Adaptation of Cone Pigments Found in Green Rods for Scotopic Vision Through a Single Amino Acid Mutation

Adaptation of cone pigments found in green rods for scotopic vision through a single amino acid mutation

Keiichi Kojimaa,1, Yuki Matsutania,1, Takahiro Yamashitaa, Masataka Yanagawab, Yasushi Imamotoa, Yumiko Yamanoc, Akimori Wadac, Osamu Hisatomid, Kanto Nishikawae, Keisuke Sakuraif, and Yoshinori Shichidaa,g,2

aDepartment of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan; bCellular Informatics Laboratory, RIKEN, Wako 351-0198, Japan; cDepartment of Organic Chemistry for Life Science, Kobe Pharmaceutical University, Kobe 658-8558, Japan; dDepartment of Earth and Space Science, Graduate School of Science, Osaka University, Osaka 560-0043, Japan; eGraduate School of Human and Environmental Studies, Kyoto University, Kyoto 606-8501, Japan; fFaculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba 305-8577, Japan; and gResearch Organization for Science and Technology, Ritsumeikan University, Kusatsu 525-8577, Japan

Edited by King-Wai Yau, Johns Hopkins University School of Medicine, Baltimore, MD, and approved April 17, 2017 (received for review December 5, 2016) Most vertebrate retinas contain a single type of rod for scotopic order Gymnophiona are fossorial and have small eyes covered vision and multiple types of cones for photopic and color vision. with skin, which express only rhodopsin without cone pigments The retinas of certain amphibian species uniquely contain two (11). Moreover, amphibian species belonging to Anura and sev- types of rods: red rods, which express rhodopsin, and green rods, eral genera in Urodela, including Hynobius and Ambystoma,but which express a blue-sensitive cone pigment (M1/SWS2 group). neither Cynops nor Salamandra, are unique in possessing two Spontaneous activation of rhodopsin induced by thermal isomer- types of rods, red and green rods. Red rods are the normal rods ization of the retinal chromophore has been suggested to con- that express rhodopsin, whereas green rods express a blue- tribute to the rod’s background noise, which limits the visual sensitive cone pigment (12–16). Thus, our goal was to deter- threshold for scotopic vision. Therefore, rhodopsin must exhibit mine whether the blue-sensitive cone pigments in green rods ex- low thermal isomerization rate compared with cone visual pig- hibit molecular properties similar to that of rhodopsin or to that ments to adapt to scotopic condition. In this study, we determined of a typical cone pigment in cones. In this study, we compared the whether amphibian blue-sensitive cone pigments in green rods thermal isomerization rates of blue-sensitive cone pigments from

exhibit low thermal isomerization rates to act as rhodopsin-like several amphibian species with those of rhodopsin and other cone BIOCHEMISTRY pigments for scotopic vision. Anura blue-sensitive cone pigments pigments. Our biochemical analysis revealed that Anura blue- exhibit low thermal isomerization rates similar to rhodopsin, sensitive cone pigments (Anura blues) acquired low thermal whereas Urodela pigments exhibit high rates like other vertebrate isomerization rates similar to rhodopsin through a single amino cone pigments present in cones. Furthermore, by mutational anal- acid mutation. We discuss the specialization process of blue- ysis, we identified a key amino acid residue, Thr47, that is respon- sensitive cone pigments in anuran green rods for scotopic vision. sible for the low thermal isomerization rates of Anura blue-sensitive cone pigments. These results strongly suggest that, through this Results and Discussion mutation, anurans acquired special blue-sensitive cone pigments Comparison of Thermal Isomerization Rates of Amphibian Blue- in their green rods, which could form the molecular basis for sco- Sensitive Cone Pigments. The thermal isomerization rates of the topic color vision with normal red rods containing green-sensitive retinal chromophore of visual pigments have been extensively rhodopsin. measured by electrophysiological analyses of photoreceptor

photoreceptor cell | visual pigment | amphibian | molecular evolution | color discrimination Significance

