Proc. Natl. Acad. Sci. USAVol. 92, pp. 3036-3040, March 1995Biochemistry

The primary structure of sensory rhodopsin II: A member of anadditional retinal protein subgroup is coexpressed with itstransducer, the halobacterial transducer of rhodopsin II

(archaea/phototaxis and chemotaxis/signal transduction/phoboreceptor/photorhodopsin)

RALF SEIDEL*, BIRGIT SCHARF*, MATHLAS GAUTELt, KARL KLEINEt, DIETER OESTERHELTt,AND MARTIN ENGELHARD*§*Max-Planck-Institut fur Molekulare Physiologie, Rheinlanddamm 201, 44139 Dortmund, Germany; tEuropean Molecular Biology Laboratory, Meyerhofstrasse 1,69117 Heidelberg, Germany; and tMax-Planck-Institut fur Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany

Communicated by Howard C. Berg, Harvard University, Cambridge, MA, November 15, 1994

ABSTRACT The blue-light receptor genes (sopII) of sen-sory rhodopsin (SR) II were cloned from two species, thehalophilic bacteria Haloarcula vallismortis (vSR-II) and Na-tronobacterium pharaonis (pSR-II). Upstream of both soplIgene loci, sequences corresponding to the halobacterial trans-ducer of rhodopsin (Htr) II were recognized. In N. pharaonis,psopIl and phtrll are transcribed as a single transcript.Comparison of the amino acid sequences of vHtr-II andpHtr-II with Htr-I and the chemotactic methyl-acceptingproteins from Escherichia coli revealed considerable identitiesin the signal domain and methyl-accepting sites. Similaritieswith Htr-I in Halobacterium salinarium suggest a commonprinciple in the phototaxis of extreme halophiles. Alignmentof all known retinal protein sequences from Archaea identifiesboth SR-Ils as an additional subgroup of the family. Positionsdefining the retinal binding site are usually identical with theexception of Met-118 (numbering is according to the bacte-riorhodopsin sequence), which might explain the typical bluecolor shift of SR-II to -490 nm. In archaeal retinal proteins,the function can be deduced from amino acids in positions 85and 96. Proton pumps are characterized by Asp-85 andAsp-96; chloride pumps by Thr-85 and Ala-96; and sensors byAsp-85 and Tyr-96 or Phe-96.

In bacterial taxis, the cell recognizes and reacts actively tonumerous chemical and physical stimuli. A well-studied ex-ample is found in the chemotactic behavior ofEscherichia coli.The chemoreceptors of E. coli display two functional proper-ties, the reception of the signal on the external side of the celland the signal transduction to cytoplasmic proteins (reviewedin ref. 1). Characteristic of transducer proteins is their abilityto adapt to a constant signal input by reversible methylation ofspecific sites flanking a cytoplasmic domain (signal domain),which links the incoming signal with cytoplasmic components.

In a similar signal transduction system, the phototacticactivity of the archaeon Halobacterium salinarium is mediatedby photoreceptors (reviewed in refs. 2 and 3) that have a closestructural relationship to the ion pumps of bacteriorhodopsin(BR) and halorhodopsin (reviewed in ref. 4). The bacteria areattracted to light >520 nm and repelled by light <500 nm. Thephotoattractant response of Halobacteria is mediated by sen-sory rhodopsin (SR) I. In a two-photon reaction, it can alsotrigger a negative response toward near-UV light. Moreover,repellent light of wavelengths <500 nm is recognized by asecond pigment, SR-II, which absorbs at -490 nm.

Quite early it was shown that methylation/demethylationreactions are involved in phototaxis in H. salinarium (5); thesubstrate is a 94-kDa protein (6). Recently, the amino acid

sequence of SR-I (7) as well as the primary structure of amethyl-accepting protein, the halobacterial transducer of sen-sory rhodopsin (Htr) I (8), became available. The open readingframe (ORF) of the latter protein was found upstream of thegene encoding SR-I. Both ORFs are transcribed as a single unit(9) and function as a stoichiometric complex in signal transduc-tion (10). Htr-I shows distinct similarities to the bacterial chemo-receptors, especially in the signal domain region and in theflanking methylation/demethylation sites. Apparently, Archaeahave developed information transfer machineries that possessextended structural similarities to bacteria.

