Archaeal-type rhodopsins in Chlamydomonas: model structure and intracellular localization

BBRC Biochemical and Biophysical Research Communications 301 (2003) 711–717 www.elsevier.com/locate/ybbrc

Archaeal-type rhodopsins in Chlamydomonas: model structure and intracellular localization Takeshi Suzuki,a Kenta Yamasaki,a Satoshi Fujita,a Kazushi Oda,a Mineo Iseki,b Kazuichi Yoshida,b Masakatsu Watanabe,b Hiromi Daiyasu,c Hiroyuki Toh,c Eriko Asamizu,d Satoshi Tabata,d Kenji Miura,e Hideya Fukuzawa,e Shogo Nakamura,f and Tetsuo Takahashia,*,1 a

School of Materials Science, Japan Advanced Institute of Science and Technology, Asahidai, Tatsunokuchi, Nomi-gun, Ishikawa 923-1292, Japan b National Institute for Basic Biology, Okazaki, Aichi 444-8585, Japan c Biomolecular Engineering Research Institute, 6-2-3 Furuedai, Suita-shi, Osaka 565-0874, Japan d Kazusa DNA Research Institute, 1532-2, Yana, Kisarazu, Chiba 292-0812, Japan e Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan f Faculty of Science, Toyama University, 3190 Gofuku, Toyama 930-8555, Japan Received 19 December 2002

Abstract Phototaxis in the unicellular green alga Chlamydomonas reinhardtii is mediated by rhodopsin-type photoreceptor(s). Recent expressed sequence tag database from the Kazusa DNA Research Institute has provided the basis for unequivocal identification of two archaeal-type rhodopsins in it. Here we demonstrate that one is located near the eyespot, wherein the photoreceptor(s) has long been thought to be enriched, along with the results of bioinformatic analyses. Secondary structure prediction showed that the second putative transmembrane helices (helix B) of these rhodopsins are rich in glutamate residues, and homology modeling suggested that some additional intra- or intermolecular interactions are necessary for opsin-like folding of the N-terminal ca. 300-aa membrane spanning domains of 712 and 737-aa polypeptides. These results complement physiological and electrophysiological experiments combined with the manipulation of their expression [O.A. Sineshchekov, K.H. Jung, J.H. Spudich, Proc. Natl. Sci. USA 99 (2002) 8689; G. Nagel, D. Olig, M. Fuhrmann, S. Kateriya, A.M. Musti, E. Bamberg, P. Hegemann, Science 296 (2002) 2395]. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Retinal; Photomovement; Bacteriorhodopsin; Sensory rhodopsin; Ion channel; Photoreceptor

In the phototaxis of the unicellular green alga Chlamydomonas reinhardtii (C. reinhardtii), light is perceived by rhodopsin-type photoreceptor(s) which are believed to be localized in a small part of the cytoplasmic membrane confined in the eyespot region. The eyespot is a carotenoid-rich disk-shaped peripheral organelle whose location is close to but beneath the cytoplasmic membrane. Owing to its reflective property, the eyespot confers the directivity of the photoreceptor molecules * Corresponding author. Fax: +81-47-472-1404. E-mail address: [email protected] (T. Takahashi). 1 Present address: School of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan.

towards the exterior of the cell body. This directivity is crucial to track the light beam in phototaxis [1]. Rhodopsins are composed of the chromophore retinal and an opsin apoprotein. Based on their sequence similarity, opsins are currently categorized into two groups [2]. Archaeal-type opsins are widely distributed in various taxa except land plants and animals, e.g., halophilic archaea, marine eubacteria [3,4], and eukaryotic fungi [5,6], and the chromoproteins are referred to as type-I rhodopsins [2]. The other, the so-called typeII opsins, have so far been exclusively found in animals. Although both type-I and type-II rhodopsins show strikingly similar molecular architecture, the two groups are distinct in amino acid sequences, chromophoric

