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• W2013710848 abstract "Previous studies on bovine opsin folding and assembly have identified an amino-terminal fragment, EF(1–232), which folds and inserts into a membrane only after coexpression with its complementary carboxyl-terminal fragment, EF(233–348). To further characterize this interaction, EF(1–232) production was examined upon coexpression with carboxyl-terminal fragments of varying length and/or amino acid composition. These included fragments with incremental deletions of the third cytoplasmic loop (TH(241–348) and EF(249–348)), a fragment composed of the third cytoplasmic loop and sixth transmembrane helix (HF(233–280)), a fragment composed of the sixth and seventh transmembrane helices (FG(249–312)), and EF(233–348) and TH(241–348) fragments with Pro-267 or Trp-265 mutations. Although EF(1–232) production was independent of the third cytoplasmic loop and carboxyl-terminal tail, both the sixth and seventh transmembrane helices were essential. The effects of mutations in the sixth transmembrane helix on EF(1–232) expression were dependent on the length of the third cytoplasmic loop. Although Pro-267 mutations in EF(233–348) failed to stabilize EF(1–232) expression, their introduction into TH(241–348) was without discernible effects. However, Trp-265 substitutions in the EF(233–348) and TH(241–348) fragments conferred significant EF(1–232) production. Therefore, key residues in the transmembrane helices may exert their effects on opsin folding, assembly, and/or function by influencing the conformation of the connecting loops. Previous studies on bovine opsin folding and assembly have identified an amino-terminal fragment, EF(1–232), which folds and inserts into a membrane only after coexpression with its complementary carboxyl-terminal fragment, EF(233–348). To further characterize this interaction, EF(1–232) production was examined upon coexpression with carboxyl-terminal fragments of varying length and/or amino acid composition. These included fragments with incremental deletions of the third cytoplasmic loop (TH(241–348) and EF(249–348)), a fragment composed of the third cytoplasmic loop and sixth transmembrane helix (HF(233–280)), a fragment composed of the sixth and seventh transmembrane helices (FG(249–312)), and EF(233–348) and TH(241–348) fragments with Pro-267 or Trp-265 mutations. Although EF(1–232) production was independent of the third cytoplasmic loop and carboxyl-terminal tail, both the sixth and seventh transmembrane helices were essential. The effects of mutations in the sixth transmembrane helix on EF(1–232) expression were dependent on the length of the third cytoplasmic loop. Although Pro-267 mutations in EF(233–348) failed to stabilize EF(1–232) expression, their introduction into TH(241–348) was without discernible effects. However, Trp-265 substitutions in the EF(233–348) and TH(241–348) fragments conferred significant EF(1–232) production. Therefore, key residues in the transmembrane helices may exert their effects on opsin folding, assembly, and/or function by influencing the conformation of the connecting loops. Rhodopsin is the photoreceptor that mediates vision in dim light. Bovine rhodopsin is composed of the apoprotein opsin, a single polypeptide chain of 348 amino acids, and an 11-cis-retinal chromophore (1Ovchinnikov Y.A. Abdulaev N.G. Feigina M.Y. Artamonov I.D. Zolotarev A.S. Kostina M.B. Bogachuck A.S. Moroshinikov A.I. Martinov V.I. Kudelin A.B. Bioorg. Khim. 1982; 8: 1011-1014Google Scholar, 2Hargrave P.A. McDowell J.H. Curtis D.R. Wang J.K. Juszczak E. Fong S.-L. Rao J.K.M. Argos P. Biophys. Struct. Mech. 1983; 9: 235-244Crossref PubMed Scopus (383) Google Scholar, 3Nathans J. Hogness D.S. Cell. 1983; 34: 807-814Abstract Full Text PDF PubMed Scopus (473) Google Scholar, 4Wang J.K. McDowell J.H. Hargrave P.A. Biochemistry. 