Anurans are unique in possessing two types of rod photore- ertebrate vision consists of scotopic and photopic vision, ceptor cells, red and green rods. Red rods express rhodopsin, triggered, respectively, by light absorption by the rod and V whereas green rods express blue-sensitive cone visual pig- cone photoreceptor cells of the retina (1). Scotopic vision re- ment. Rhodopsin exhibits a low rate of thermal isomerization quires high sensitivity and low threshold to be able to detect a of the retinal chromophore, which enables rods to detect few photons (2–4). Thus, electrical signals generated by single- photons with extremely high signal-to-noise for scotopic vi- photon absorptions in rods need to be reliably transmitted to sion. Here, we show that anuran blue-sensitive cone pigments higher-order retinal neurons in the presence of overwhelming acquired a rhodopsin-like property through a single amino acid intrinsic noise in rods, which is composed of two components: mutation at position 47 in the evolutionary process from other discrete and continuous noise (5). The former originates from cone pigments. Thus, anurans have special blue-sensitive cone thermal activation of the visual pigment, rhodopsin, and the pigments for the contribution of green rods to the low threshold latter results from spontaneous activation of the phosphodies- of light detection, which could form the molecular basis in tan- terase in the phototransduction cascade. Although the continuous dem with red rods containing rhodopsin in scotopic color vision. noise can be separated from the background noise, the discrete noise events are indistinguishable from true single-photon re- Author contributions: K.K., Y.M., T.Y., and Y.S. designed research; K.K. and Y.M. per- sponses (4, 6). Thus, the discrete noise sets a limit for the ab- formed research; M.Y., Y.I., Y.Y., A.W., O.H., K.N., and K.S. contributed new reagents/ solute visual threshold in scotopic vision (4, 7, 8). Thermal analytic tools; K.K., Y.M., T.Y., and Y.S. analyzed data; and K.K., T.Y., and Y.S. wrote activation of rhodopsin originates from the thermal isomeriza- the paper. tion of the retinal chromophore (9, 10). Taken together, sup- The authors declare no conflict of interest. pressing the thermal isomerization rates of the retinal in This article is a PNAS Direct Submission. rhodopsin improves the threshold of light detection. Data deposition: The sequences reported in this paper have been deposited in the GenBank Most vertebrates have one type of rod containing rhodopsin database (accession nos. LC180360, LC180361,andLC180362). and multiple types of cones containing different cone visual 1K.K. and Y.M. contributed equally to this work. pigments. Thus, they have the ability to discriminate color only 2To whom correspondence should be addressed. Email: [email protected]. under photopic conditions. By contrast, amphibians have diver- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. sified photoreceptor systems in their retinas. Most species in the 1073/pnas.1620010114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1620010114 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 Fig. 1. Two-step reaction scheme of thermal activation and deactivation of visual pigments. R and R* indicate visual pigments in the inactive and active states, respectively. An opsin regenerated with normal 11-cis-retinal spontaneously converts to R* by thermal isomerization of retinal in the dark. After the first reaction, R* is degraded into opsin and retinal.