0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(02)03079-6

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configuration/conformation, and their photochemistry. Photosignal transduction in animal eyes is initiated by the isomerization of the chromophore from 11-cis to alltrans upon absorption of a photon by type-II rhodopsins. Type-I rhodopsins in their ground state, on the other hand, contain the all-trans retinylidene chromophore whose conformation about 6–7 single bond, that is, between the cyclohexene ring and the polyene chain, is coplanar 6s-trans instead of the highly twisted 6s-cis conformation in type-I rhodopsins [7,8]. The alltrans!13-cis photoisomerization triggers a variety of functions of type-I rhodopsins including light-driven ion pumping in bacteriorhodopsin, or sensory transduction to their cognate transducers in haloarchaeal sensory rhodopsins [2,9]. Recent expressed sequence tag (EST) project on C. reinhardtii [10] has provided a hint that this green alga also represents a taxon expressing type-I opsins. Two open reading frames coding for archaeal-type opsins are identified, and they are supposed to function as phototaxis receptors on the basis of physiological and electrophysiological experiments combined with the manipulation of their expression [11,12]. As an essential step towards understanding how these archaeal-type structures of the photoreceptors function, we report here the putative molecular architecture and the intracellular location of a Chlamydomonas type-I opsin.

Materials and methods Sequencing. Two cDNA sequences for Acop-1 and Acop-2 (GenBank Accession Nos. AB058890 and AB058891, respectively) were obtained from the following EST clones (Kazusa DNA Research Institute): (Acop-1) CL49h12, CL44c03, and CL65f02; (Acop-2) HCL063f11, HCL057g02. All DNA sequencing experiments were carried out by the dye-primer method with a SQ-5500 sequencer (Hitachi) on both strands. Northern and Southern blot analyses. Probes used for Northern blot hybridization analyses on polyðAÞþ RNA were 0.7 kbp DNA fragment of CL44c03 digested with XhoI and KpnI and 1.2 kbp DNA fragment of HCL063f11 digested with SacI and SmaI, for Acop-1 and Acop-2, respectively. Southern blot analyses were performed on 5 lg of genomic DNA digested with BamHI, HindIII, NheI, or SacI, fractionated on a 0.75% agarose gel, and transferred to a Hybond Nþ membrane (Amersham) by conventional capillary method [13]. Probes were prepared by PCR (primer pairs: 5-GATTACTTTTGCGCTTTCAGCG CTCT/5-CTCGATCGTGGCCACGTAAATCTC and 5-GCTTGC TGCTGGCTTCTCCATC/5-CTCGATAGCGCACACATAGATCTC CTC, for Acop-1 and Acop-2, respectively) and labeled with ECL Direct Nucleic Acid Labelling kit (Amersham). Hybridization was carried out at 42 °C overnight, followed by 20 min washes in 0.1 SSC, 6 M urea, and 0.4% SDS at 42 °C, and in 2 SSC at room temperature. Reactive bands were visualized on BIOMAX ML film (Kodak) with a chemiluminescent HRP substrate (ECL, Amersham). Plasmid construction. The C-terminal region of Acop-1 gene encoding 374-residues was cloned into NcoI/XhoI site of Escherichia coli expression vector pET-21d (Novagen) as a translation fusion to a hexahistidine tag. XhoI site was created at the 30 ends by PCR with primers (50 -GGTCACTccaTGgGTGTCAAGATCCACGAG) and (50 GTTTACTCctCgAGCTCGTTCTTCAGGCGGTTGAT). The cod-

