Heterologous Expression of Limulus Rhodopsin
2003; Elsevier BV; Volume: 278; Issue: 42 Linguagem: Inglês
10.1074/jbc.m304567200
ISSN1083-351X
AutoresBarry E. Knox, Ernesto Salcedo, Katherine Mathiesz, Jodi Schaefer, Wen-Hai Chou, Linda V. Chadwell, W. Clay Smith, Steven G. Britt, Robert B. Barlow,
Tópico(s)Neurobiology and Insect Physiology Research
ResumoInvertebrates such as Drosophila or Limulus assemble their visual pigment into the specialized rhabdomeric membranes of photoreceptors where phototransduction occurs. We have investigated the biosynthesis of rhodopsin from the Limulus lateral eye with three cell culture expression systems: mammalian COS1 cells, insect Sf9 cells, and amphibian Xenopus oocytes. We extracted and affinity-purified epitope-tagged Limulus rhodopsin expressed from a cDNA or cRNA from these systems. We found that all three culture systems could efficiently synthesize the opsin polypeptide in quantities comparable with that found for bovine opsin. However, none of the systems expressed a protein that stably bound 11-cis-retinal. The protein expressed in COS1 and Sf9 cells appeared to be misfolded, improperly localized, and proteolytically degraded. Similarly, Xenopus oocytes injected with Limulus opsin cRNA did not evoke light-sensitive currents after incubation with 11-cis-retinal. However, injecting Xenopus oocytes with mRNA from Limulus lateral eyes yielded light-dependent conductance changes after incubation with 11-cis-retinal. Also, expressing Limulus opsin cDNA in the R1-R6 photoreceptors of transgenic Drosophila yielded a visual pigment that bound retinal, had normal spectral properties, and coupled to the endogenous phototransduction cascade. These results indicate that Limulus opsin may require one or more photoreceptor-specific proteins for correct folding and/or chromophore binding. This may be a general property of invertebrate opsins and may underlie some of the functional differences between invertebrate and vertebrate visual pigments. Invertebrates such as Drosophila or Limulus assemble their visual pigment into the specialized rhabdomeric membranes of photoreceptors where phototransduction occurs. We have investigated the biosynthesis of rhodopsin from the Limulus lateral eye with three cell culture expression systems: mammalian COS1 cells, insect Sf9 cells, and amphibian Xenopus oocytes. We extracted and affinity-purified epitope-tagged Limulus rhodopsin expressed from a cDNA or cRNA from these systems. We found that all three culture systems could efficiently synthesize the opsin polypeptide in quantities comparable with that found for bovine opsin. However, none of the systems expressed a protein that stably bound 11-cis-retinal. The protein expressed in COS1 and Sf9 cells appeared to be misfolded, improperly localized, and proteolytically degraded. Similarly, Xenopus oocytes injected with Limulus opsin cRNA did not evoke light-sensitive currents after incubation with 11-cis-retinal. However, injecting Xenopus oocytes with mRNA from Limulus lateral eyes yielded light-dependent conductance changes after incubation with 11-cis-retinal. Also, expressing Limulus opsin cDNA in the R1-R6 photoreceptors of transgenic Drosophila yielded a visual pigment that bound retinal, had normal spectral properties, and coupled to the endogenous phototransduction cascade. These results indicate that Limulus opsin may require one or more photoreceptor-specific proteins for correct folding and/or chromophore binding. This may be a general property of invertebrate opsins and may underlie some of the functional differences between invertebrate and vertebrate visual pigments. In all animals, visual pigments consist of an apoprotein and bound chromophore, usually 11-cis-retinal (most animals), 3,4-didehydro-11-cis-retinal (some fish, amphibia, and reptiles), or 3-hydroxy-11-cis-retinal (some insects) (1Seki T. Vogt K. Comp. Biochem. Physiol. B. 1998; 119: 53-64Crossref Scopus (43) Google Scholar, 2Wald G. Nature. 