Less is known about SR-I1, which was first described byTakahashi et al. (11). This receptor eluded a thorough analysisdue to its minute concentration in the cell and its instability atlow ionic strength and in the presence of certain detergents.Although a partial purification was accomplished (12), infor-mation was mostly restricted to spectroscopic data and phys-iological properties (13, 14). The absorption maximum occursat 490 nm with a shoulder at 460 nm, suggesting a planarstructure of the retinal chromophore (15). Its molecular massof 24 kDa is typical for the bacterial rhodopsins. The photo-cycle of SR-II, like that of BR and SR-I, includes an interme-diate with a blue-shifted absorption maximum characteristic ofa deprotonated Schiff base (12, 16).The difficulties encountered in isolating SR-II from H.

salinarium led to a search for SR-II-like pigments in otherspecies. Such a blue-light receptor was detected in Natronobac-terium pharaonis (17), which was characterized spectroscopi-cally (18). This SR from N. pharaonis (pSR-II) was found to befunctionally identical to SR-II and to be stable under low saltconcentrations (19, 20), which allowed its purification and thedetermination of its N-terminal amino acid sequence (21).By screening a wide variety of Archaea with suitable oligo-

nucleotides, further information about the primary structureof SR-II from Haloarcula vallismortis (vSR-II) was gained.The present work resulted in the sequence determination$

of SR-II from two archaeal species and their correspondingHtr-IIs. The transducers have considerable sequence identityto the chemotactic receptors described in E. coli, indicating acommon principle of signal transduction in Bacteria andArchaea. The SR-IIs from the two Archaea are members of anadditional subgroup of the retinal protein family.

MATERIALS AND METHODSBacterial Strains, Plasmids, and Culture Conditions. N.

pharaonis strain SP1[28] (17) and H. vallismortis (22) were usedfor isolation of DNA and preparation of genomic clones.

Abbreviations: SR, sensory rhodopsin; Htr, halobacterial transducerof sensory rhodopsin; ORF, open reading frame; Tsr, transducer ofserine receptor; pSR-II and vSR-II, SR-II from Natronobacteriumpharaonis and Haloarcula vallismortis, respectively.§To whom reprint requests should be addressed.IThe nucleotide sequences reported in this paper have been depositedin the GenBank data base (accession nos. Z35086 and Z35308).

3036

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad Sci. USA 92 (1995) 3037

N. pharaonis SP1[28] and H. vallismortis were grown in asynthetic medium as described (23) and in a yeast/sea watermedium (22, 24), respectively. E. coli strain XL1-Blue (Strat-agene) was grown in 2x YT (25) medium containing theappropriate antibiotics and transformed by electroporation(Gene Pulser, Bio-Rad).N. pharaonis DNA was cloned into the plasmid vector

pBluescript I SK(-), and H. vallismortis DNA was cloned intopBluescript II KS(+) (both from Stratagene).

Determination of the N-Terminal Sequence. pSR-II wassolubilized in dodecyl maltoside and purified using severalchromatographic steps (21). The final purification step in-cluded gel filtration (Superose 12; Pharmacia) in organicsolvents (formic acid/acetonitrile/isopropyl alcohol/H20,50:25:15:10, vol/vol). The N-terminal peptide was sequencedby automated Edman degradation (Applied Biosystems 473Asequencer).Molecular Biological Procedures. Unless indicated other-

wise, standard molecular biological methods were used (25).Oligonucleotides. Oligonucleotides were synthesized on an

automatic DNA synthesizer (Gene Assembler; Pharmacia/LKB) and purified by 15% polyacrylamide/7 M urea gel elec-trophoresis and reversed-phase chromatography using SepPakC18 cartridges (Waters). Oligonucleotides were end-labeled withT4 polynucleotide kinase (Boehringer Mannheim) and[y32P]dATP (6000 Ci/mmol; Amersham; 1 Ci = 37 GBq).

Isolation of Genomic DNA and Southern Analysis. GenomicDNA was isolated and purified (26). For Southern blot anal-ysis, 10 ,ug of total N. pharaonis DNA was digested withdifferent restriction endonucleases. After electrophoresis on a0.9% agarose gel, the DNA was transferred to a nylon mem-brane (Biodyne A; Pall) by capillary blotting under alkalineconditions (27). Southern blots were hybridized in sealedplastic bags. The hybridization conditions were as follows: 6xSSC/5x Denhardt's solution/0.1% SDS/100 ,ug of sonicatedsalmon sperm DNA per ml of hybridization solution/2-4 x105 cpm of the radioactive oligonucleotide per ml. Afterincubation at 45°C for 12-20 h, the filters were washed in 2xSSC/0.1% SDS starting at room temperature and increasingthe stringency by washing at 50°C, 55°C, and 60°C.