cells from a series of genetically modified animals (17–19). that of rhodopsin in the course of molecular evolution after Recently, to better understand the molecular mechanism that anurans had acquired green rods. regulates the retinal thermal isomerization, we developed a Some urodelans also have green rods in their retina that biochemical method to estimate thermal isomerization rates by contain blue-sensitive cone pigments. Therefore, we determined using recombinant visual pigment proteins (10). Thermal isom- the thermal isomerization rates of blue-sensitive cone pigments erization rates (kth) are calculated using three experimentally from three urodelan species (Figs. S1−S3 and S4B). The tiger determined values in the following manner. First, we assumed a salamander (Ambystoma tigrinum) was reported to have green simplified two-step reaction as shown in Fig. 1, where R is the rods that express blue-sensitive cone pigments (14). The Mexican pigment in the inactive state and R* is the activated pigment by salamander (Ambystoma mexicanum) is a closely related species the thermal isomerization of the retinal chromophore. Given to the tiger salamander (23). The Japanese fire-bellied newt that the thermal isomerization occurs much slower than the (Cynops pyrrhogaster), however, has no green rods, and its blue- decay of R*(kth << kd), a steady-state approximation can be sensitive cone pigments are expressed only in blue cones (12). applied to the concentration of R*. (Here, kd is the spontaneous Our results show that the tiger salamander and Mexican sala- decay rate of the activated pigment.) Therefore, we obtain the mander blue-sensitive cone pigments (tiger salamander blue and following: Mexican salamander blue, respectively) exhibit thermal isomerization rates about 50-fold higher than that of bovine rhodopsin, * = kth½ [1] whereas Japanese fire-bellied newt blue-sensitive cone pigment R R 0, kd exhibits a rate about 300-fold higher than that of bovine rhodopsin (Fig. 2B). Although the rates of the former two species where [R]0 is the initial concentration of the visual pigment. are sixfold lower than that of the latter, these rates are similar to Namely, it can be considered that a small but constant concen- those of typical cone pigments but considerably different from tration of R* exists in a solution of the purified pigments in the dark. Because R* is essentially the same as the light-induced Meta II state (20), the initial rate of G protein activation by a pigment in the dark (vdark) can be approximated by

kth vdark = vlight, [2] kd

where vlight is the initial rate of G protein activation by an activated pigment. Therefore, we can estimate the kth from three experimentally determined values (vdark, vlight, and kd)as

vdark kth = kd. [3] vlight

By using this biochemical method, we have previously analyzed the thermal isomerization rate of Xenopus tropicalis blue- sensitive cone pigment (Xenopus blue) (10). The measured thermal isomerization rate was about 100-fold lower than rates of normal cone pigments in mouse and chicken green-sensitive cones. Moreover, the measured rate was closer to that of rhodopsin. This finding is consistent with electrophysiological evi-

dence that green rods in a toad, Rhinella marina, formerly Bufo Fig. 2. Comparison of the thermal isomerization rates (kth) of wild-type marinus, exhibit low discrete noise like normal rods (21, 22). To visual pigments. (A) Comparison of the thermal isomerization rates (kth)of examine whether Xenopus blue is a special case exhibiting low bovine and Xenopus rhodopsin and Xenopus, American bullfrog, man- thermal isomerization rate, we determined the thermal isomer- telline frog, and zebrafish blue estimated from the data presented in Fig. ization rates of the blue-sensitive cone pigments of other anuran S4A.(B) Comparison of the thermal isomerization rates (kth)ofbovineand Lithobates catesbeianus Xenopus rhodopsin, tiger salamander, Mexican salamander, newt, and species: the American bullfrog, , formerly zebrafish blue estimated from the data in Fig. S4B. All error bars in Fig. 2 Rana catesbeiana, and the mantelline frog, Mantella baroni (Figs. − represent the SEM of more than three independent measurements. An S1 S3 and S4A). Our results show that these pigments also exhibit asterisk (*) indicates a significant difference in relative rate constants be- rhodopsin-like thermal isomerization rates (Fig. 2A). Therefore, we tween visual pigments and Xenopus rhodopsin (P < 0.05; Student’s t test, concluded that Anura blues changed their properties to mimic two-tailed).