1980; 19: 5111-5117Crossref PubMed Scopus (136) Google Scholar). The apoprotein folds into a structure of seven transmembrane (TM) 1The abbreviations used are: TM, transmembrane; DM, n-dodecyl β-d-maltoside; PAGE, polyacrylamide gel electrophoresis1The abbreviations used are: TM, transmembrane; DM, n-dodecyl β-d-maltoside; PAGE, polyacrylamide gel electrophoresis helices connected by solvent exposed polypeptide segments on the intradiscal and cytoplasmic surfaces. These seven membrane-spanning helices form a binding pocket for the retinal chromophore (5Khorana H.G. J. Biol. Chem. 1992; 267: 1-4Abstract Full Text PDF PubMed Google Scholar). Light-induced conformational changes in rhodopsin mediated by retinal isomerization expose cytoplasmic binding sites for the heterotrimeric guanine nucleotide-binding protein (G-protein), the interface between the receptor and effector molecules in visual transduction (6Hargrave P.A. McDowell J.H. Int. Rev. Cytol. 1992; 137B: 49-97PubMed Google Scholar, 7Chabre M. Breton J. Vision Res. 1979; 19: 1005-1018Crossref PubMed Scopus (64) Google Scholar).An understanding of how rhodopsin adopts its tertiary structure is important not only to clarify details of the folding and assembly process but also to gain insight into the severe visual impairments occurring as an immediate consequence of natural mutations affecting opsin structure and function. One approach that has been used successfully to study the mechanism of protein folding and assembly is to use fragments of a polypeptide. In earlier studies (8Ridge K.D. Lee S.S.J. Yao L.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3204-3208Crossref PubMed Scopus (111) Google Scholar, 9Ridge K.D. Lee S.S.J. Abdulaev N.G. J. Biol. Chem. 1996; 271: 7860-7867Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), we have examined whether expressed complementary bovine opsin fragments separated in the intradiscal, membrane-embedded, and cytoplasmic regions contain sufficient information to independently fold, insert into a membrane, and assemble into a functional pigment. Virtually all of the singly expressed fragments fold to a conformation that allows for membrane insertion and, in some cases, form the rhodopsin chromophore with 11-cis-retinal when coexpressed with their complementary partners. Thus, these results suggest that the functional assembly of bovine rhodopsin is mediated by the association of multiple folding domains and demonstrate the utility of defined polypeptide fragments for studying the mechanism of bovine opsin folding and assembly.We have now focused on the nature and specificity of the fragment interaction(s) by identifying determinants that lead to proper folding, membrane insertion, and assembly. For this purpose, we utilized an amino-terminal five-helix opsin fragment, EF(1–232), which is stably produced only upon coexpression with its corresponding carboxyl-terminal partner, EF(233–348) (9Ridge K.D. Lee S.S.J. Abdulaev N.G. J. Biol. Chem. 1996; 271: 7860-7867Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). This finding suggests that regions within the 1–232 opsin polypeptide do not fold independently of interactions with other domains present in the complementary portion of the polypeptide chain (amino acids 233–348). To study this in greater detail, eight additional carboxyl-terminal bovine opsin fragments were constructed and expressed (Fig.1). These include a fragment composed of the third cytoplasmic loop and sixth TM helix (HF(233–280)), a fragment that lacks the third cytoplasmic loop and carboxyl-terminal tail (FG(249–312)), and EF(233–348) as well as TH(241–348) fragments with Pro-267 → Gly/Leu or Trp-265 → Phe mutations. Two previously reported (8Ridge K.D. Lee S.S.J. Yao L.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3204-3208Crossref PubMed Scopus (111) Google Scholar, 9Ridge K.D. Lee S.S.J. Abdulaev N.G. J. Biol. Chem. 1996; 271: 7860-7867Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) bovine opsin fragments with incremental deletions of the third cytoplasmic loop (TH(241–348) and EF(249–348)), were also utilized. The opsin gene fragments were expressed in COS-1 cells singly or in combination with EF(1–232), and the polypeptide fragments were examined for stable production and their ability to form the rhodopsin chromophore with 11-cis-retinal. Coupled with our earlier findings, the present results suggest that specific amino acid residues in the TM