFIEAIICAL Co., and BOLTON-HUNTERreagent for protem lodination from the RADIOCHEBCICAL CENTERLtd , Amersham Optical density

measuremetrk

Optical density 1~1the vrsible region was measured w&r a CARY 15 spectrophotometer, using quartz cells of o I cm and I o cm light path.

LabeUzng procedure

wdh

BOLTON-HUNTER

reagenf

The BOLTON-HUNTER reagentr’ (N-succinimi

_

~

k-

, ia L -

dyl-3-(4-hydroxy)&

726

Tsuji

and

Rosenheck

(rLji)-iodophen_\-1 propionate) was used for protein iodination. The specific activity was more than 1375 Ci/mmol. Prior to iodination of the purple membrane, the solvent of the reagent - benzene-containing 0.2 o/Odimethylforrnamide - was removed at room temperature by directing a gentle stream of dry nitrogen on to Purple membrane, which included 3-76 mg of bacteriothe surface. rhodopsin in 3 cm3 of 0.1: ~11borate buffer, pH=S.3, was added to I mCi of the dried BOLTOS-HUSTER reagent and the reaction misture was agitated for 15 minutes at 0 OC. After reaction, the membranes were recol-ered by- ccntrifugation at 30,000 x g for 30 min and washed with 0.1 dI borate buffer, pH = S-5, by centrifugation. The washing process Then the pellet of the purple membrane was suswas repeated twice. pended m water, dialyzed against the 0.1 o/o bovine serum albumin solution overnight and washed with water t\\ice. All these steps after labelling were carried out at _+oC. The final molar ratio of the BOLTOKHUSTER reagent to the bacteriorhodopsin molecules was 0.6.

To the purple membrane suspensions of I-IS mg bacteriorhodopsin/ cm” (ODjio = 2.5s) in 20 mM Tris buffer at pH = 7-4. either try-psin, pronase or acti\-ated papain were added. The w/w ratio of each enzyme to the bacteriorhodopsin was O-I_ The preparations were incubated at 37 OC overnight for the try-p& and papain treatments and 3 hours for pronase treatment_ Enzymatic digestion was carried out both in dark and ItgIlt conditions, i.e. ain the absence and the presence of the acti\-e proton pump, respectively. In the dark condition, the purple membrane-enz~me mistures were covered with aluminium foil, from one day before the addition of enzyme to the end of incub‘rtion, and in the Z#zt condition the mistures were steadily irradiated with a slide projector (Q-L. Lamp 3x0 \I\‘att ; GXF, Belgium) Proteolysis with pronase was carried and 3 parallel fluorescent tubes. out also \\-hiltthe suspension was illuminated intermittently for ~5 seconds with 15 min dark inter\-als, in order to keep bacteriorhodopsin in the light-adapted form without showing proton pump activity.s After incubation, the preparations were centrifuged at 30,ooo xg for 30 min at 4 OC. The supematant was removed and used for amino acid analysis or radioactive counting (in the case of labelled purple membrane) _ The pellet was washed with water twice and then used for the SDS-pal_\-acrJ-l-amide gel electrophoresis, the studies of the proton pump activity- and the radioactil-e counting_

Sodium dodec)-1 sulfate-polyacrylamide gel electrophoresis was carried out according to L_AEJIJILI.~~ The samples of purple membrane and its digestion products were boiled for 7 min with a misture of 25 mIl Tris buffer, pH = 6-S, 2 o/o sodium dodecJ-1 sulfate, 5 “,L mercaptoethanol, IO y/oglvcerol and a small quantitv of bromophenol blue before electrod