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1620010114 Kojima et al. Downloaded by guest on September 30, 2021 that of rhodopsin. This finding is consistent with electrophysio- 122 and 189 (Fig. S5). Thus, our working hypothesis is that logical evidence that blue cones in tiger salamander exhibit higher amino acid residue(s) at position(s) other than 122 or 189 may be discrete noise than rods although a rod contains ∼30 times as responsible for the low thermal isomerization rates of Anura many pigment molecules as a cone (24). Therefore, unlike an- blues. We compared amino acid residues located within 10 Å of urans, urodelans express typical blue-sensitive cone pigment the retinal chromophore in the crystal structure of bovine rho- irrespective of its presence or absence in green rods. dopsin between Anura and other animals’ blue-sensitive cone pigments and found that threonine at position 47 is conserved Identification of a Key Amino Acid Residue Responsible for the Low only in Anura blues (25) (Fig. 3 A and B and Fig. S5). Mutation Thermal Isomerization Rates of Anura Blue-Sensitive Cone Pigments. of this threonine in Xenopus blue increased the thermal isom- To investigate the mechanism underlying the low thermal isomerization rate about 60-fold (Fig. 3C), which is comparable to erization rates of Anura blues, we sought to identify the amino that of zebrafish blue. Therefore, the threonine at position acid residue(s) responsible for the low thermal isomerization 47 appears to be a determinant of the low thermal isomerization rates. We previously identified two amino acid residues, at po- rates of Anura blues. sitions 122 and 189, which are responsible for the low thermal To study whether or not introduction of threonine at position isomerization rate of rhodopsin (10). However, Anura blues con- 47 causes a decrease of thermal isomerization rate of tiger sal- tain amino acid residues common in cone pigments at positions amander blue, we prepared an M47T mutant of tiger salamander BIOCHEMISTRY

Fig. 3. A key amino acid residue regulates the thermal isomerization rates (kth) of amphibian blue-sensitive cone pigments. (A) Comparison of amino acid residue at position 47. The residues of Xenopus, American bullfrog, tiger salamander, newt, and zebrafish blue and bovine rhodopsin are shown. (B) Ho- mology model of Xenopus blue that was constructed based on the crystal structure of the dark state of bovine rhodopsin (PDB 1U19). Thr47, Lys296, and 11-

cis-retinal are colored blue, black, and red, respectively. (C) Comparison of the thermal isomerization rates (kth) of bovine rhodopsin, wild-type, and T47L mutant of Xenopus blue and zebrafish blue estimated from the data in Fig. S4C.(D) Comparison of the thermal isomerization rates (kth) of bovine rhodopsin, wild-type, and M47T mutant of tiger salamander blue estimated from the data in Fig. S4C. All error bars in Fig. 3 represent the SEM of more than three independent measurements. An asterisk (*) indicates a significant difference in relative rate constants between visual pigments and Xenopus blue in C or tiger salamander blue in D (P < 0.05; Student’s t test, two-tailed).

Kojima et al. PNAS Early Edition | 3of6 Downloaded by guest on September 30, 2021 blue and determined its thermal isomerization rate. This mutation decreases the thermal isomerization rate by only a quarter (Fig. 3D). This finding suggests that the introduction of threonine at position 47 together with other mutations in Urodela blue-sensitive cone pigments can suppress the thermal isomerization rates to the same level as Anura blues.

Mechanistic Implication of the Low Thermal Isomerization Rates. Previous electrophysiological and theoretical studies showed that the thermal isomerization rates can be expressed as Xm m−1 −Ea 1 Ea k = A × e RT th ðm − 1Þ! RT 1