1968; 219: 800-808Crossref PubMed Scopus (575) Google Scholar, 3Ebrey T. Koutalos Y. Prog. Retinal Eye Res. 2001; 20: 49-94Crossref PubMed Scopus (349) Google Scholar). Typically, in both vertebrates and invertebrates the visual pigment is very stable in the dark, with thermal isomerization rates measured in years (4Birge R.R. Barlow R.B. Biophys. Chem. 1995; 55: 115-126Crossref PubMed Scopus (36) Google Scholar, 5Barlow R.B. Birge R.R. Kaplan E. Tallent J.R. Nature. 1993; 366: 64-66Crossref PubMed Scopus (109) Google Scholar). This stability contributes significantly to the overall limit of visual sensitivity. In addition, the mechanism of visual pigment activation is similar in vertebrates and invertebrates, in that absorption of a photon causes the isomerization of the chromophore from the cis to the trans conformation. This event initiates a series of conformational changes in the chromophore-protein complex that lead to the formation of an active state that interacts with G-proteins to regulate membrane conductance (6Menon S.T. Han M. Sakmar T.P. Physiol. Rev. 2001; 81: 1659-1688Crossref PubMed Scopus (279) Google Scholar, 7Filipek S. Stenkamp R.E. Teller D.C. Palczewski K. Annu. Rev. Physiol. 2003; 65: 851-879Crossref PubMed Scopus (196) Google Scholar, 8Shichida Y. Imai H. Cell Mol. Life Sci. 1998; 54: 1299-1315Crossref PubMed Scopus (214) Google Scholar). Despite these similarities, the invertebrate and vertebrate pigments differ in at least two significant ways. First, the active states have different thermal stabilities. In vertebrates, the active state, metarhodopsin II bleaches (or decomposes) rapidly into the apoprotein and free all-trans-retinal. In contrast, invertebrate metarhodopsin (M) is stable under physiological conditions (for a review, see Ref. 9Hillman P. Hochstein S. Minke B. Physiol. Rev. 1983; 63: 668-772Crossref PubMed Scopus (109) Google Scholar). Second, the regeneration of pigment containing the 11-cis-chromophore following exposure to light differs between vertebrates and invertebrates. Vertebrate opsins can bind 11-cis-retinal, synthesized enzymatically in the adjacent pigment epithelium, and spontaneously form the Schiff's base linkage, apparently without any additional protein cofactors (2Wald G. Nature. 1968; 219: 800-808Crossref PubMed Scopus (575) Google Scholar). In invertebrates, the pigment can be efficiently converted back to the dark-adapted state by light at ambient temperature, a process called photoregeneration, or by thermal isomerization of the chromophore (9Hillman P. Hochstein S. Minke B. Physiol. Rev. 1983; 63: 668-772Crossref PubMed Scopus (109) Google Scholar). Photoregeneration is the main mechanism by which the rhodopsin concentration is maintained in bright light (10Hamdorf K. Autrum H. Handbook of Sensory Physiology. Vol. VII/6A. Springer, Berlin1979: 145-224Google Scholar, 11Hamdorf K. Paulsen R. Schwemer J. Langer H. Biochemistry and Physiology of Visual Pigments. Springer, Berlin1973: 155-166Crossref Google Scholar, 12Schwemer J. J. Comp. Physiol. A. 1984; 154: 535-547Crossref Scopus (66) Google Scholar). Studies to investigate the molecular bases for the differences between vertebrate and invertebrate pigments have been limited by the lack of a suitable expression system for invertebrate opsins that would be comparable with those available for vertebrate opsins (13Oprian D. Molday R. Kaufman R. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8874-8878Crossref PubMed Scopus (385) Google Scholar, 14Nathans J. Weitz C.J. Agarwal N. Nir I. Papermaster D.S. Vision Res. 1989; 29: 907-914Crossref PubMed Scopus (41) Google Scholar, 15Max M. Surya A. Takahashi J. Margolskee R. Knox B. J. Biol. Chem. 1998; 273: 26820-26826Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 16Starace D.M. Knox B.E. Exp. Eye Res. 1998; 67: 209-220Crossref PubMed Scopus (52) Google Scholar, 17Abdulaev N.G. Ridge K.D. Methods Enzymol. 2000; 315: 3-11Crossref PubMed Scopus (7) Google Scholar, 18Klaassen C.H. DeGrip W.J. Methods Enzymol. 2000; 315: 12-29Crossref PubMed Google Scholar, 19Mollaaghababa R. Davidson F.F. Kaiser C. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11482-11486Crossref PubMed Scopus (39) Google Scholar). Here, we have evaluated a number of these systems for their suitability to express Limulus rhodopsin. We find that none of the common systems used for the expression of vertebrate opsin produce a Limulus opsin capable of binding retinal. In fact, our findings suggest that one or more protein cofactors in invertebrate photoreceptors are necessary for the functional expression of the Limulus visual pigment. COS1 Expression Vector—The Limulus rhodopsin cDNA, Lim Rh1 (20Smith W.C. Price D.A. Greenberg R.M. Battelle B.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6150-6154Crossref PubMed Scopus (53) Google Scholar), was cloned into the mammalian expression vector, pMT3 (21Khorana H.G. Knox B.E. Nasi E. Swanson R. Thompson D.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7917-7921Crossref PubMed Scopus (80) Google Scholar). In order to facilitate detection and purification of the expressed protein, the carboxyl five amino acids of Limulus rhodopsin were replaced with 14 amino acids from bovine rhodopsin, thus introducing the epitope recognized by the 1D4 monoclonal antibody (22Molday R.S. MacKenzie D. Biochemistry. 1983; 22: 653-660Crossref PubMed Scopus (351) Google Scholar). The resulting cDNA, Lim Rh1-1D4, contained 180 base pairs of 5′-untranslated sequence, the coding region for amino acids 1-362 (of 367 total) from Lim Rh1 (including an alanine at position 105, which is a polymorphic site in Limulus (20Smith W.C. Price D.A. Greenberg R.M. Battelle B.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6150-6154Crossref PubMed Scopus (53) Google Scholar)), and the cDNA for amino acids 330-348 from bovine rhodopsin. Sf9 Expression Vector—Lim Rh1-1D4 was transferred into the SmaI-NotI restriction sites of the baculovirus expression vector pVL1393 (Pharmingen). To form the infecting virus, this construct was recombined with Baculogold DNA (Pharmingen) in Sf9 cells according to the manufacturer's instructions. Drosophila Expression Vector—An expression construct was generated in which either the Lim Rh1-1D4 cDNA (referred to as P{Rh1+LimMT} or wild type Lim Rh1 cDNA (referred to as P{Rh1+Lim}) was cloned into an expression cassette containing 2.5 kb of Drosophila ninaE promoter sequence, the transcription initiation site, 33 bp of the 5′-untranslated region, a short polylinker, and 650 bp from the 3′-end of the ninaE gene that includes the polyadenylation signal (23Townson S.M. Chang B.S. Salcedo E. Chadwell L.V. Pierce N.E. Britt S.G. J. Neurosci. 1998; 18: 2412-2422Crossref PubMed Google Scholar). Both constructs were subcloned into the y + marked P-element vector “C4” (24Chou W.H. Hall K.J. Wilson D.B. Wideman C.L. Townson S.M. Chadwell L.V. Britt S.G. Neuron. 