Proteolysis

of Bacteriorhodopsin

‘7’7

phoresis. -4 slab gel consisted of two parts: the upper gel (15 cm X_F cm x 0.16 cm) was made with 3 o/o acrylamide - 0.0s o/o methylene bisacrylamide and 0.125 M Tris buffer, pH = 6.S, and the lower gel (15 cm x S cm x0.16 cm) with 15 oh acrylamide - 0.4 oh methylene bisacrylamide and o-375 M Tris buffer, pH = S.S. The electrophoresis was performed in 0.1 o/o sodium dodecyl sulfate (0.05 M Tris-base + 0.375 AI glycine) buffer, pH = S-5. for a.5 hours at IOO \-olt d.c. The protein bands were detected by staining with o._0 O/, Coomassie Brilliant Blue, followed by A plot of migration distance “JS. log destaining with 7.3 o/O acetic acid. M.W. gave an cstimatc of the molecuiar weight of each sample. Pepsin (hl.\V. = 35,000), trypsin (AI.\V. = 23,ooo), myoglobin (AI.\V. = 17,000) and iyzozyme (At.IV. = 14,300) were used as markers. For the radioactive counting, each channel of the gel was cut into 5 mm wide slices. For the amino acid analysis, the proteins of each band were extracted from the gel according to the method of TR..~VHURN et nl.1s The bands containing the required protein were sliced into small-pieces and placed on a 7-5 o/o acvlamide disc gel (0-S cm diameter xg cm length)_ A dialysis sac was fitted onto the bottom of the gel tube and the protein collected in the sac bv again electrophoresing overnight. The protem solutions in the dialJ-$s tubes were dial)-zed against distilled water for $3 hours, concentrated by gentle evaporation and acid hydrolyzed. Meas2~rement.s

of the proton

pump

activity

Light-induced pH changes in reconstituted proteoliposomes were measured according to EISESBACH et nl. lo Suspensions of the proteoliposomes incorporating the bacteriorhodopsin or the enzymatically modified protein were illuminated by a slide projector with halogen lamp, IVidioscope (AS IVIKTORS MEKANISKI, Sweden) and pH changes were measured at ~5 OC with a RADIOMETER GK a321 C combined glass clecThe outputs were trode connected to a RADIOMETER 64 pH-meter. display-ed on a high-speed VARIXS A-25 recorder ; response time - 0.5 s. The recorder scale. was calibrated bJ- addition of 5 mm3 of IO mM HCl to The initial pH of the suspension was 5.5 for the comthe suspension. parison of untreated and trypsin-treated samples, and 7 for that of _ 5 untreated, pronase-treated and papain-treated samples.

Results The molecular weights of the bacteriorhodopsin chain fragments after enzymatic digestion were estimated by SDS-polyacq-lamide gel Fig. I shows the electrophoretic patterns of the enzymaelectrophoresis. and Table 2 summarizes the results. tically treated purple membrane, After trypsin treatment bacteriorhodopsin which originally runs as a single band corresponding in our experiments to a molecular weight of ~4,000 daltons gives rise to a strong band at ~3,000 daltons and two weak bands at ~2,000 and ao,ooo daltons, respectively. There was no difference

Tsuji

728

O(d)

O(l)

1

and

2

Rosenheck

5

6

9

10

Fig. I. Gel-electrophoretlc patterns of enzymatically modified protein of purple membrane. Sample numbers refer to those III Table 3. O(d) and 0 (I) refer to duvk and It&, rcspcctn-ely-. _\ppro_ximate molecular wexghts are mdxcated

in kilodaltons

between digestion in the dark and in the light. Chromophore loss calculated from the decrease in optical density at 570 run was about IO “,/oand 15 o/o in the dark and light conditions, respectively. Pronase digestion in the dark reduced the protein to fragments of molecular weight zz,ooo (weak band) and 17,400 (main band). After digestion in the light the main band was shifted to about 16,600 daltons, and weak bands only- were There was also a remarkable observed at az,ooo and 17,400 daltons. difference in the chromophore loss between the dark condition (- 15 y&) and the light condition (-55 “/)_ Thus there is a significant increase in the sensitivity to degradation by_ pronase when the bacteriorhodopsin proton pump is activated by illurnmation. There is, however, also the possibility that this increase in sensiti\Gty is due to the change from the dark-adapted to the light-adapted form without being linked to the activation of the proton pump_ In order to check this point proteolysis was carried out in the dark, i.e. in the absence of proton pump activity, keeping, howeI.-er, the bacteriorhodopsin in the light-adapted form bJshort flashes of light with large dark intervals in between. Table 2 shows that in such a condition the proteolysis proceeded just like that of the dark-adapted form of bacteriorhodopsin. Proteolysis with papain resulted in fragments of mean molecular weight of 21,ooo (main band) and 16,600 (weak band), and was accompanied by a chromophore loss of 15 o/o in the dark and 20 o/o in the light. In order to obtain quantitative estimates of the amounts of cleaved protein in each fraction and also a better resolution of the lower molecular weights, purple membrane was labelled with BOLTOX-HUSTER reagent The electrophoretic patterns were prior to the enzymatic treatment. quite similar to those of Fig. I, except that the aa,ooo-dalton band of the pronase-treated sample was stronger. The same applied also to the