Size-Enriched N. pharaonis DNA Libraries. After prepara-tive digestion of genomic DNA and electrophoresis on low-melting-point agarose gels, DNA fragments of the desired sizewere isolated from the gel by extraction methods (28) andligated into pBluescript SK(-). Positive clones were detectedby colony hybridization with end-labeled oligonucleotide orrandom-labeled DNA fragments {by random priming withhexanucleotides and [a-32P]dATP or digoxigenin-labeleddUTP (DIG-11-dUTP)} as a probe.

Cloning ofH. vallismortis DNA. For PCR, a sense (bopseql7:5'-GACTGGMTSKTSACSACSCCG) and an antisense (bop-rev33: 5'-GACCTTCGCGADCACGTC) oligonucleotide (M= A or C; S = C or G; K = G or T; D = A, G, or T) weredesigned based on well-conserved sequences of helix C andhelix G in the retinal proteins bop and sopl (24). GenomicDNA purified from H. vallismortis (24) was used as template;the reaction was performed at 95°C for 45 sec, at 58°C for 45sec, and at 72°C for 60 sec. After 25 cycles, a PCR product of400 bp was obtained, purified, and subcloned. Southern anal-ysis of Sma I-digested H. vallismortis DNA probed with theamplified 400-bp fragment resulted in a single band at -3.1kbp. The detected fragment was cloned, and positive cloneswere identified by colony hybridization and analyzed bydouble-strand sequencing.

Sequencing. Double-stranded DNA was sequenced by thechain-termination method by using a Sequenase version 2.0 kitfrom United States Biochemical. The ends of fragments weresequenced with universal primers, and the remainder wassequenced by primer walking. Compression artifacts wereresolved using 7-deaza-dGTP (Pharmacia) and Taq poly-

merase (Boehringer Mannheim). Both strands of the geneswere sequenced.

Isolation of Total RNA and Northern Analysis. Total N.pharaonis RNA was isolated according to ref. 29. Ten micro-grams of total RNA was subjected to electrophoresis in a 2.2M formaldehyde/1.2% agarose gel, transferred to a nylonmembrane (Biodyne A; Pall), and probed with the appropriateDNA fragments. Hybridization conditions were 42°C in 50%formamide/5X SSC/0.1% SDS. Washing conditions were 65°Cin 0.1x SSC/0.1% SDS.

Alignments. Protein sequences were initially aligned usingthe program PILEUP (version 7.0) from Genetics ComputerGroup and then further aligned manually. Sequence similar-ities were established using the BESTFIT program (GeneticsComputer Group) operating under default parameters.

RESULTSGene Isolation and Sequencing. pSR-II was purified essen-

tially as described (21). A partial amino acid sequence con-sisting of the first 30 N-terminal amino acid residues wasdetermined: (A, V)GLTTLF(F, W)LGAIGMLVGTLAF(F,A)(I, W)AGR(R, D)AG(G, S). The parentheses denote po-sitions where the correct amino acid could not be establishedunequivocally, but the residues underlined were confirmed byDNA sequencing. To identify the psopII gene, a partiallydegenerate 25-mer oligonucleotide (5 '-TTCTTCCTCG-GCGCMATCGGCATGC-3'; where M = A or C) coding forFFLGAIGML (aa 7-15) was synthesized using the codonpreference described in ref. 30.With the above oligonucleotide, a single band in the region

of 1.6 kbp hybridized in a genomic Sac I digest. This 1.6-kbpfragment was isolated from preparative gel electrophoresisand cloned in pBluescript. Several positive clones were found,and one clone (pRS-1.6SacI; Fig. 1A) containing the 1.6-kbpinsertion was used for restriction mapping and double-strandsequencing. An overlapping clone (pRS-1.7SalI) was isolatedfrom the Sal I genomic library probed with the 516-nt SacI-Cla I terminal fragment from pRS-1.6SacI. Both clonescovered 3058 bp (GenBank accession number Z35086).The overlapping clones contain two ORFs. A 1602-nt ORF

starts at position 505 with an ATG and ends at position 2107 with

A

a

8 8PRS-1.78tII

0 S~~~~~~~~co

S Kb i -

0 394

1475m

& SSS C Sp AC east37 1708 I -9 2 25 2 Z70

1471 177 1j839 2524 B

1487 1792

3058

Al SC

283082988

ATG TAA ATG TM

phtr1802bp) I pso 1)7A

505 210 2112 2831

8m Sm

I pKKMOPpKKb

c

Ba

0 so Av 2978

S5EV SeN 58 EN SPS B AV E B AvSb 4 11111 ir11 402486 97 I32 18281 178 2033 2294'1 2823 12978as 44 1388 in1 2339 27921