based on the Hinshelwood distribution (R is the gas constant, T is absolute temperature, and m is the number of molecular vibrational modes contributing thermal energy to pigment activation), where Ea is inversely proportional to the pigment’s absorption maximum (λmax), and the preexponential factor, A,is independent of the pigment’s λmax but dependent on the chromophore binding site structure (open vs. closed) (22, 26). To assess the molecular mechanism about the regulation of kth,we previously determined that there is a strong relationship between thermal isomerization rates of the retinal chromophore in the Fig. 4. Correlation between the thermal isomerization rates (k ) and k of dark state and decay rates of the active state (k ) in visual pig- th d d the visual pigments and their mutants. We plotted lnkth and lnkd of the ments and their mutants (10). These results indicated that the visual pigments. Red circles indicate data from this study, and black circles two rates are regulated by a common molecular feature such indicate data from our previous study (10). Error bars represent the SEM of as flexibility of the protein moiety. Therefore, we proposed a more than three independent measurements. The regression line derived molecular model in which the flexibility of the chromophore from the data in this study (red circles) is shown by a red dashed line; r = 0.96 binding site is responsible for the thermal isomerization rates (P < 0.05). of the retinal chromophore and decay rates of the active state (10, 27, 28). Interestingly, we found that the results we obtained in this study can be plotted on a correlation line as before (Fig. pigments suggests that, after the divergence of Anura and Uro- 4). In particular, the mutations at position 47 caused little spec- dela, Anura blues specifically acquired threonine at position tral shift in Xenopus and tiger salamander blues (Fig. S6) but 47 to suppress thermal isomerization rates (Fig. 5). Electro- significantly changed the thermal isomerization rates, probably physiological evidence from frogs shows that green rods trans- by interacting with Lys296 (Fig. 3B). This suggests that the ac- duce signals to most retinal ganglion cells, some of which can – quisition of threonine at position 47 in Anura blues decreased A also receive inputs from red rods (31 34). Thus, the decrease of to suppress their thermal isomerization rates. This effect is com- the thermal isomerization rates of anuran blue-sensitive cone monly observed between rhodopsin and green-sensitive cone pig- pigments in green rods enables them to function in tandem with ments (i.e., A of rhodopsin is 2 orders of magnitude lower than green-sensitive red (normal) rods for scotopic vision, which those of green-sensitive cone pigments) (10). Taken together, could provide a molecular basis for color discrimination under this structural flexibility could also be an important factor in the dark conditions. This theory is consistent with behavioral regulating the difference in A. evidence that indicates that frogs can distinguish the colors blue A recent study suggests that the ability to exchange of 11-cis- and gray at low illumination levels, probably using green rods, in retinal in a pigment with 9-cis-retinal in solution in the dark can addition to color vision in photopic condition (35–37). Scotopic be explained by the differences between the structures of the color vision may be advantageous for nocturnality that is ob- chromophore binding site (open or closed) (29). We assessed the served in many anuran species (such as X. tropicalis and the degree of retinal exchange in rhodopsin, zebrafish blue, and American bullfrog). On the other hand, a few anuran species Xenopus blue (Fig. S7). As shown previously (29), we observed (such as the mantelline frog) are exceptionally diurnal. They are the spectral shift induced by the chromophore exchange in thought to have changed their behavioral rhythms in several zebrafish blue, but not in rhodopsin. However, we could also phylogenetic branches after the divergence of nocturnal species detect the spectral shift in wild-type and T47L mutant of Xen- (23). Our analysis of mantelline frog blue-sensitive cone pigment opus blue. This finding suggests that the low thermal isomeri- showed that the pigment maintains the low thermal isomeriza- zation rate of the retinal in Xenopus blue cannot be explained tion rate. Therefore, diurnal frogs may have adapted to photopic simply by a more open or closed chromophore binding pocket conditions using a cone photoreceptor system containing red- that would be predicted by the ability to exchange retinal. and violet-sensitive cone pigments without changing the molecular properties of the blue-sensitive cone pigment. Diversity of the Molecular Properties of Blue-Sensitive Cone Pigments in Amphibians. Green rods are uniquely observed in amphibian Conclusion species belonging to Anura and several genera in Urodela, in- Anuran species acquired the low thermal isomerization rates cluding Hynobius and Ambystoma, but not Cynops nor Salaman- of the retinal chromophore of blue-sensitive cone pigments dra. Considering the phylogenetic relationship of amphibians (23, expressed in green rods by a single amino acid replacement at 30), it has been proposed that green rods emerged before the position 47 in the evolutionary process. We previously deter- divergence of Anura and Urodela and were lost from several mined that the low thermal isomerization rate of rhodopsin was genera of Urodela, including Cynops and Salamandra (12). Our achieved by two amino acid replacements at positions 122 and analysis of the thermal isomerization rates of blue-sensitive cone 189 (10). Therefore, we propose that the contribution of normal

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1620010114 Kojima et al. Downloaded by guest on September 30, 2021 Fig. 5. Evolutionary relationship of the molecular properties of blue-sensitive cone pigments.