ing sequence of the resulting plasmid pETAcop1C1-1 was confirmed by DNA sequencing. A hexahistidine fusion of the entire coding region of Acop-1gene was cloned into BamHI/EcoRI site of the Pichia pastoris (P. pastoris) expression vector pPIC3.5 (Invitrogen), by inserting BamHI/EcoRI fragment of pBAcop1, in which pBluescript II SK()) (Strategene) was inserted at first by a 6 his-containing PCR fragment amplified from pETAcop1DXhoDSph harboring 460-bp of Acop-1 gene that lacks 1.7kbp SphI/SphI region, and thereafter by a partially digested 1.7-kbp SphI fragment of CL44c03. The coding region in pBAcop1 was confirmed by DNA sequencing. Sequence analyses. The amino acid sequence alignment of type-I opsins (including Acop-1 and Acop-2) was performed with CLUSTAL W program [14] and positions of gaps were manually corrected as described in [15]. The secondary structures of Acop-1 and Acop-2 were predicted by use of several programs: Prof [16] (http://www.aber.ac.uk/ phiwww/prof/), PHD [17] (http://cubic.bioc.columbia.edu/predictprotein/), SSpro [18] (http://promoter.ics.uci.edu/BRNN-PRED/), SAM T-99 [19] (http://www.cse.ucsc.edu/research/compbio/HMMapps/T99-query.html), and PSIPRED [20] (http://bioinf.cs.ucl.ac.uk/ psiform.html). Unrooted phylogenetic tree was constructed by the neighbor joining (NJ) method [21] on the basis of the distance calculated on every pair of aligned type-I opsins by the maximum likelihood method. The reliability of each node of the tree was evaluated by bootstrap analysis with 1000 tree reconstructions. The software packages PHYLIP (J. Felsenstein, University of Washington, WA) and MOLPHY (J. Adachi, M. Hasegawa, Institute of Statistical Mathematics, Japan) were used for molecular phylogenetic analysis. The homology model of Acop-1 was constructed with the described method [22] using the structure of bacteriorhodopsin [23] as a template (PDB Accession No.: 1CQW). Eyespot preparation. The C. reinhardtii cells were centrifuged at 1000g for 10 min and the pellet was resuspended in disruption buffer (10 mM MOPS, 1 mM EDTA, 1 lM leupeptin, 1 lM pepstatin A, and 50 mM sucrose). The suspension was incubated under nitrogen at 1200 psi in a Parr cell disruption bomb for 30 min. The cell lysate was centrifuged at 400g for 15 min and the supernatant was subjected to high-speed centrifugation at 10000g for 20 min at 4 °C. The collected pellet was resuspended in disruption buffer with 1.25 M sucrose and overlaid on discontinuous sucrose density gradients (0.3, 0.6, and 1 M). Gradients were developed at 113,000g for 2 h at 4 °C. The resulting orange colored band was collected as eyespot-rich fraction. Immunostaining. Rabbit polyclonal antibody was raised against KLH-conjugated synthetic peptide CSLDGDPNGD (Hokkaido System Science, Japan). The antiserum was adsorbed to and eluted from a protein A column (Amersham) and purified with KLH–agarose (Sigma). Total membrane fraction of P. pastoris and the eyespot fraction of C. reinhardtii each containing 5 lg of protein were blotted on Sequi-Blot PVDF membrane (Bio-Rad) as described in [24]. The purified antibody at a 1:1000 dilution was used as the primary antibody and goat anti-rabbit IgG AP conjugate (Promega) at a 1:5000 dilution was used as the secondary antibody. Reactive bands were visualized using Western Blue reagent (Promega). Anti-His-tag antibody (Qiagen), sheep anti-mouse IgG HRR conjugate (Amersham), and ECL plus Western blotting kit (Amersham) were also used for the detection of the recombinant Acop-1 expressed in P. pastoris. Immunocytochemical staining of C. reinhardtii cells was conducted with FITC-conjugated goat anti-rabbit IgG (Cederlane, Canada) as the secondary antibody as described [25].