1996; 17: 1101-1115Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). In Vitro Transcription—Lim Rh1-1D4 was cloned into the EcoRI-NotI restriction sites of pALTER1 (Invitrogen). Capped RNA was prepared by run-off transcription using NotI-linearized plasmid and T7 polymerase (either RiboMax or RiboMax kit from Promega or Ambion, respectively). Transcripts were polyadenylated in vitro, purified using oligo(dT) chromatography (PolyATtract, Promega, Inc.), and quantified using UV spectroscopy. Final RNA samples were suspended in water. mRNA—Retinas from adult Limulus polyphemus (200 animals), collected in summer and fall near Woods Hole, Massachusetts, were isolated and used either immediately for RNA extraction or frozen in dry ice and stored at -70 °C until use. RNA was extracted using the TRIzol reagent (Invitrogen) and poly(A)+ RNA isolated using oligo(dT) chromatography (PolyATtract; Promega) and eluted in water at a concentration of ∼0.5 mg/ml. COS1 cells were grown and transfected using the DEAE-dextran protocol (25Karnik S.S. Sakmar T.P. Chen H.-B. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8459-8463Crossref PubMed Scopus (347) Google Scholar). All Limulus rhodopsin transfection experiments were performed in parallel with bovine rhodopsin transfections as a positive control. Transfected cells were allowed to incubate 48-72 h post-transfection. Three methods were used for regeneration of visual pigment. In method 1 (26Starace D.M. Knox B.E. J. Biol. Chem. 1997; 272: 1095-1100Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), reconstitution was carried out in the dark by incubation of cells suspended in Buffer Y (50 mm HEPES, pH 6.6, l40 mm NaCl, 3 mm MgCl2, and 1 μg/ml protease inhibitors (aprotinin, pepstatin, leupeptin, and benzamidine) with 5 μm 11-cis-retinal (in ethanol) for 3-18 h at 4 °C. In method 2, cell membranes were first isolated (27Robinson P. Cohen G. Zhukovsky E. Oprian D. Neuron. 1992; 9: 719-725Abstract Full Text PDF PubMed Scopus (440) Google Scholar) and then resuspended in 67 mm NaPO4, pH 7.0, containing protease inhibitors. Reconstitution was carried out with 2 μm 11-cis-retinal at 4 °C for 3 h. To remove retinal, membranes were washed with 4% BSA 1The abbreviations used are: BSA, bovine serum albumin; ERG, electroretinogram; DM, dodecyl maltoside. in 67 mm NaPO4. In method 3, COS1 cells were grown in the dark in the presence of 5 μm 11-cis-retinal, beginning 24 h after DNA addition. The medium was changed twice prior to harvest of the cells. Pigment formation was determined by taking UV-visible spectra of protein purified using immunoaffinity chromatography (26Starace D.M. Knox B.E. J. Biol. Chem. 1997; 272: 1095-1100Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) or by measuring light-dark difference visible spectra on solubilized membranes. Membranes were solubilized using 2% digitonin or 1% DM on ice for 20 min with occasional homogenization with a Teflon-glass homogenizer. Insoluble material was removed by centrifugation in a JA-20 (Beckman) rotor at 19,000 rpm for 45 min. The top 9/10 of the supernatant was removed and transferred to a new tube for spectral analysis. The insect cell line, Sf9, from Invitrogen was cultured in Sf-900II SFM (Invitrogen) with 10 units/ml penicillin and 10 μg/ml streptomycin. The titer of the amplified Baculo/LimRh1-1D4 was about 108 plaque-forming units as estimated by end-point assay. Sf9 cells at 5-6 × 106 cells/ml were infected with the Baculo/LimRh1-1D4 by incubating cells, recombinant viruses and fresh medium, mixed in the ratio of 10:1:20 (v/v/v). Sf9 cells were harvested 1-4 days after infection. To reconstitute the opsin with chromophore, Sf9 cells were harvested 2 days after infection. The chromophore, 11-cis-retinal or all-trans-retinal, was added to a final concentration of 2 μm 1 day after infection and again at each of the following steps: cell harvesting, membrane solubilization, and immunoaffinity purification. Infected cells were harvested as previously described (28DeCaluwe G.L. DeGrip W.J. Biochem. J. 1996; 320: 807-815Crossref PubMed Scopus (9) Google Scholar). Cell membranes were solubilized