helices can exert their effects on opsin folding, membrane insertion, and/or assembly by influencing the conformation of the solvent exposed connecting loop regions.EXPERIMENTAL PROCEDURESMaterialsRestriction endonucleases were from New England Biolabs or Roche Molecular Biochemicals, and horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG was from Promega. The enhanced chemiluminescence detection system was from Amersham Pharmacia Biotech, and the 3, 3′,5,5′-tetramethylbenzidine Peroxidase Substrate was from Kirkegaard and Perry Laboratories. The B6–30N, rho 4D2, K42–41L, and rho 1D4 monoclonal antibodies, which are specific for amino acid sequences 3–14, 2–39, 276–286, and 341–348 of bovine opsin, respectively, have been described (10Molday R.S. McKenzie D. Biochemistry. 1983; 22: 653-660Crossref PubMed Scopus (351) Google Scholar, 11Laird D.W. Wong S.Y.C. Molday R.S. Goheen S.C. Membrane Proteins:Proceedings of the Membrane Symposium. Bio-Rad, Richmond, CA1987: 45-70Google Scholar, 12Adamus G. Zam Z.S. Arendt A. Palczewski K. McDowell J.H. Hargrave P.A. Vision Res. 1991; 31: 17-31Crossref PubMed Scopus (168) Google Scholar). An anti-rhodopsin polyclonal antibody, opi, was a gift from B. Knox (SUNY Medical School, Syracuse, NY). The sources of other materials used in this investigation have been reported (8Ridge K.D. Lee S.S.J. Yao L.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3204-3208Crossref PubMed Scopus (111) Google Scholar, 9Ridge K.D. Lee S.S.J. Abdulaev N.G. J. Biol. Chem. 1996; 271: 7860-7867Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar).MethodsConstruction of Opsin Gene FragmentsAll opsin gene fragments were constructed by restriction fragment replacement of the bovine opsin gene in the pMT-3 expression vector (13Franke R.R. Sakmar T.P. Oprian D.D. Khorana H.G. J. Biol. Chem. 1988; 263: 2119-2122Abstract Full Text PDF PubMed Google Scholar, 14Ferretti L. Karnik S.S. Khorana H.G. Nassal M. Oprian D.D. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 599-603Crossref PubMed Scopus (160) Google Scholar). The HF(233–280), EF(233–348/P267G), EF(233–348/P267L), EF(233–348/W265F), TH(241–348/P267G), TH(241–348/P267L), TH(241–348/W265F), and FG(249–312) gene fragments (see Table I) were constructed by replacing the appropriate restriction fragment with a synthetic oligonucleotide duplex containing a CCACC consensus sequence (15Kozak M. Nucleic Acids Res. 1984; 12: 857-872Crossref PubMed Scopus (2378) Google Scholar) and a Met codon (ATG) to provide a translation initiation site. Construction of the EF(1–232), EF(233–348), TH(241–348), and EF(249–348) gene fragments has been described (8Ridge K.D. Lee S.S.J. Yao L.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3204-3208Crossref PubMed Scopus (111) Google Scholar, 9Ridge K.D. Lee S.S.J. Abdulaev N.G. J. Biol. Chem. 1996; 271: 7860-7867Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The sequences of the opsin gene fragments were confirmed by the dideoxynucleotide chain termination method of DNA sequencing (16Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52348) Google Scholar).Table IGene construction and expression levels of the opsin fragmentsOpsin or opsin fragmentAmino acids encodedRestriction fragment(s) replacedOpsin or opsin fragment/107 cells1-aThe values were obtained using the anti-rhodopsin rho 1D4, K42–41L, or opi antibodies. The data shown are averages from at least two independent determinations and are ± 0.2–0.5 μg.μgWild type1–3484.0HF(233–280)233–280EcoRI-PstI/NdeI-NotI2.1EF(233–348)233–348EcoRI-PstI5.3EF(233–348/W265F)233–348EcoRI-PstI3.8EF(233–348/P267G)233–348EcoRI-PstI4.2EF(233–348/P267L)233–348EcoRI-PstI4.3TH(241–348)241–348EcoRI-MluI4.4TH(241–348/W265F)241–348EcoRI-MluI3.6TH(241–348/P267G)241–348EcoRI-MluI4.2TH(241–348/P267L)241–348EcoRI-MluI4.9EF(249–312)249–312EcoRI-MluI2.9EF(249–348)249–348EcoRI-MluI5.01-a The values were obtained using the anti-rhodopsin rho 1D4, K42–41L, or opi antibodies. The data shown are averages from at least two independent determinations and are ± 0.2–0.5 μg. Open table in a new tab Expression and Purification of Opsin Polypeptide FragmentsProcedures for the transient transfection of COS-1 cells with the opsin genes and gene fragments have been described (8Ridge K.D. Lee S.S.J. Yao L.