Proteolysis

of Bacteriorhodopsin

7’9

0D5;e! and molecular weight after various enzymatm treatments Tabie 2. Purple membrane suspensions of 1.1s mg bacteriorhodopsm/cm3 In 20 m&1 Tris buffer at pH 7 4 were dIgested wth trypsin, pronase and papam. The ratro of temperature, each enzyme to bacterIorhodopsIn was o I (w/w) ; incubation Lidabelled and labelled refer to treatment with the BOLTOS-HUSTER 37 OCreagent. Mean values + s-d. are derived from at least 4 esperlments. Sample NO.

Conditions of enzymatic treatment

-_

Unlabelled

A1.W. (kDaltons)

*abelled

IO0

-

0

-- (%I

ODSio

IO0

24-O

- _ I

Trypsm.

overnight,

dark

QI+2

91

23 o (mam badd) 23.0 20.0

- _ Trypsm. overnight, IllummatIon

2

steady

96

23 o (main band) 22-O 20

0

96

22

0

-

22 0 17 4 (maIn band)

7-l

22 0

- _ 5

Pronase,

3 hr., dark

-

5-5

-_ Pronase, 3 hr , intermittent Illummation

-I_

--

. Pronase, 3 hr., stead! Illumination

17-4 . 16 6 (main band)

j -_

/ Papain,

I

overnight,

3651

dark

!

94

I

I

17 4 (mam band)

2x.0 (main band) 19.0

9

16 6

I Papain, overnlght. illummatIon

steady

-I- 79f4

a-

l

!

I I

I

L

79

21.0 (main band) IQ.0 16.6

730

Tsuji

and

Rosenheck

chromophore loss in the labelled samples (see Table a), which was reduced significantly only for the pronase treated sample. Thus, the BOLTOS-HUETER reagent seems to protect to some extent the protein from cleavage by pronase. The kinetics of the light-induced pH change, to be described below, is similar in unlabelled and labelled purple membranes that have not undergone proteolysis, suggesting that the function of the membrane itself is not si,tificantlv changed by the labelling proprofiles of labelled membranes cedure. Fig. a shows the gel-electrophoretk after enzymatic treatment, in terms of radioactive counts normalized The specificity of to IOO %, includin, 0‘ the counts in the supematant. the labelling for the protein in the purple membrane was high (-go %) and no lipid was labelled. The figure shows the presence of a large fraction of counts in the region of the gel corresponding to fragments having molecular weights ran,$ng from - 6,000 to - 10,000 daltons.

10

Gel-electrophoretic profiles of enz?;matically mod&4 protein of purple membrane Sample numbers are the same as those m labelled \\lth Bolton-Hunter reagent Shce numbers in each Table 2 Sup. represents the [*‘5i] counts in the supematant. sampIe correspond to molecular lbeelght regons as follows _ 36k > (I) > 31k > (2) > > 26k > (3) > zzk > (4) > Igk > (5) > r6k > (6) > r3k > (7) > r~k > (S) > > g jk > (9) > S.3k > (IO) > 6.gk > (I I) > 5 Sk > (12) > 4 gk > (13) > _i 2k. according to a plot of nu,gation distance vs. lo,- _WW- of pepsin. trypsm. myo,olobin and lyzozyme.