in buffer A (5 mm PIPES, pH 6.8, 10 mm EDTA containing protease inhibitors) with 1% DM on ice for 20 min. The following 14 protease inhibitors were used in each step of the purification: aprotinin, benzamidine, calpain, chymostatin, leupeptin, pepstatin A, phenanthroline, phenylalanine, phenylmethylsulfonyl fluoride, tosyl arginine methyl ester, tosyl lysine chloromethyl ketone, tosyl phenylethyochloromethylketone, antipain, and p-chloromercuriphenyl sulfonic acid. All of these inhibitors were from Sigma and were used in a final concentration of 5 μg/ml, with the exception of phenylmethylsulfonyl fluoride (0.1 mm). The unsolubilized material was removed by centrifugation at 43,000 × g at 4 °C for 30 min. The expressed opsin was purified by immunoaffinity chromatography and used for spectral analysis. P{Rh1+LimRh1} and P{Rh1+LimRh1-1D4} were injected into yw;sr ninaE17 mutant embryos, and multiple independent P-element-mediated germ line transformants were obtained, as previously described (24Chou W.H. Hall K.J. Wilson D.B. Wideman C.L. Townson S.M. Chadwell L.V. Britt S.G. Neuron. 1996; 17: 1101-1115Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Four homozygous lines containing P{Rh1+Lim1} on the X (line 4), second (line 242), and third (lines 30 and 155) chromosomes were retained. Six lines of flies that contained P{Rh1+Lim-1D4} on the X (lines 63 and 69), second (lines 133 and 147), and third (lines (93 and 179) chromosomes were retained. The strains used in this study were constructed using visible markers and balancer chromosomes. The following mutant alleles were used: ninaE17 , norpAP24 , and Gαq1 . Xenopus oocytes were isolated as previously described (29Knox B.E. Barlow R.B. Thompson D.A. Swanson R. Nasi E. Methods Enzymol. 2000; 316: 41-49Crossref PubMed Google Scholar). Stage V and VI oocytes were injected with 50-100 nl of RNA solution and cultured for 3-6 days in MBS solution (88 mm NaCl, 1 mm KCl, 2.4 mm NaHCO3, 15 mm NaHEPES, pH 7.6, 0.3 mm Ca(NO3)2, 0.41 mm CaCl2, 0.41 mm MgSO4, 5 mm sodium pyruvate, and 10 μg/ml gentamycin). Limulus lateral eyes were dissected from 150 adult animals over a period of 3 days. Dissections of the retina from the cornea were done in ambient room light, and animals were not dark-adapted beforehand. Retinas were placed in saline solution (275 mm NaCl, 3.7 mm KCl, 8.0 mm CaCl2, 7.0 mm MgCl2, and 23 mm MgSO4), covered in foil, and allowed to dark-adapt for 3 h. Retinas were divided into batches of 40 eyes, spun briefly to remove saline, flushed with N2, quick frozen on dry ice and stored at -70 °C. All procedures were carried out in the dark. Limulus eyes (40Pearn M.T. Randall L.L. Shortridge R.D. Burg M.G. Pak W.L. J. Biol. Chem. 1996; 271: 4937-4945Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar) were allowed to thaw on ice, suspended in 67 mm NaPO4, pH 6.5, containing 5 μg/ml protease inhibitors (benzamidine, pepstatin, leupeptin), and homogenized in a glass tissue grinder. This homogenate was layered on a 45% sucrose cushion and spun in an SW-27 rotor at 25,000 rpm at 4 °C for 25 min. The membrane band was collected, and the pellet was rehomogenized. The membrane fractions were pooled and diluted 10-fold with phosphate buffer to lower the sucrose concentration, and membranes were collected by centrifugation in a Ti35 rotor at 30,000 rpm for 25 min. The pellet was solubilized with 0.3 ml of a solution containing 2% digitonin or 1% DM in phosphate buffer and incubated in ice for 1 h. The solubilized solution was centrifuged in a JA-20 rotor at 19,000 rpm for 30 min. The top portion of the supernatant was removed and used for spectral analysis. Bleached membranes were prepared by suspending in 10 ml of 0.1 m NH2OH in phosphate buffer. The membranes were put