rods and anuran green rods for scotopic vision results from the in HEK293 cells and sample preparation of the visual pigments for the convergent evolution of the visual pigments to suppress the measurements of vdark, vlight,andkd were performed as previously de- thermal isomerization rates of retinal chromophore. scribed (10). The cell membranes expressing the visual pigments were di- vided into two aliquots. One was regenerated with 11-cis-retinal and Materials and Methods 7-membered-ring 11-cis-retinal (7mr) (10, 40), and the other was regenerated by only 7mr. After regeneration, they were solubilized with Buffer A

Animals. X. tropicalis were obtained from The Institute for Amphibian Biol- BIOCHEMISTRY ogy (Hiroshima University). Mantelline frog (M. baroni), tiger salamander (50 mM Hepes, 140 mM NaCl, pH 6.5) containing 1% dodecyl maltoside (A. tigrinum) and Mexican salamander (A. mexicanum) were obtained (DDM) and purified using Rho1D4-conjugated agarose. The purified visual from local pet shops. The use of animals was in accordance with guidelines pigments were eluted with 0.02% DDM in Buffer A containing the synthetic established by the Ministry of Education, Culture, Sports, Science, and C-terminal peptide of bovine rhodopsin. All of the experiments after re- Technology, Japan. The Amphibians of the World database of amphibian constitution of the visual pigments with 11-cis-retinal were performed under species (38) was used for the most current taxonomy of amphibians. infrared light. We refer to the purified samples regenerated by both 11-cis- retinal and 7mr, or only 7mr, as “pigment name-n” or “pigment name-7mr,” Heterologous Expression and Purification of the Visual Pigments. The cDNAs of respectively. We verified that the concentrations of the visual pigments X. tropicalis rhodopsin (NM_001097334) and blue-sensitive cone pigment contained in the two samples were similar by Western blot analysis (Fig. S1). (XM_002937226) were isolated by PCR from the first strand cDNA from eyes. The samples of the visual pigments for the analysis of the chromophore- The cDNA of tiger salamander (A. tigrinum) blue-sensitive cone pigment was exchange reaction were regenerated with 11-cis-retinal or 9-cis-retinal and also isolated from the first strand cDNA from eyes based on the sequence purified as described above. information (AF038946) from the National Center for Biotechnology In- 35 formation (NCBI) database. The ORF sequence of our clone had several Measurement of vdark, vlight, and kd. The vdark was measured by a [ S]GTPγS different amino acid residues, including position 47, reported previously binding assay in the dark under infrared light as previously described (10). (AF038946). Thus, we deposited the sequence of our clone in the NCBI da- The assay mixture consisted of 300 nM pigments, 1 μM transducin (Gt), 5 μM tabase under accession number LC180360. Mexican salamander (A. mexicanum) GTPγS, 25 nM [35S]GTPγS, 0.015% DDM, 50 mM Hepes (pH 6.5), 140 mM

blue-sensitive cone pigment (LC180361) was cloned by PCR from the first NaCl, 5.8 mM MgCl2, and 1mM DTT. Experimental data were fitted by a