TMATG TAG

C vhtr7rlI>1304 I vso(I1M |A1304 107 217 200bp

FIG. 1. The restriction map of the gene loci of phtr-II/psop-II (A)and vhtr-II/vsop-II (B). (a) Isolated clones. (b) Restriction maps. (c)ORFs. At, Aat II; Ac, Acc I; Av, Ava I; Bg, Bgl II; Bs, BssHII; C, CiaI; E, EcoRI; EII, EcoRII; EV, EcoRV; K, Kpn I; N, Not I; P, Pst I; Sc,Sac I; S, Sal I; Sm, Sma I; Sp, Sph I.

Biochemistry: Seidel et al.

Proc. NatL Acad Sci USA 92 (1995)

a TAA stop codon. The following 717-nt ORF begins twonucleotides after the stop codon. It starts with anATG at position2112 and ends at position 2829 with a TAA stop codon. A similararrangement of the genes is observed in the vhtrII/vsopII gene

region (GenBank accession number Z35308; Fig. 1B).The amino acid sequence deduced from the second ORF

(positions 2112-2829) contained the N-terminal amino acidsof the previously determined pSR-II protein sequence andadditionally the N-terminal methionine, confirming the iden-tity of the genomic sequences with psopII. It encodes a proteinof 239 aa with a calculated Mr of 25,355, whereas the corre-

sponding vsopII gene encodes 236 aa with a Mr of 25,025.Sequence analysis upstream of psopII revealed a 1602-nt

(positions 505-2107) ORF, analogous to the gene arrangementin the recently reported H. salinarium htr-I-sopI operon (8).The ORF coding for the putative Htr-II contains two possibleATG initiation codons (corresponding to Met-1 and Met-22).Because the amino acid sequence between these positions ishighly similar to Htr-I, the first ATG is more likely to be theinitiation codon. The entire protein would then consist of 534amino acid residues with a calculated Mr of 56,622.Twenty-nine base pairs upstream from the predicted initi-

ator methionine codon (position 505) the sequence TT-TATGT is observed, which is in agreement with the box Aconsensus sequence of archaeal promoters (31). Neither a

clearly defined box B-related sequence nor a ribosome bindingsite could be identified.The 3'-noncoding region of psopII gene contains a short

inverted repeat, which could be a possible termination site. InvsopII a significant palindromic structure following the stopcodon was found (24).

Coexpression of pHtr-II with pSR-II. To investigate whetherthe two detected genes, which are separated by 2 nt, are tran-scribed as a single unit as recently reported for the H. salinariumhtr-I-sopI operon (9), we performed Northern analysis of total N.pharaonis RNA (data not shown). The presence of a -2.3-kbspecific mRNA is detectable with two probes representingphtrII-and psopII-specific sequences. These data suggest that the genes

are cotranscribed as a bicistronic mRNA.

DISCUSSIONSR-II. The sequence alignment of the two photophobic

receptors (vSR-II and pSR-II) with members of the otherbacterial rhodopsins demonstrates that certain positions are

specific for their functional classification (Fig. 2; colored ingreen; unless indicated otherwise, numbering is according tothe BR sequence). For BR, these residues are Asp-85 andAsp-96, which are crucially involved in the proton pump

mechanism (reviewed in ref. 4). In halorhodopsin these aminoacids are characteristically changed to Thr and Ala, respec-

tively. For the SRs, the position corresponding to Asp-85(Asp-76 in SR-I; Asp-75 in SR-II) is conserved, while Phe or

Tyr occupies the second position.A further distinctive assignment is found in BR positions 115

and 118, which both belong to the retinal binding pocket, withMet-118 being close to the /-ionone ring (4). If the pigmentsare absorbing at wavelengths >550 nm, Asp and Met are

characteristic in these positions. In both SR-IIs the corre-sponding sites are replaced by Gln and Thr (vSR-II) or Asn andVal (pSR-II), and their absorption maxima are found at <500nm. A similar blue shift of about 2800 cm-' was described forthe BR mutant Met-118 -- Ala. However, this shift is not

observed if Met-118 is replaced by Glu, which has about thesame molecular volume as Met (32). From these data, it wassuggested that the size of the amino acid might be involved indetermining the color of the chromophore. Whether thisassumption is connected to the hypsochromic shift and the finestructure of the absorption band of SR-Il and whether it can

be reconciled with current models (15, 33) of wavelengthregulation in SR-II and BR remain to be elucidated.