on ice and exposed to the light projector (Eastman Kodak Co. projector equipped with a 300-watt tungsten bulb and a high pass 515-nm cut-off filter) for 3 h with occasional mixing. Membranes were centrifuged in a 70Ti rotor at 50,000 rpm for 15 min. To remove the retinal oxime, the membranes were washed twice in phosphate buffer containing 2% BSA and resuspended in phosphate buffer. Bleached membranes (from 40 eyes) were reconstituted with retinal using two methods. In method 1, membranes were incubated with 5 μm 11-cis-retinal at 4 °C. The incubated membranes were collected by centrifugation, washed twice with 2% BSA in phosphate buffer, and each solubilized with 0.6 ml of 2% digitonin in phosphate buffer for 1 h. The insoluble material was removed by centrifugation, and the top portion of the supernatant was taken for spectral analysis. In method 2, a Limulus eye extract, containing soluble proteins including putative retinal photoisomerase activity, was prepared (30Smith W.C. Friedman M.A. Goldsmith T.H. Vis. Neurosci. 1992; 8: 329-336Crossref PubMed Scopus (12) Google Scholar). Limulus retina (∼48) were homogenized in 5 ml of PI buffer (0.1 m NaPO4, pH 6.8). The homogenate was spun at 8,000 × g at 4 °C for 40 min. The supernatant was saved, and the pellet was re-extracted in another 5 ml of buffer PI and respun. The two supernatants were pooled (10 ml total) and used as a soluble Limulus eye extract. For reconstitution, membranes were prepared from Limulus retina as usual. The resuspended membranes were divided into two. One aliquot was bleached with NH2OH and subsequently washed with 2% BSA as described above. The other aliquot was not bleached. Membranes were resuspended in either 2.5 ml of buffer PI or 2.5 ml of eye extract, and a retinal solution containing a 2:1 molar ratio of all-trans- to 11-cis-retinal) was added to a final 5 μm concentration. Samples were mixed at 4 °C overnight, washed two times with 2% BSA in phosphate buffer, and finally solubilized in 0.7 ml of 2% digitonin in phosphate buffer for spectral analysis. Protein samples were digested with N-glycanase as described by Kaushal et al. (31Kaushal S. Ridge K. Khorana H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4024-4028Crossref PubMed Scopus (192) Google Scholar). Solubilized protein samples were run on 10% SDS-PAGE gels and transferred to nitrocellulose membranes. LimRh1-1D4 was detected using 1D4 IgG and anti-mouse horseradish peroxidase secondary antibody. Horseradish peroxidase was visualized using DAB/NiCl2. Blots were scanned, and figures were prepared using Photoshop (Adobe). COS1 cells were grown on gelatin-coated cover slips and transfected as usual. After 48 h, cells were fixed with cold (-20 °C) methanol followed by acetone. Opsin was detected using 1D4 antibody and a fluorescein-conjugated secondary antibody. UV-visible absorption spectra of purified visual pigments and solubilized membranes were recorded in the indicated buffers at 20 °C with a Beckman DU 640 single beam spectrophotometer. Bleaching illumination was provided with light from a 300-watt projector (Kodak) at a distance of 50-60 cm. Unfiltered light from the projector was termed white light, and a high pass colored glass cut-off filter (>515 nm; Edmund Scientific, Inc.) was used as indicated. Total radiant energy of the light stimuli was measured using an optical power meter (3M Photodyne) equipped with a calibrated silicon photodiode (EGG, Inc.). Unfiltered light delivered 4.0 milliwatts. Spectra (4-6 scans) were obtained from the sample before and after illumination. All spectra were averaged and analyzed using SigmaPlot Software (Jandel). Microscopy—The immunohistochemistry using the 1D4 antibody and confocal imaging were performed as previously described (24Chou W.H. Hall K.J. Wilson D.B. Wideman C.L. Townson S.M. Chadwell L.V. Britt S.G. Neuron. 