strand cDNA from eyes based on the homology to the sequences of tiger single exponential function, and the vdark was estimated by the difference salamander and Japanese fire-bellied newt (C. pyrrhogaster) blue-sensitive between the initial rates between two samples (‘pigment name-n’ and ‘pigment ′ cone pigments. Primer sequences are as follows: 5 -GCTTTGGAGTCAGACC- name-7mr’). The v was measured by fluorescence assay as previously ′ ′ ′ light GGAG-3 (forward) and 5 -GGTTTGTGTGAGGGTGCCCTA-3 (reverse). The described (41, 42). The assay mixture consisted of 20 nM pigments, 0 or 1 μM isolated clone contained the full-length ORF sequence. To isolate the clone Gt, 5 μM GTPγS, 0.015% DDM, 50 mM Hepes (pH 6.5), 140 mM NaCl, 5.8 mM of mantelline frog (M. baroni) blue-sensitive cone pigment, we first MgCl2, and 1 mM DTT. Experimental data were fitted by a single exponential obtained the sequences of exons 1 and 5 (including start and stop codons of function, and the vlight was estimated as previously described (41, 42). The kd ORF) by PCR from genomic DNA based on the homology to the sequences of was measured by a fluorescence assay as previously described (42, 43). other frog blue-sensitive cone pigments. Primer sequences are as follows: 5′- The assay mixture consisted of 20 nM pigments (60 nM for zebrafish, tiger GGGCCCAGGCCAGACCTTTTC-3′ (forward) and 5′-ACCAGGATGTAGTTAAG- salamander, Mexican salamander and newt blue, Xenopus blue T47L, and GTGGG-3′ (reverse) for exon 1 and 5′-ATGATGAAGCTGATCTTCTGTGG-3′ tiger salamander blue M47T to increase signal to noise ratio), 5 μM GTPγS, (forward) and 5′-GCGTGGCAGAACAGGAGCTG-3′ (reverse) for exon 5. After 0.015% DDM, 50 mM Hepes (pH 6.5), 140 mM NaCl, 5.8 mM MgCl ,and identification of start and stop codons, the full-length ORF sequence of 2 1 mM DTT. Experimental data were fitted by a single exponential function mantelline frog blue-sensitive cone pigment (LC180362) was obtained by to estimate k . PCR from the first strand cDNA from eyes. Primer sequences are as follows: d 5′-ATGAGCAAAGGTCGACCAGA-3′ (forward) and 5′-TTATGCAGGAGTCACC- TGGCTGC-3′ (reverse). American bullfrog (L. catesbeianus) blue-sensitive Western Blotting. Extracts from visual pigment-transfected HEK293 cells were cone pigment (AB010085) and Japanese fire-bellied newt (C. pyrrhogaster) subjected to SDS/PAGE, transferred onto a polyvinylidene difluoride mem- blue-sensitive cone pigment (AB040148) were isolated as described pre- brane, and probed with Rho1D4. Immunoreactive proteins were detected by viously (12, 13). The cDNAs containing a point mutation were constructed ECL (GE Healthcare) and visualized by a luminescent image analyzer (LAS using an In-Fusion cloning kit (Clontech) according to the manufacturer’s 4000mini; GE. Healthcare). instructions. The cDNA of bovine rhodopsin (K00506) was inserted into the mammalian expression vector, pUSRα. The cDNAs of other visual pigments Spectroscopic Measurements. Absorption spectra of the samples were recorded were tagged with the epitope sequence of the anti-bovine rhodopsin with a UV–visible spectrophotometer (Shimadzu UV-2450, UV-2400). Samples monoclonal antibody Rho1D4 at the C terminus and inserted into the were kept at 0 °C using a cell holder equipped with a temperature-controlled mammalian expression vector pMT4 (39). Expression of the visual pigments circulating water bath.

Kojima et al. PNAS Early Edition | 5of6 Downloaded by guest on September 30, 2021 Retinal Exchange Reaction. The samples of visual pigments purified after ACKNOWLEDGMENTS. We thank Dr. M. Kono for a critical reading of the reconstitution with 11-cis-retinal were incubated with a 10-fold molar excess manuscript. We also thank Prof. R. S. Molday for the generous gift of a Rho1D4- of 9-cis-retinal in the dark for 15 h at 20 °C. Then, after the addition of producing hybridoma and The Institute for Amphibian Biology (Hiroshima neutralized hydroxylamine (final concentration: 5 mM in blue-sensitive cone University) for X. tropicalis through the National Bio-Resource Project of the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT). pigments or 50 mM in mouse rhodopsin), the samples were incubated in the This work was supported in part by MEXT Grants-in Aid for Scientific Research dark for 5 h. Absorption spectra were recorded before and after yellow light 26650119 and 16H02515 (to Y.S.) and 15H00812 (to T.Y.), and a grant from the (>410-nm light in blue-sensitive cone pigments or >500-nm light in rhodopsin) Takeda Science Foundation (to T.Y.). K.K. was supported by a Japan Society for irradiation. the Promotion of Science Research Fellowship for Young Scientists (15J02054).

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