It is known from site-directed mutagenesis experiments thatBR mutants such as Asp-96 -* Asn do pump protons, thoughless efficiently (34). Because SR-I and SR-II possess aromaticresidues in position 96 but keep Asp-85, they should, inprinciple, be able to translocate protons. Indeed, Olson andSpudich (35) observed that upon light excitation of SR-I andin the absence of the transducer Htr-I, protons are released tothe bulk phase. Furthermore, the capability of SR-I to pumpprotons has been' demonstrated using black lipid membranes(U. Haupt, D.O., and E. Bamberg, unpublished data).For both sensory pigments, changes in the Fourier transform

IR spectra occur in the region of carboxylic group(s) duringformation of the long-lived intermediates (36, 37), which arecharacterized by a deprotonated Schiff base. Whereas in SR-Ionly an environmental change of Asp-76 is observed (37), thephotoactivation of pSR-II clearly leads to a protonation of acarboxylate group, most probably Asp-75 (36). Whether thesedifferences bear significance for the type of signal transducedstill remains to be determined.From the amino acid sequences of the four bacterial rho-

dopsins, it is now possible to identify the amino acids that arefundamentally important for the binding of retinal to theprotein (Fig. 2, colored in red). It becomes evident that mainlyhelices C and F contain conserved residues. The motif of helixC consists of the sequence in BR of 82RYXXWXXXTP.According to the model of BR (4), Arg-82 is a member of theretinylidene counter ion complex, whereas Trp-86 belongs tothe layers that intercalate retinal. The other two componentsare two Trp residues from the second conserved sequencemotif in helix F: 182WXXYPXXWXXGXXG. In helix D onlyGly-122, which provides space for the cyclohexene ring ofretinal, is fully conserved. Interestingly, in helix G, the se-quence DXXXK corresponding to the binding site of retinal iswell conserved.A calculation of the phylogenic relationship of all known 22

amino acid sequences of bacterial rhodopsins establishes fourdifferent members of the family: (i) proton pumps, (ii) chloridepumps, (iii) SR-I, and (iv) the additional distinct member SR-II.The sequence homologies between both SR-I and SR-II

(Fig. 2, colored in blue) and the two SR-IIs (colored in yellow)might be indicative of the sensory function. Identical residuesfound in the helices of the SRs in addition to universally con-served residues (in red and green) are similar to the correspond-ing residues of the other retinal pigments [e.g., BR positions 49and 60 (hydrophobic), 89 (hydrophilic), and 135 and 138 (aro-matic)]. On the other hand, the helix connections of the sensorypigments show specifically conserved residues, although thehomology of the three cytoplasmic loops and of the C termini,where one would expect possible zones of contact between theSRs and their transducers to be present, is not really pronounced.It might be conceivable that the protein-protein interaction isregulated mainly by surface hydrophobicity or that an interactionbetween the helices of SR and Htr occurs (10).

Htr-II. SR activity involves specific transducer molecules.The halobacterial transducer I has been identified as a proteinand subsequently sequenced (8). Independently, a geneticapproach led to its identification (9). The physical interactionof Htr-I with SR-I was demonstrated by genetic and physio-logical methods (9, 10, 35, 38).

In the case of the SR-II-Htr-II pair, such a functionalrelationship has not been demonstrated. The data presentedhere show that SR-II and Htr-II are transcribed as a singletranscript, and polyclonal antibodies raised against the C-terminal peptide detected proteins with apparent molecularmasses of 64 kDa and 96 kDa in a Western blot analysis (datanot shown). The calculated mass of pHtr-II is 56,622 Da, whichwould coincide with the band at 64 kDa. The higher molecularmass compound might be caused by dimerization on treatment

3038 Biochemistry: Seidel et al.

Proc. NatL Acad Sci USA 92 (1995)

BRHRSR IvSR IpSR U

-13....... MLE LLPTAVEGVSM SITSVPGVV DAGVLGAOSA

51 61BR AIAFTM LSM LLGYGLTMVPHR LVSISS LGL LSGLTVGM IESkRI VF GLS VG YD TVIV NvSR| G VA A L L GF SIOQS EpSR II G I A VA VVY L GV WV PVYA

45 55

GAO TORPEWAAVRENAL L S.. MDAVA.MAT TTWMMVGLTTL

71F GG.. EMPAGHAL AGEN Q.