1996; 17: 1101-1115Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). The confocal image was collected using a Zeiss LSM-310 microscope (Thornwood, NY). Electrophysiology—Electroretinograms (ERGs) were recorded from immobilized white-eyed flies using glass microelectrodes filled with normal saline (0.9% NaCl, w/v) as previously described (23Townson S.M. Chang B.S. Salcedo E. Chadwell L.V. Pierce N.E. Britt S.G. J. Neurosci. 1998; 18: 2412-2422Crossref PubMed Google Scholar, 24Chou W.H. Hall K.J. Wilson D.B. Wideman C.L. Townson S.M. Chadwell L.V. Britt S.G. Neuron. 1996; 17: 1101-1115Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Spectral sensitivity was measured using a modification of the voltage clamp method of Franceschini (32Franceschini N. Invest. Opthalmol. 1979; 5Google Scholar, 33Franceschini N. Borsellino A. Cervetto L. Photoreceptors. Plenum, New York1984: 319-350Crossref Google Scholar), which we have described in detail elsewhere (23Townson S.M. Chang B.S. Salcedo E. Chadwell L.V. Pierce N.E. Britt S.G. J. Neurosci. 1998; 18: 2412-2422Crossref PubMed Google Scholar). Briefly, the amplitude of the ERG response to a flickering (10 Hz) monochromatic stimulus was maintained at a criterion level while the wavelength of stimulating light was varied during a scan. Throughout the scan, the criterion response was maintained by constantly adjusting the light intensity using a proportional integral-derivative algorithm (34Corripio A.B. Tuning of Industrial Control Systems. Instrument Society of, Research Triangle Park, NC1990Google Scholar). Spectral sensitivity was defined as the inverse of the light flux required to produce the criterion response, taking into account the wavelength and intensity of the stimulating light (i.e. spectral sensitivity ∝ 1/(light intensity × wavelength)). Sensitivity data were normalized to an amplitude of 1.0 at the wavelength of maximal sensitivity, averaged, and smoothed with a window of 10 nm. Rhodopsin Nomogram Modeling—Spectral sensitivity measured in vivo in the fly's eye was curve-fit to the theoretical rhodopsin absorption using the exponential function described by Stavenga et al. (35Stavenga D.G. Smits R.P. Hoenders B.J. Vision Res. 1993; 33: 1011-1017Crossref PubMed Scopus (260) Google Scholar). Briefly, the spectral shape of the rhodopsin α-band absorption can be described by the following log normal function: α = A × exp[-a 0 x 2(1 + a 1 x + a 2 x 2)], where x = 10log(λ/λmax), A = 1, a 0 = 380, a 1 = 6.09, and a 2 = 3a 12/8. The curve-fitting routine was implemented in KaleidaGraph™ (version 3.08d, Synergy Software, Reading, PA) using the Levenberg-Marquardt (nonlinear least-squares) algorithm. The computer solved for the λmax and amplitude of the rhodopsin absorption spectrum and calculated the S.D. value for each variable and the correlation coefficient (Pearson's r). The effect of β-band absorption (λmax = 340 nm) was ignored, because this is likely to be minimal in the measured region (450-600 nm). Immunoprecipitation—Oocytes were injected with ∼50 ng of LimRh1-1D4 cRNA and cultured in labeling medium (MBS containing 10% heat-inactivated newborn calf serum and 10 μCi/μl [35S] TRANS label (ICN, Inc.)) for 4 days. The medium was replaced each day with fresh labeling medium. Oocytes were washed in modified Barth's saline and homogenized in 150 mm NaCl, 10 mm NaPO4, pH 7.0, and 0.5% dodecyl maltoside (10 μl/oocyte). Insoluble material was removed by centrifugation at 14,000 rpm for 15 min at 4 °C. Opsin was immunoprecipitated using 1D4-Sepharose and eluted from the resin with competing peptide. 