Proteolysis

of Bacteriorhodopsin

of hydrophobic amino Table 3_ Distribution Sample Nos. are same as those in Table 2. Band

Sample

(kDaltons)

-~

NO. --

acids

731

after

proteolytic

% of the original Val

1

--

Supematant Band I (23.0. 22.0) L**

II

20

9I -2

89 -9

-I

quantitv

T _

LIu

i I

cleavage.

9

I i

Phe 7 91 2

70

21

II i

3

Supea-natant

Band I (32 o) 1 Band II (;7:4) I

IL**

8 +

22

I7

IO

25

26

20

I7 36

24 33

4s

24 -

29

II IS

3I I5 22

I9 IO 20

I5 I4 25

42

32

51

46

21

39 Ii 9 35

13

II

IS

23

22

9 I9 _ 22 50

I --l I

6

-____ g

, SupemaEant 1 Band I (22.0) Band II (17-4. 16.6) I j L** I

21 IO

I9 50

j ( Supernatant Band I (21 o) Band II (16.6) L**

14

7 58

S 6I

IO

I4 19 S

56

59

-10

/

Supernatant Band I. (21.0) I Band II (16.6) ! L**

25

I

I

I3 7 55

_

45 I7 II 27

* Value is uncertam due to an error m determination. ** Fragments of low molecular weight which remained enzymatic

I5 I5 9 61

in the membrane

16 I3 IO 61

after

treatment.

Table 3 shows the distribution of some of the hydrophobic amino acids between the supernatant and the various membrane-bound fragments, obtained by proteolytic cleavage, e.xpressed as percent of the original quantity of the untreated purple membrane. The amino acids in the supematant and in the bands of high molecular weight (band I and II) were analyzed by the method described in the experimental section. The amino acids of the fragments of lower molecular weight, Iocated near the migration front of the gel (L), were estimated as the

Tsuji

and

Rosenheck

d

6 ‘Light-on’

6 - Ilqht -off =

-4

Fig.

3

Kinetic anal~is of the hght-Induced proton concentration change in proteoliposome suspensions mcorporatmg treated and untreated purple membrane for lighten and (0) Experilight-off reactions. Sample numbers are the same as those in Table 2 (a) The rapld process, calculated bv subtraction of the line for the mental values. (0) The slow process after the con&ion of the leak effect. slogprocess

6

2,5&oo.I

55

4-o& 0.3 (0072)

(o 81)

3*7&o I (o-85)

5

(0.41)

’

,

-

(0.42)

1_ I

“I . ’

(0136) I

1~8 k 0.1~- F

2” ’ ~

II&b,2

(o-43)

I.O& 0,I

-- I

I

1I

(0.82)

2,7&o

\

20

(0 71)

I

1,7i 0.2 (00’38)

0.63

9

5

7.6f I 8 (0.28)

bI9)

0857

3

(

1

’ Y

I

1.2f 0,I (0.62) “‘1.‘, -1 F o:g4$ c&8 I 1 (0.64)~ I’

\

2.6&0,1 (0.18)

’

3 o&o,1 (0.29)

I

o 52

1,4fo

2.1fo

(0~15)

29io5 (0 40)

(6 69)

o 28&o.o1

(0155) ’

2.7 10.2 (0060)

I

18

(0.64)

1.6

088&006

1,3fo

(0 45)

o,g6

o-53 (0 36)

-

i2, &f-IXIO4 (9-I)

Light-01 R process

(0 31)

I

5

I

%

leak

contributions

(o 59)

0053

o 56koo2

1,5& 0.1

4mo

.-

-

fractIonal

x IO2V)

0

!’2,011

61

, ’

x IO2W1)

4#7 (0.54)

2,011

process

and

706 (o 52)

i

I

constants

204 (o 48)

(o-46)

.

x 10 V)

1~6

‘r1,011

Light-on

Rate

2’

0'