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3204-3208Crossref PubMed Scopus (111) Google Scholar,17Oprian D.D. Molday R.S. Kaufmann R.J. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8874-8878Crossref PubMed Scopus (382) Google Scholar). The transfected cells were harvested 55–72 h after addition of DNA and washed with phosphate-buffered saline (10 mmNaH2PO4, pH 7.0/150 mm NaCl). The cells were either solubilized with 1% (w/v) DM in phosphate-buffered saline/0.1 mm phenylmethylsulfonyl fluoride or incubated with 5 μm 11-cis-retinal for 3 h at 4 °C in the dark. The retinal reconstituted proteins were solubilized with 1% DM in phosphate-buffered saline/0.1 mmphenylmethylsulfonyl fluoride and purified on immobilized rho 1D4 antibody or concanavalin A as described (8Ridge K.D. Lee S.S.J. Yao L.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3204-3208Crossref PubMed Scopus (111) Google Scholar, 17Oprian D.D. Molday R.S. Kaufmann R.J. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8874-8878Crossref PubMed Scopus (382) Google Scholar).SDS-PAGE Analysis of Opsin Polypeptide FragmentsProtein samples were analyzed by nonreducing SDS/Tris-glycine PAGE (18Laemmli U.K. Nature. 1970; 277: 680-685Crossref Scopus (205973) Google Scholar) with a 5% stacking and a 15 or 16% resolving gel and electroblotted onto poly(vinyldifluoride) membranes (19Matsudaira P. J. Biol. Chem. 1987; 262: 10035-10038Abstract Full Text PDF PubMed Google Scholar). In some cases, the proteins were analyzed by nonreducing SDS/Tris-tricine PAGE (20Schagger H. Von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10436) Google Scholar) with a 4% stacking and a 10 or 12% resolving gel. Immunoreactive protein was detected using the B6–30N, rho 4D2, K42–41L, rho 1D4, or opi primary antibodies and horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG as the second antibody. The protein bands were visualized by chemiluminescence.Other MethodsEnzyme-linked immunosorbent assays were carried out as described (9Ridge K.D. Lee S.S.J. Abdulaev N.G. J. Biol. Chem. 1996; 271: 7860-7867Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). COS-1 cell membranes were prepared by hypotonic lysis essentially as described (21Bhattacharya S. Ridge K.D. Knox B.E. Khorana H.G. J. Biol. Chem. 1992; 267: 6763-6769Abstract Full Text PDF PubMed Google Scholar). Membrane integration of wild-type opsin or the opsin fragments was determined by incubating the crude membrane preparations with 100 mmNa2CO3, pH 11.0, for 1 h at 4 °C (22Howell K.E. Palade G.E. J. Cell Biol. 1982; 92: 822-832Crossref PubMed Scopus (146) Google Scholar). Protein determinations were done using the method of Peterson (23Peterson G.L. Anal. Biochem. 1977; 83: 346-356Crossref PubMed Scopus (7088) Google Scholar) with bovine serum albumin as the standard.RESULTSExpression of the Opsin Fragments in COS-1 CellsThe Singly Expressed Carboxyl-terminal Bovine Opsin FragmentsCellular expression of the opsin fragments (TableI) was examined by immunoblotting whole cell detergent extracts with the rho 1D4, K42–41L, or opi primary antibodies. All of the singly expressed carboxyl-terminal fragments were stably produced in COS-1 cells (Fig.2). A polypeptide of ∼15 kDa was noted for the HF(233–280) fragment (Fig. 2 A), which is considerably higher than the calculated molecular mass of ∼5.5 kDa. Similarly, the EF(249–312) polypeptide (molecular mass = ∼17 kDa) also migrated at a position much higher than the calculated molecular mass (∼7.2 kDa). This most likely arises from an intrinsic property of many membrane proteins (and membrane protein fragments) to show anomalous migration on SDS-PAGE (9Ridge K.D. Lee S.S.J. Abdulaev N.G. J. Biol. Chem. 1996; 271: 7860-7867Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 24Tanford C. Reynolds J.A. Biochim. Biophys. Acta. 1976; 457: 133-170Crossref PubMed Scopus (664) Google Scholar). Importantly, both of these fragments required detergent for cellular extraction, suggesting that they are membrane integrated. Although the