35S-Labeled proteins were resolved on 12% acrylamide gels and exposed to x-ray film. Electrophysiology—The procedure for recording light responses has been described previously (29Knox B.E. Barlow R.B. Thompson D.A. Swanson R. Nasi E. Methods Enzymol. 2000; 316: 41-49Crossref PubMed Google Scholar). Briefly, oocytes were selected 4-7 days postinjection and analyzed using a two-electrode (2-5 megaohms) voltage clamp. Pigment generation was performed following impalement of the oocytes by switching the bath solution to one containing 5-10 μm 11-cis-retinal. Light responses were elicited by exposing the cell to a stimulus delivered through an optical light guide. Responses were digitized and collected using pCLAMP. Native Pigment—As a preliminary study for heterologous expression of a Limulus opsin cDNA, we characterized the photobleaching and regeneration properties of detergent extracts from lateral eye membranes (Fig. 1). In digitonin, there was a broad absorbance between 400 and 500 nm that arose from screening pigments, thus preventing direct observation of the visual pigment. However, exposure of the extract to light caused a loss of absorbance at ∼540 nm and an increase at ∼370 nm (Fig. 1B). The difference spectrum fit the profile expected for an 11-cis-retinal-based rhodopsin. The visual pigment was quite stable in detergent extracts when kept in the dark, with only a minor loss of absorbance after 18 h (Fig. 1B). The same results were obtained with membranes that were solubilized in dodecyl maltoside (data not shown). Rhodopsin could be isolated from dark-adapted and light-adapted animals as long as the retina was incubated in the dark prior to extraction (Fig. 1C). This shows that the M form of the visual pigment efficiently thermally converts back to R in the dark. The chromophore could be completely removed from the visual pigment by exposing to light in the presence of hydroxylamine, resulting in no light-sensitive spectra. We attempted to regenerate the visual pigment by two methods. In the first, membranes were incubated with 11-cis-retinal in the dark before solubilization with digitonin. There were no light-sensitive changes detectable (Fig. 1C). In the second method, membranes were incubated with an extract of soluble proteins from Limulus retina and either 11-cis- or all-trans-retinal. Again, no detectable visual pigment was regenerated. The reason that chromophore did not bind apoprotein is not clear. One possibility is that the chromophore must bind to the opsin co-translationally, prior to conformational changes that form the final pigment. Alternatively, there might be accessory proteins not extracted or stable under our conditions (e.g. integral or peripheral membrane proteins) required for insertion of chromophore into the apoprotein. Expression in COS1 Cells—COS1 cells were transfected with an epitope-tagged Limulus opsin expression plasmid. Western analysis (Fig. 2A) of the purified protein showed that the expressed Limulus opsin was found in a number of different sized bands: a major broad band at 39 kDa, aggregated species of the size of dimers and trimers, and a proteolytic fragment at 21 kDa. Treatment of Limulus opsin with N-glycanase caused a mobility increase of monomer and higher aggregates, indicative of the removal of N-linked carbohydrate. Transfected COS1 cells were incubated with 11-cis-retinal, and the protein was solubilized in 1% dodecyl maltoside and then purified using immunoaffinity chromatography. The recovered protein did not have any associated retinal as measured by absorbance spectroscopy (Fig. 2B) or by dark-light difference. In order to determine whether the purification protocol caused the protein to release retinal, 11-cis-retinal was added directly to the purified protein sample. No l
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