No

siample

M.W. of main pro& band (kDaltons)

proton concentration change In proteohposome suspensions mcorporating Table 4, Kinetrcs of tha bght-Induced purple membrane, with or wthout enzymatm treatments (InItIal pH 7,o In o 5 M K&SO,) Sample numbers arc those Leak oh 13 equal to -100 A', (see Appendix). In Table 2. Standard devrations are dewed from at least 4 experlmcnts

Tsuji

and

Rosenheck

M

3

s

0

d

b

CI

0 0

0 d

-l-I

+I

z 6-

-* 0

6

b I-Q

d

4

-

0

rl

d

9 0

-H

+I

\c, T

-2 lr.

d

d

0

;;

6

0

-4

0 0. +I 0

a

0

N

0 0-

+I t WY

o-

rr

0

-

0

6

d

d

r3 l-4

d +-I

0 0

Proteolysis of Bacteriorhodopsin

735

difference between IOO yO and the total percentage for each amino acid in the other fractions. A marked effect of irradiation was observed during pronase digestion ; the percentage of non-polar amino acids in band I was about twice as large in dark than in light, while, on the contrary, that in the supematant was less in dark than in light. In the papain case, no significant difference between the dark and light conditions was observed. Reconstituted bacteriorhodopsin-containing proteoliposomes have a reversed membrane orientation,21 so that proton uptake by the proteoliposomes occurs and a decrease of the proton concentration in the environment is measured during the lig&-on process. In the dark, the pH returns to the original value, the total estent of the pH change in the light-08 process being only slightly less than that in the light-on process. Using suspensions of proteoliposomes containing the various enzymatitally modified purple membrane preparations, this cycle could be repeated 5 to 6 times without any significant changes in the BpH values measured. The kinetic analysis of the light-on and light-off processes is shown in Fig. 3 for two representative cases. Plotted are the proton concentrations on a semilogarithmic scale ZLS.time for the untreated purple membrane [Fig. 3(a)] and for a membrane sample that had been treated with pronase in the light [Fig. 3(b)]. Both the build-up and the decay transients were found to be almost biphasic, i.e. they could be fitted by two first-order processes with the respective rate constants differing by a factor of about IO or more, the anti-logarithms of the zero-time extrapolations correIt should be noted that the time sponding to the partial amplitudes. scale in Fig. 3(b) is ten-fold expanded compared to that in Fig. 3(a), i.e. both processes become faster after enzymatic treatment. Table 4 lists the rate constants and Table 5 the amplitudes corrected for chromophore concentration, as measured by 0D570. Trypsin treatment makes both the fast and the slow processes a little faster than those of the untreated purple membrane, and the total amplitude of the proton concentration change is reduced by IO o/o_ On the other hand, pronase treated samples exhibit much larger alterations in kinetic behaviour which are dependent on the experimental conditions. The values of kn,., and k,_,,f increase strongly after the pronase treatment, while k,,.. and khom are %onIy twice as large. While kl, o,l for dark-adapted and light-adapted membranes are almost the same, there is a vast difference between the values of There is no difference in k,,r and k 2.0n for the three different states. k 2,0ff between the dark-adapted and the two types of light-adapted membranes_ The amplitudes of the fast phase, both in the light-on and light-off processes of pronase-treated membranes, are almost equal to those However, the slow phase amplitudes decrease of untreated samples. by about So %_ The transformation from the dark-adapted to the lightadapted form is seen to have no influence on the amplitudes of the various processes. The modification of the kinetic behaviour induced by papain treatment is slight and rather similar to that of trypsin, except for k,on which increases by a factor of about four. No effect of the illumination The amplitudes of the fast phase in both light-on and is observed.