EF(233–348) and TH(241–348) fragments showed the appropriate size by SDS-PAGE, the corresponding fragments with Pro-267 mutations migrated at a slightly higher apparent molecular mass than their wild-type counterparts (Fig.2, C and D). Replacement of Pro-267 by Gly and Leu presumably alters the conformation of the polypeptide fragment even in the presence of high concentrations of SDS. The spectral and functional properties of these Pro-267 mutations in context of the entire polypeptide chain have been previously reported (25Nakayama T.A. Khorana H.G. J. Biol. Chem. 1991; 266: 4269-4275Abstract Full Text PDF PubMed Google Scholar, 26Sung C.-H. Davenport C.M. Nathans J. J. Biol. Chem. 1993; 268: 26645-26649Abstract Full Text PDF PubMed Google Scholar, 27Hwa J. Garriga P. Liu X. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10571-10576Crossref PubMed Scopus (81) Google Scholar). Notably, the Pro-267→ Leu mutation is associated with autosomal dominant retinitis pigmentosa (26Sung C.-H. Davenport C.M. Nathans J. J. Biol. Chem. 1993; 268: 26645-26649Abstract Full Text PDF PubMed Google Scholar). Substitution of Trp-265 by Phe did not alter the migration of the EF(233–348) or Th(241–348) polypeptides. This mutation has been shown to have a profound effect on the spectral properties of rhodopsin (25Nakayama T.A. Khorana H.G. J. Biol. Chem. 1991; 266: 4269-4275Abstract Full Text PDF PubMed Google Scholar, 28Ridge K.D. Bhattacharya S. Nakayama T.A. Khorana H.G. J. Biol. Chem. 1992; 267: 6770-6775Abstract Full Text PDF PubMed Google Scholar). The levels of carboxyl-terminal fragment production relative to wild-type opsin were estimated from enzyme-linked immunosorbent assays. With the exception of the HF(233–348) fragment, the carboxyl-terminal fragments were present at levels equivalent to or higher than that of wild-type opsin (Table I).Figure 2Expression of opsin polypeptide fragments in COS-1 cells. Transiently transfected cells expressing the indicated opsin polypeptides were solubilized in DM detergent, and equivalent amounts of protein (∼25 μg) were analyzed by immunoblotting following nonreducing SDS-PAGE. Opsin and the carboxyl-terminal opsin fragments were detected using the anti-rhodopsin antibodies K42–41L (A), opi (B), and rho 1D4 (C–E). Immunoreactive protein was visualized by chemiluminescence. Detergent extracts prepared from COS-1 cells transfected with the expression vector minus the opsin or opsin fragment genes (pMT-3) served as control. Positions of molecular size standards are shown at the left in kilodaltons.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The Coexpressed EF(1–232) Bovine Opsin FragmentsStable expression of the EF(1–232) opsin fragment in cells transfected with the various carboxyl-terminal fragments was examined by immunoblotting of whole cell detergent extracts with the rho 4D2 or B6–30N primary antibodies. As shown previously (9Ridge K.D. Lee S.S.J. Abdulaev N.G. J. Biol. Chem. 1996; 271: 7860-7867Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), although single expression of the EF(1–232) fragment results in two faint polypeptides that are observed sporadically, coexpression of this opsin fragment with EF(233–348) results in stable and reproducible production of a high mannoseN-glycosylated doublet (Fig.3 A). Coexpression of EF(1–232) with TH(241–348) and EF(249–348) resulted in the appearance of the two polypeptides at a level similar to that of coexpression with EF(233–348) (Fig. 2 B and TableII). These findings suggest that the correct folding and membrane insertion of EF(1–232) does not depend on the presence of the third cytoplasmic loop (amino acids 233–249). Coexpression with HF(233–280) did not afford stable production of the EF(1–232) fragment (Fig. 2 B). Similarly, removal of the third cytoplasmic loop and the sixth TM (FG(281–348)) also did not confer stable and reproducible production (Fig. 2 C). However, the EF(249–312) fragment, which lacks the third cytoplasmic loop and carboxyl-terminal tail, resulted in significant EF(1–232) production (Fig. 2 C and Table II). Taken together, the above results suggest that structural elements within the sixth and/or