Results The two archaeal-type Chlamydomonas opsins, which we refer to as Acop-1 and Acop-2 (equivalents of CSOA and CSOB [11], and channelopsin-1 and channelopsin-2

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[12,26], respectively), are both comprised of N-terminal 300 residues showing resemblance to membranespanning regions of archaeal-type opsins and a massive (400-aa) C-terminal region that has no similarity to known protein families. Northern hybridization analyses have suggested the expression of both opsins in fulllength in C. reinhardtii cells (Fig. 1A). Among exons coding for the well-conserved membrane spanning re-

Fig. 1. (A) Northern blot analyses for mRNA expression of Acop-1 (lane 1) and Acop-2 (lane 2). (B) Southern blot analysis of restriction enzyme-digested genomic DNA hybridized at high stringency with probes for Acop-1 (left) and Acop-2 (right). Lane 1: BamHI, lane 2: HindIII, lane 3: NheI, and lane 4: SacI.

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gion, we selected the longest 100-bp nucleotides for using as a probe for the genomic Southern blot analyses to test whether there is any other archaeal-type one (Fig. 1B). Multiple alignment performed with established 34 type-I opsin sequences suggests relative positions of seven transmembrane helices in the N-terminal region (Fig. 2), in which the residues surrounding the chromophore in haloarchaeal opsins are conserved but putative helix B is located in a more hydrophilic region than what has been previously analyzed [11,12]. A search for protein sorting signal with iPSORT program [27] (http://hypothesiscreator.net/iPSORT/) strongly suggested that the first hydrophobic segment in the Nterminus of Acop-1 (Fig. 3A) is plasma membrane directed. We then predicted the secondary structure using several programs, i.e., PROF, PHD, SSpro SAM T-99, and PSIPRED, which provided essentially similar results for the N-terminal domain. The positions of the seven sufficiently long putative a-helical segments coincided well with those of hydrophobic peaks except for a region between putative helices D and E (Fig. 3A), where considerable similarity is observable between Acop-1 and bacteriorhodopsin. Taking into account that the above results confirmed the relative positions of putative helices A to G in the alignment shown in Fig. 2, we constructed a homology model using the structure of bacteriorhodopsin as the template (Fig. 4). This model suggests a striking feature that polar residues in the middle of the glutamate-rich helix B point outwardly

Fig. 2. Alignment of Archaeal rhodopsin-like domains with known 34 type-I opsin sequences. Only Acop-1 and Acop-2 (GenBank Accession Nos. AB058890 and AB058891), bacteriorhodopsin (bR) from Halobacterium salinarum, proteorhodopsin from uncultured marine eubacterium, and Nop1 from Neurospora crassa are indicated. Transmembrane helices and residues in contact with the chromophore retinal in the crystal structure of bR are marked with horizontal lines and asterisks (*), respectively, and any residues conserved in at least three of the indicated five amino acid sequences are marked with boxes.

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Fig. 3. (A) Hydropathy plots of N-terminus of Acop-1 compared with the putative secondary structure predicted by Prof program. Essentially similar results were obtained for Acop-2. (B) Hydropathy plots of C-terminus of Acop-1 compared with the putative helices predicted by several programs: Prof, PHD, SSpro, SAM T-99, and PSIPRED.

Fig. 4. Homology model for the putative membrane-spanning region of Acop-1 (stereo view). Crystal structure of bR [23] was used as the template. Only the backbone of the peptides in putative transmembrane helices, chromophore retinal, and the polar side chains in putative helix B are shown with well-conserved residues throughout type-I opsins or the corresponding one (e.g., C167 ). Retinal and oxygen atoms are shown in red. Nitrogen and sulfur atoms are colored in cyan and yellow, respectively.

with respect to the chromophore-accommodating central pore surrounded by the seven transmembrane helices.