736

Tsuji

and

Rosenheck

light-off processes are not affected by papain of the slow phase decrease by about 40 %_

treatment,

however

those

Discussion In the preceding section w-e have presented some of the consequences of the proteolytic treatment of purple membranes on the fragmentation of the bacteriorhodopsin chain, the loss of chromophore and the proton pump activity_ Our emphasis was on the comparison of the susceptibility to enzymatic attack of the protein in its different forms : the dark-adapted, the light-adapted and the proton pumping state. Very recently two related papers dealing with the proteolytic cleavage of purple membrane, in its presumably light-adapted but non-pumping state, have appeared.z2.23 Our experiments with trypsin show that this enzyme cleaves off a part of the chain ends that are exposed on the surface of the membrane. This is indicated by the fact that the molecular weight of the main band is only by about 1,000 smaller than that of the intact bacteriorhodopsin, which in our gels runs at ~4,000 (Table 2). The latter value is in fair agreement with other obser\-ations. If the enzyme would be cleaving also one or more of the turns which link sequential helical segments and are situated near the membrane surfacesa.% then smaller molecular weight fragments would result, as indeed is the case with the other two enzymes used. The small number of radioactive counts in the supematant Similar ( -5 o/o of the total) provides further evidence for this conclusion. findings were reported by GERBER et CZZ.~~ who place the site of cleavage by trypsin in the carboxyl end of the protein chain. It is to be noted also that the chromophore concentration is largely unaffected by trypsin treatment_ Furthermore, activation of the proton pump by steady illumination does not alter these results, and thus does not seem to lead to the exposure of any further trypsin-sensitive sites. Pronase treatment of bacteriorhodopsin in the dark-adapted and in the light-adapted, non-activated states, on the other hand, is seen to produce at least one cut in the interior of the protein chain, probably the region of one of the turning points, in addition to possible cleavage at the chain ends, since the molecular weight of the major fragment is smaller bv m 7,000 than the original one. If the three-dimensional model of bactegorhodopsin by USU-IN and HENDERSOS"" is taken as a basis, and it is assumed that the se\-en helical segments are of about equal length, i.e. comprisin, = about 30 amino acid residues each, this reduction in moIecular weight could mean that cleavage occurs at a single site about two segments removed from one of the chain ends. Alternatively, cleavage could occur also at two sites, separated one and two segments, respectively, from one of the chain ends, or one segment each removed from both of the chain ends. The observation, however, of a second broad peak in radioactivity (Fig. a), running at fairly high molecular weight (t-e. about 6,000-10,000), is in favour of there being olnly one single cleavage

Proteolysis

of Bacteriorhodopsin

737

site. It is to be noted (Table 3) that, as with trypsin, proteolysis by pronase does not markedly affect the chromophore concentration, as long as the proton pump is not activated. It thus seems that the interactions within the membrane are sufficient to stabilize the three-dimensional structure, so that in spite of the cut in the chain, the environment of the chromophore site and therefore its spectroscopic behaviour is not appreciably altered. GERBER et n1.23 have reported that pronase digestion liberated a high molecular weight fragment (M.W. 6,300) when the apomembrane was used. The situation is seen to be quite different when the purple membrane proton pump is activated by steady illumination_ As shown in Table 3. the chromophore loss reaches more than 50 y0 and there is a further drop in the molecular weight of the major The data in Table 3 seem to indicate fragment by about 1,000 daltons. that the additional small molecular weight fragment cleaved off the bacteriorhodopsin chain under illumination is largely composed of hydrophobic amino acids, being possibly a short segment of one of the helical regions adjacent to the site of cleavage observed when the purple membrane is in the dark-adapted state. Illumination thus seems to induce a shift in position of the protein chain leading to a slight increase in the degree of exposure in the region of the original cleavage site. Alternatively, this latter site may become shielded due to this Aft in position, while another site further along the chain may become esposed to the enzymatic attack. Our results also show that changes in the configuration of the chromophore and possibly, conformation of the protein, that may occur in the transition from the dark-adapted to the light-adapted, but non-pumping form, do not influence the esposure of the bacterioconcentration and the rhodopsin chain, since both the chromophore fragments recovered from the enzymatic treatment are practically identical for these two states. Proteolytic digestion with papain results mainly in a drop of the The fraction of radioactivity in the molecular weight by about 3,000. supernatant is larger than those in both the trypsin and the pronase digestions_ It,. therefore, seems that papain cleaves off a somewhat larger part of the exposed chain ends than in the two aforementioned cases. Papain attacks, to a small ektent, also some other regions, as can be seen from the presence of two additional weak bands of’loaver molecular weights (Table z), as well as from a peak in radioactivity (Fig. a) in the 7,000-10,000 dalton region. This peak is similar in shape, but smaller However, although the fragin size than that in the case of pronase. ments removed from the protein chain are larger than those for the trypsin case, and thus the location of the sites of cleavage is different, this has little consequence on the chromophore loss and sensitivity to illumination_ It is reasonable to assume that the limited amount of chromophore loss observed is correlated to the appearance of the limited quantity of r6,6oo-dalton fragment, just as in the pronase case the large drop in chromophore concentration upon illumination and the appearance of a large amount of 16,6oo-dalton fragment go together. We now turn to a consideration of the consequences of enzyme