G H A.E R T.65

A B1.1 21 31 41IWLALGTALM GLGTLYFLVK GMGVSDPDAK KFYAITTLVPSSLWVNVALA GIAILVFVYM GRTIRPGRPR LIWGATLMIPTAYLGGAVAL IVGVAFVWLL YRSLDGSPHO SALAP Al PF TL GL L GE LL G TA VL AY .. G YT LV PE E TR KR Y LL PFWLGAIGMLV GTLAFAW... AGRDAGSGER RYYVT VG S8 1 8 25 35

C75 85 95 105QNPIYWA A LFT LLL L LALLVDAD QGTILALVGAMV RS QWGG L A LS _M IL L L GL LA DV D L GS LF TVYIA A

.I VGLI LV IL G VGYA_AS RRS IGVMVAVFA V L_LPN W LAL AS REDTVKLVVL

...V F A P_I L L Y LG L L L D S R E F G V T L75 85 95

D E F115 125 134 144 154 163 173

BR GI I3GT LV GALTKVY.SY RFVWWAISTA AMLYILYV F FGFTSKA.ES MRPEVASTFK V RNVTVVLHR IG CCVT LA AAMTTSALLF RWAFYAISCA FFVVVLSA V TDWAASA.SS AGT..AEIFD T RVLTV VL

SRI AL IAV AG AVVTDGT ..L KW GVSSI FHLSLFAY Y VIFP.RVVPD VPEQIG FN L K HIGL

vSRII AL IVf FA GAVTPSP ..V SY AVGGA LFGGVIYL Y RNIAVAAKST LSDIEVS YR T R FVVV

pSRI TV MLA FA GAMVPGI .E RY GMGAV AFLGLVYY V GPMTESASQR SSGIK.S YV R R LTVI

105 115 123 133 143 153 162

G183 193 202 212 222

BR SA V L SE AG. IVPL NIETLLFMVL *VSAE GFGL ILLRSRAIFGHR LG IV AV VE LALVQSV GVTSWAYSVL VFAYVFAF ILLRWVANNE

SRI LA LV F PA GEATA AGVALTY F VLA PYVY FFYARRRVFM

vSRI LVV AL AGA VG.LMDV ETATLVV Y VVT_GFGV IALLAMIDLG

pSRlI Al F1 LEPP VA.LLTP TVDVALI Y LV GFGF IALDAAATLR172 182 191 201 211

232EAEAPEPSAGRTVAVAGQT LHSESPPAPEQSAGE TAE EPTAEHGE SLAG V221

242DGAAATSDGTMSSDDATVEATAADA V A G DDTDAPAVAD231

FIG. 2. The amino acid sequences of pSR-II and vSR-II derived from the DNA sequence of their gene in comparison with SR-I, HR, and BR.The colors indicate sequence homologies between (i) pSR-II and vSR-II (yellow), (ii) both SR-IIs and SR-I (blue), and (iii) all known sequencesof bacterial rhodopsins (red). Amino acids with a green background are positions of functional importance (see text). The helical regions are markedby dotted lines, and the connections between helices are marked by solid lines. The sequence numbers are given for BR (top) and pSR-II (bottom).

with heat and/or detergents. A similar effect was also observedfor halocyanin, a blue copper protein from N. pharaonis (39).The observation of a single transcript indicates that similar

to SR-I-Htr-I a SR-II-Htr-II protein complex exists. Thisassumption is substantiated by the observation that the pho-tocycle of SR-II in membranes is faster than in solubilizedpreparations (12) pointing to, as it is the case for SR-I-Htr-I(10, 38), a physical interaction of SR-II with Htr-II, which leadsto the physiologically important construct.The alignment of the transducers pHtr-II and vHtr-II and

the serine receptor (Tsr) from E. coli is shown in Fig. 3. Fourdifferent regions can be differentiated. The first two areunique for the archaeal proteins and consist of the membrane-spanning helices and the intervening loop between membraneand methyl-accepting sites. These two areas clearly showhomologies not only between the two SR-II-related transduc-ers but also with Htr-I. According to a hydropathy plot, thetransmembrane-spanning helices are found at the N terminus.This coincides with a cluster of conserved amino acids.The following sequence of about 140 aa has been considered