A phylogenetic tree of type-I opsins was constructed by the NJ method (Fig. 5). The topology of the tree, which was also supported by the maximum likelihood

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(ML) method, indicated that Acops were closer to haloarchaeal and eubacterial opsins than to fungal opsins, which had previously been known as the only archaeal-type opsin subfamily in eukaryotes. Polyclonal antibody raised against a synthetic peptide corresponding to the amino acid residues 377–385 of Acop-1 sufficiently stained 374-aa C-terminal domain expressed in Escherichia coli (Fig. 6A). This antibody also stained a ca. 66 kDa protein on Western blot of the eyespot-rich fraction from cell lysate of C. reinhardtii cells, as well as heterologously expressed full-length Acop-1 in the total membrane fraction of P. pastoris (Fig. 6B). To confirm the localization of Acop-1 in individual C. reinhardtii cells, indirect immunofluorescence analysis was performed using the same antibody (Fig. 6C). The fluorescence spot size along with their position with respect to that of the flagella in each cell indicated localization of Acop-1 near the eyespot, wherein Chlamydomonas phototaxis receptors are believed to be enriched.

Fig. 5. Phylogenetic relationship between membrane spanning domains from a variety of type-I opsins represented as an unrooted dendrogram. The number associated with each node indicates the bootstrap probability of the cluster at the node. Accession Nos. are indicated except for haloarchaeal proton pumping group (e.g., bR) and chloride ion pumps. Sensor I and Sensor II represent sensory rhodopsin-I and phoborhodopsin (sensory rhodopsin-II) [2,8], respectively. The scale represents 0.1 estimated amino acid substitutions per site.

Discussion Our demonstration of the localization of Acop-1 to the eyespot region (Fig. 6C) strongly supported the hypothesis that the member of this new subfamily of archaeal-type rhodopsins functions as a phototaxis receptor. Also, successful homology modeling suggested that the folding of the N-terminal domains of the

Fig. 6. Expression of Acop-1 in P. pastoris and in C. reinhardtii cells. (A) Anti-peptide antibody raised against amino acid residues 377-385 of Acop-1 was used for staining the truncated Acop-1 (C-terminal 38 kDa protein) expressed in E. coli (lane 1). Lane 2, control transformant containing vector without the Acop-1 gene. (B) The same antibody was used for staining an eyespot-rich fraction of C. reinhardtii (lane 1) and full-length Acop-1 expressed in P. pastoris (lane 2). Lane 3, control transformant containing vector without the Acop-1 gene. (C) Immunocytochemical staining of individual C. reinhardtii cells. Arrows indicate the stained spot corresponding to the eyespot. Scale bar, 5 lm.

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polypeptides is similar to that of haloarchaeal rhodopsins, although putative helix B in both Acops has an exceptionally hydrophilic character. To reconcile our results with energetical difficulty for these polar residues lying in the middle of helix B to be buried inside the hydrophobic lipid bilayer, we think it necessary to assume some additional intra- or intermolecular interactions. A likely candidate for the putative counterpart of such interaction might be one of the two putative transmembrane helices in the C-terminal region of Chlamydomonas sensory rhodopsin A mentioned in [11]. Even in such cases, molecular mechanisms of the interaction must be different from those found in haloarchaeal photosignal transducers, because the C-terminal half of this protein shows neither amino acid sequence similarity to haloarchaeal transducers nor a high possibility of having hydrophobic transmembrane helices (Fig. 3B). Another tempting speculation accounting both for the extraordinarily hydrophobic character and for the established role of the Chlamydomonas phototaxis receptor, i.e., photoregulation of cation conductance [28,29], is to assume inter- or intramolecular interaction through which, for example, the glutamate residues near the center of helix B provide membraneembedded divalent cation-binding sites as in the case of the known Ca2þ -transporting proteins [30]. It should be also noted that although our homology modeling has successfully provided a putative three-dimensional structure of archaeal-type Chlamydomonas rhodopsins, other possibilities have not been entirely excluded; for example, a model in which the polar residues in the putative helix B face the center of the seven transmembrane helices. Further studies are necessary for elucidating the structure–function relationship of this new type-I rhodopsin subfamily with remarkably unique features.

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Acknowledgments

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We thank Dr. Takahisa Yamato, Graduate School of Science Nagoya University, for providing us with a topology file for calculation of the model structure of retinylidene proteins. This work is supported in part by Grants-in Aid for Scientific Research from Japanese Ministry of Education, Science and Culture.

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