735

Tsuji

and

Rosenheck

cleavage on the functional properties of the purple membrane.

The pH

during the Light-on period have been ascribed to uptake of protons by the proteoliposomes, while the relaxation of these pH changes

changes

in the light-off period has been attributed to passive back-diffusion of the protons through the vesicle membrane.‘l The kinetics of these changes was found to be accounted for by at least two first-order processes.20s25 \Ve find that the kinetics in both light-on and light-off processes appears to be a function of estent of enzymatic degradation of the bacteriorhodopsin. Thus, as shown in the Reszrlts section, the rate constants and amplitudes show gradually increasing changes as one goes from trypsin to papain, and on to pronase. The most pronounced effects are in pronase on kc. o~f which increase by about one order of magnitude digested membranes. At present, it seems most reasonable to attribute these changes in k,,ff to an increased rate of passive back-diffusion of

protons in the dark. It is interesting to note that this process is practically independent of whether the enzymatic treatment was carried out

in the dark-adapted or in any one of the two light-adapted states. kc,,, does, however, strongly depend on the state of adaptation of the purple membrane during proteolysis, especially for the pronase treated samples. If one considers the various processes, i.e. proton uptake and proton back-diffusion, as going on simultaneously also during the light-on period, the apparent lvariations in the values of kl_., can be shown to arise from the increased amounts of proton leakage. The values of k2,0,, corrected for leakage, are listed in Table 4, together with the extents of the leak, obtained by the simple analysis given in the Afi~e~zd~x. It then turns out that the values of k 2.on are all within a narrow range. irrespective of the kind of enqme used and the state of adaptation of the purple membrane, and that, furthermore. they differ from those of the untreated membrane by a factor of at the most 1.5. It should be

mentioned that the vaIues of klwo,, are not affected by the correction. In contrast to the relative insensitivity of the rate constants to proteolytic cleavage, the amplitudes characterizing the proton pump actil-ity a.re dramatically- changed. Even taking the percentage of leak

(ranging from 2 to 20 o/O ; Table 4) into account the amplitudes remain smaller than those of the untreated membrane, by a factor of 2 and 4 for papain and pronase, respectively. It should be stressed that the values in Table 5 are normalized for chromophore concentration, and thus these losses in activity cannot be accounted for simply by chromophore loss. As mentioned earlier the cleavage near one of the turning points does not affect the spectral properties of the bacteriorhodopsin in some instances, such as by pronase treatment in the dark, so that the conformation of the chromophore site seems to remain intact. Nevertheless, It is conceivable such cleavage is seen to abolish the pump activity. that the continuity of the protein chain, at least for those segments in the vicinity of the retinal site is essential for the working of the proton

pump.

Proteolysis

of Bacteriorhodopsin

739

Acknowledgements We wish to thank Prof. S. R. CAPLAX and Dr. bl. EISEXBACH for a critical discussion of this work. \Ve are also grateful to Mr. H. GARTT for his advice on the technique for measuring the proton pump activity, and to Miss A. Z_-\K_~REA for her technical assistance. This research was supported by a grant from the Stiftung I’olkswagenwerk. Appendix Caldation

of the e#ect of the leak OILk,so,,

As mentioned above, both the light-on and the light-off processes have been approximated by two first-order reactions such as :