seventh TM influences the stable production of EF(1–232).Figure 3Effect of carboxyl-terminal fragment coexpression on EF(1–232) production. Transiently transfected cells expressing the indicated opsin polypeptides were solubilized in DM detergent, and equivalent amounts of protein (∼25 μg) were analyzed by immunoblotting following nonreducing SDS-PAGE. EF(1–232) expression was detected using the rho 4D2 or B6–30N antibodies. Immunoreactive protein was visualized by chemiluminescence. Detergent extracts prepared from COS-1 cells transfected with the expression vector minus the opsin or opsin fragment genes (pMT-3) served as control. Positions of molecular size standards are shown at theleft in kilodaltons.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IIExpression levels of EF(1–232) in the presence of various carboxyl-terminal opsin fragmentsCoexpressed opsin fragmentEF(1–232) opsin fragment/107 cells2-aThe values were obtained using the anti-rhodopsin rho 4D2 or B6–30N monoclonal antibodies. The data shown are averages from two independent determinations and are ± 0.2–0.4 μg. ND, no detectable expression.μgHF(233–280)NDEF(233–348)3.1EF(233–348/W265F)3.3EF(233–348/P267G)NDEF(233–348/P267L)NDTH(241–348)3.4TH(241–348/W265F)3.1TH(241–348/P267G)2.8TH(241–348/P267L)3.0EF(249–348)3.6EF(249–312)3.4FG(281–348)ND2-a The values were obtained using the anti-rhodopsin rho 4D2 or B6–30N monoclonal antibodies. The data shown are averages from two independent determinations and are ± 0.2–0.4 μg. ND, no detectable expression. Open table in a new tab To further examine the role of the sixth TM in this process, EF(1–232) was coexpressed with EF(233–348) fragments containing Pro-267→ Gly/Leu or Trp-265→Phe mutations. As shown in Fig. 3 D, the presence of the Pro-267 to Gly or Leu mutations in the EF(233–348) fragment essentially abolishes EF(1–232) expression. Surprisingly, introduction of the Pro-267 mutations into the TH(241–348) fragment resulted in the expression of EF(1–232) at levels similar to those of the wild-type fragment (Fig. 3 E and Table II). Similarly, introduction of the Trp-265→ Phe mutation into either the EF(233–348) and TH(241–348) fragments had essentially no effect on the level of EF(1–232) expression (Fig. 3 F and Table II). These findings suggest that the Pro-267 mutations alter the conformation of the third cytoplasmic loop in the region extending from amino acids 233–240 and that this portion of the loop, if present, exerts a stabilizing effect on EF(1–232) expression.Spectral Characterization of the Complexes Formed from the Coexpressed Opsin FragmentsThe EF(1–232) + EF(233–348) fragment complex, like wild-type rhodopsin, shows a 500-nm chromophore after reconstitution with 11-cis-retinal (Fig. 4). The yield of this fragment complex relative to wild-type rhodopsin varied from ∼25 to 40%. Previous attempts to isolate a rhodopsin-like pigment composed of these fragments on immobilized rho 1D4 were unsuccessful (9Ridge K.D. Lee S.S.J. Abdulaev N.G. J. Biol. Chem. 1996; 271: 7860-7867Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The reason(s) for the discrepancy between the present results and those reported earlier is not clear. Both the EF(1–232) + TH(241–348) and EF(1–232) + EF(249–312) fragment complexes formed chromophores to essentially the same level as the EF(1–232) + EF(233–348) complex (Fig. 4). Clearly, the complete absence of the third cytoplasmic loop does not appear to compromise the association of these fragments or their ability to bind retinal. This is consistent with the results of Franke et al. (29Franke R.R. König B. Sakmar T.P. Khorana H.G. Hofmann K.P. Science. 1990; 250: 123-125Crossref PubMed Scopus (302) Google Scholar), who showed that a 13-amino acid deletion in the third cytoplasmic loop (positions 237–249) did not abolish chromophore formation. Although the EF(1–232) + TH(241–348/P267G) complex formed the rhodopsin chromphore, the yield of regenerated pigment was ∼25% that of the full-length protein containing the P267G mutation (Fig. 4). Similarly, the EF(1–232) + TH(241–348)/P267L complex also formed a chromophore to a lesser degree than the P267L mutant. Coexpression of EF(1–232) with EF(233–348)/W265F resulted in a pigment with a blue-shifted chromophore (λmax = ∼480 nm). This is the same absorbance maximum for the full-length opsin containing the Trp-265 → Phe mutation (27Hwa J. Garriga P. Liu X. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10571-10576Crossref PubMed Scopus (81) Google Scholar). Finally, the EF(1–232) + EF(249–312) fragment complex also bound retinal to form a 500-nm chromophore. After purification on immobilized concanavalin A (Fig. 4), the yield of the complex relative to that of wild-type rhodopsin was estimated to be ∼30%. The lectin-purified pigment also showed a nearly-UV absorbing species, suggesting that a portion of the chromophore was linked as an unprotonated Schiff base. This has also been observed for the HG(1–312) fragment (9Ridge K.D. Lee S.S.J. Abdulaev N.G. J. Biol. Chem. 1996; 271: 7860-7867Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar).Figure 4UV-visible absorption spectra of rhodopsin and rhodopsin fragment complexes. COS-1 cells expressing the indicated opsin or opsin fragments were incubated with 11-cis-retinal and solubilized in DM detergent (1%, w/v), and the proteins were purified on immobilized rho 1D4 or concanavilin A. Spectra shown were recorded in the dark. WT, wild type.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONThe interactions that occur between different segments of a polypeptide chain during the folding and assembly process are the subject of numerous experimental and theoretical investigations. Although a variety of experimental strategies can be employed to gain valuable information about protein folding and assembly, we have utilized the approach of expressing fragments of a polypeptide to study this process in the integral membrane photoreceptor rhodopsin. Previous work from our and other laboratories has shown that many bovine opsin fragments contain sufficient information to fold independently, insert into a membrane, and assemble with a complementary fragment(s) in vivo to form a rhodopsin-like pigment (8Ridge K.D. Lee S.S.J. Yao L.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3204-3208Crossref PubMed Scopus (111) Google Scholar, 9Ridge K.D. Lee S.S.J. Abdulaev N.G. J. Biol. Chem. 1996; 271: 7860-7867Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 30Yu H. Kono M. McKee T.D. Oprian D.D. Biochemistry. 1995; 34: 14963-14969Crossref PubMed Scopus (119) Google Scholar, 31Heymann J.A. Subramaniam S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4966-4971Crossref PubMed Scopus (36) Google Scholar). However, not all opsin fragments are capable of folding independently and/or inserting into a membrane and appear to require other elements present in the corresponding complementary portion of the protein to adopt an appropriate conformation. Such is the case with the EF(1–232) opsin fragment, which does not fold to stable conformation unless it is coexpressed with its complementary fragment, EF(233–348). Presumably, the EF(1–232) fragment lacks the necessary information for proper folding and/or membrane insertion and is digested by cellular proteases that eliminate misfolded proteins from the cell. The purpose of this study was to examine whether truncated and/or mutated versions of EF(233–348) could stabilize expression of the EF(1–232) fragment.Coexpression experiments with the various carboxyl-terminal fragments revealed that EF(1–232) production was independent of the third cytoplasmic loop and carboxyl-terminal tail but required both the sixth and seventh TM helices (Fig. 3, A–C). These findings suggest that helix-helix interactions between complementary TMs are sufficient to confer significant EF(1–232) fragment stabilization. However, mutations in the sixth TM showed that in some cases, the presence of the third cytoplasmic loop was detrimental to EF(1–232) expression (Fig. 3, D–F). In particular, introduction of the Pro-267→ Gly or Leu mutations appeared to alter the conformation of the EF(233–348) fragment so that it no longer interacts with EF(1–232) in a productive manner. However, the same" @default.
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