functionally important and possibly capable of interactionswith the photoreceptor (10). Several interesting sequencemotifs can be recognized in all three Htrs. The first spans astretch of 20 aa with the consensus sequence 113RXDEXGX-LXXXXXXMRXXXR. Taking similarities into account also,this motif appears even more conserved. It is noteworthy thatthe intervening sequence between Leu-120 and Met-127 (yel-low) is only conserved in Htr-II. Two more clusters of se-quence homology are found further downstream: 145AEX-AXXXA and 174AXGDXTXR.The loop region is followed by areas (Val-240 to Thr-481) very

similar to those of the bacterial transducers like Tsr presentingseveral methylation sites (green) and the signal domain (red) (41).Four methylation sites are congruent with Tsr, although quiteoften Glu or Gln is changed to Asp or Asn. A fifth site at the Cterminus (Glu-480; light green) is not clearly reproduced in Htr.The signal domain (red) of the E. coli transducers is evidently

maintained in the archaeal counterparts. These extensive homo-

logies raise the question about the possible phylogenetic origin ofthe transducers and their receptors. Retinal protein structures areshared between Archaea and Eucarya. The photochemistry,however, an 11-cis/trans isomerization found in several vertebraterhodopsins versus a trans/13-cis thermoreversible photoexcita-tion for the bacterial rhodopsins, is different, as are the coupledproton reactions. The "transducers" in many Eucarya are Gproteins, whereas archaeal transducers resemble bacterial che-moreceptor/transducers. Recent studies (40) analyzing transcrip-tion factors in Bacteria, Archaea, and Eucarya suggest that theArchaea may have branched from the main tree later than theBacteria. If this is true, it would mean that rhodopsins haveevolved after Bacteria branched from the main stem but beforeArchaea appeared. The archaeal branch could have combinedthe bacterial signal transduction with the newly invented photo-receptors. In the further course of evolution, a mechanismdeveloped that relied on G-protein-coupled receptors, making itunnecessary to conserve the bacterial transducer molecules.

We thank R. Wittenberg for support in membrane and antibodypreparations, A. Scholz for the peptide synthesis, and A.-M. Voie, H.Erfle, and V. Adam for DNA sequencing and oligonucleotide syn-thesis support. The technical help of M. Hiilseweh, M. Wischnewski,and K.-H. Wuster is gratefully acknowledged. This workwas supportedby a grant from the Bundesministerium fiir Forschung und Technolo-gie.

1. Hazelbauer, G. L. (1992) Curr. Opin. Struct. Biol. 2, 505-510.2. Oesterhelt, D. & Marwan, W. (1993) in The Biochemistry of

Archaea, eds. Kates, M., Kushner, B. J. & Matheson, A. T,(Elsevier Science, Amsterdam), pp. 173-187.

3. Spudich, J. L. (1993) J. Bacteriol. 175, 7755-7761.4. Henderson, R., Baldwin, J. M., Ceska, T. A., Zemlin, F., Beck-

mann, E. & Downing, K H. (1990) J. Mol. Bio. 213, 899-929.5. Schimz, A. (1981) FEBS Lett. 125, 205-207.6. Spudich, E. N., Takahashi, T. & Spudich, J. L. (1989) Proc. Natl.

Acad. Sci. USA 86, 7746-7750.7. Blanck, A., Oesterhelt, D., Ferrando, E., Schegk, E. S. & Lotts-

peich, F. (1989) EMBO J. 8, 3963-3971.

Biochemistry: Seidel et aL 3039

3040 Biochemistry: Seidel et al. Proc. NatL Acad Sci USA 92 (1995)

1 1 21 31 41 51 61MSLNVSRLLL PSRVRH SYTG KMGAVFIFVG ALTVLFG:AIA YGEVTAAAAT GDAAAVCQEAA Y,SA[A.LG:LII:L

MT IAWARRRYGV

71 81LGINLGLVAA TLGGDT AASL

HtrICI S i N

141pHtr 11 A R. EvHtr 1 A K. QHtr TRADLEETQA

KLGLGY ATA GLL.YGVAiVTTelix 1

91 101S T L AAK ASRM G D:GD L.D v.E:L E

AAET VAS K E AAQ T E.RV A N ,QN,L EO.E.V T

144DA.EQ;AQKRA .EAEAAK E QAQE,AE T AR E EA E

200EMMOG EALVTTLDALEDTQMMOD LQATV

260 270pHtr Q A L E M-D.D V S T T EEV A S AvHtr 1 SAAAEMQ,L S T AQ VY S S AHtr I S A AD D VbQOV-j SAEEIA ATTsr D L S S R T,E. t:C A SSLXET ASM