Here, both y,,,, (t) and yOr/(t) are normalized and the leak effect is neglected in the light-on orocess. If the leak effect is taken into account, and its rate constant is taken to be given by k2,0,-,-,equation (I) should be rewritten as follows : A

y,,,

(t) =

1 -

with : . and

5; 1=I

A/‘, on esp

k’ 3.on -

(-

k’,,O,

d)

(3)

k,,t.//

: -4 r 3.i

<

0

. The parameter ri Ia+,,, can be estimated, using the trial and error method, so that the linearity of the semilogarithmic plot in the slow phase is conserved after the subtraction of the leak effect given by , A’ Q.on =p (--Con 0. Then the equation of the light-on process corrected for the leak becomes :

y’,, (t) = y,, (t) + &on

=p

(-

&.o,,

t)

=

I -

i G.on 2=I

=p

(--k’,,,

(4 (4

The estimation of A’i,o,land k’, J,, from equation usual peeling-off method.

(4) is done by the

Tsuji

7-P

and

Rosenheck

References 1

D. OESTERHEL~

1

I49 (1971) D_ OESTERHELT

and and

\\-_ STOECKESIUS, \\-.STOECKESIGS,

(London)

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(1973)

R. HESDERSOS and P.S.T. Usw-rs, Xntzrre (Loxdou) R_ HESDERSOS. /_ 310[. Biob. 93, 123 (1975) _%\.E.BLAUROCK, / Moi. BZOZ. 93, 139 (1975) D. OESTERHELT, Jr. MEEXTZES and L. SCHIJHMASS, i

453 (19’73) I<_ OHSO.

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and

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Btochzrn.

New Set.

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233, 2553

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Biop/r_l,s.

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(‘9Ti) 8 9

10 11 1%

13 14 15 16 Ii 18 19 20

L-Y.‘. JAS, Iwrsiotr Res. 15, IOSI (1975) D. OESTERHELT and B. HESS, Ezir. J. Biochein. 37, 316 (1973) R.N. LOZIER. R.X Boco~ro~sr and 1;. STOECKESIUS, BiopJiys.

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D. OESTERHELT, L SCHIJHM_~SS and H. GRUBER. FEBS Lett. 44,257 (1974) P. LISDSER, A. ZXKARIX and S.R. I<. ROSESHECK, 11. BRITH-LISDSER. CAPLAS, Brophys. Struct MecJl., in press (197s) _A. DAXOS and \\-.STOECKESIUS, Proc. Nntl. dcad. SCL. US--f 71, 1234 (197-c) D. OESTERHELT and \\-.STOECKESIUS. JIetJlods E~ynzol. -31, 667 (197-I) E. RACKER. Bioclrem Bioph_vs Res Conrmu~r. 55, 224 (1973) E. RACKER and PC. HISKLE, J Membv. BioC 17, ISI (Igil) -1-E. BOLTOS and \\-_>I.HUSTER. Bzochem J_ 133, 529 (1973) U.K. LAE\I\ILI, A-ntzrre (Londox) 227, 6So (1970) P. TRAX-HURS, P. MASDEL and S. VIRVAUS. E-t-p. Eye Res. 19, zsg (1974) R. KORESSTEIS and S.R. CAPLAS, FEBS M. EISESBACH. E P. BAKKER. Lett

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RH. LOZIER, \V. XIEDERBLRCER, R-1. BOGOMOLSI. S-B HWASG and \V'.STOECKESIUS, BrocJ~rnr BropJ~ys. Accta 440, 343 (1976) \-v.A. ov CHISSIKO\-, S.G. _XBDULAE\-, J1.Y-u FEIGISA. ..\.V.RISELIZ\- and S.-A_ LOBASOV, FEBS Lett. 84, I ('977) G.E GERBER, C.P. GRAI-, D. \\-ILDESAUER and H G. KHORASA. Proc. h’alt. rlcad.

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P.S.T. Us\\-IS and R HESDERSOS, S.-B_ HWASG and \V. STOECKESIUS,

J_ Mol. Btol 94, 425 (1975) J_ Menrbr. Bzol. 33, 3~5 (1977)