N D ..Y P S T iVJ A G 6L--A ],. L T j_~~ Helix 2

1 1 1 121 131

TRR.E1,E. GDL YAAFDEM4iRC S VYT S L EDAK NE YAAFDN

STRT FG TL. YADFIDNt.N LTCRLNEMERRTfiE iAI D S E Q O$QS L .XGRL

160EEINTEL QAEAAERFGEV

AAREDVESER N £EMMEALTGHL ELKAQCQYSDAOA.KCEAQAAE R EARELAATY CDT AKRYCET

210GR ERFADAVA D M K A F.A: r N VRTVTTVADE

280DO AKTARCAOQVAD T S QSADO L A S R S E D VEQCL T A T V K N

220SEDAE AVRAN0 SSDRVNSN

EAKT ERMSET

230AESVMEASE DAERVDRASKQSAD EASAGD

170 180M C}RC^ DF1; QRL D A E T D N E

L D A A'A'NDi.L T: A,RIVKTDSMNDMEAAATfilL QOVVDVDTDHE

240VNRAVQN SODVSKSINE FETVEAVSKK ESVRNGANA YS

290 300 310IET GE AGRE T A E T T.EM:N E VE SRTEOQA AKVGEDCGR.'-EA A Q E A E,.% S A IE.AETGET E

TASDAARDS SKS LDjSS I.E TEVDDACGENARCASHL ALS SETAQR GGKVVDNV Q

330 340 350 360 368 378

pHtr 11 E GEVSEM A D A. I I RDDG NSE A VKCA EK1KAATEEvHtrn E GE I VGV T S VE HDH DCGE I K G EKEAAAD

Htr I ETD VDV T DEC.CM iS R GGNADGD S VD £ITODRANE

Tsr K ADIISV D G AF AVR GE ..QG RA G VRN QRSAQAARE

398pHtr 11 TCTTVDDIREvHtr n AGDTVETMESHtr TEDVTAS IQQTsr DSVGKVDV

468pHtr 11 S E Q:T A S D A E TvHtr 11 S CjQ:T A T E A D THtr -S.:T.T A L AS DTsr V EESAAAAAA

408T SD V S E.GV ET S T R T EIGY STR T R V E SGS EGSTLVESAGE

478AAE T T E TQA EV AC A AQ DQS AA E S A V CGR ELEEQASRL TE

418I VEDTVDAL EV E E1TV D A L EV.SjLLRD RMAE VSAVT

488SVKEVFDL IDSI EEVSDSATAA E- A A S L EAVAVFR 0Q2Q

428R IY:V)D SV E R T NT IVEYTEEVOTJ.ADS AEVSRVTD MCE IA

498G L S E Q-A D St.E L R Q R A D D.LQ F Q N T Y&V E O.LQRETSAVVKT

438 448

D GI.I:QO IN Q ST DA A;-i.- D A AIa K A

TGh.OE:D R A: EE0 R TAQ DV

N SIC HQ R Ti. S E E T VS T

SASD OSRGI DQVGLAVAEM

507SETLSRTDTESLLDRFTV.QSRVASF TVAVTPAAP

516EEEASAADLEN SAGT

TEDSETAGGS

250A A GD Q T E TV QG T T E Q. N E G L EO A N D QR T E L DCASE ATCNN

320S M E E LAN E D V RE NA'L-DDELDO V E QI R D O V ATMRD STSSQ

388CDL IGTVQ0REGR EA CEQAAVVEKVTACKSL E.

458TTMVE DMAATMGT DDLTTATSVERVAGLDRVTQONAAL

526

DDQPTLAA_GjD ,D

GTDSTAAV-DVEOPVMRA .A GG A

FIG. 3. The amino acid sequences of pHtr-II and vHtr-II derived from the DNA sequence of their gene in comparison with Htr-I and Tsr. TheTsr sequence is only depicted from the beginning of the region of homology. The helical region of vHtr-II has not yet been determined. The colorsindicate sequence homologies between (i) pHtr-II and vHtr-II (yellow), (ii) the halobacterial signal transducers (blue), and (iii) all known sequencesof halobacterial signal transducers and the eubacterial methyl-accepting proteins (red). Amino acids with a green background are positions ofmethylation in chemoreceptors (40). The sequence numbers of pHtr-II are depicted.

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9746-9753.

pHtr 11vHtr 11Htr

pHtr 11vHtr 1

pHtr 11vHtr nIHtr ITsr

190AM O S E G S F NAM A E VCE D NAM E T V G T A F N