©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Examining Rhodopsin Folding and Assembly through Expression of Polypeptide Fragments (*)

(Received for publication, October 23, 1995; and in revised form, January 6, 1996)

Kevin D. Ridge (§) Stephen S. J. Lee Najmoutin G. Abdulaev

From the Center for Advanced Research in Biotechnology, National Institute of Standards and Technology and the University of Maryland Biotechnology Institute, Rockville, Maryland 20850

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Previous work on the expression of bovine opsin fragments separated in the cytoplasmic region has allowed the identification of specific polypeptide segments that contain sufficient information to fold independently, insert into a membrane, and assemble to form a functional photoreceptor. To further examine the contributions of these and other polypeptide segments to the mechanism of opsin folding and assembly, we have constructed 20 additional opsin gene fragments where the points of separation occur in the intradiscal, transmembrane, and cytoplasmic regions. Nineteen of these fragments were stably expressed in COS-1 cells. A five-helix fragment was stably produced only after coexpression with its complementary two-helix fragment. Two fragments composed of the amino-terminal region and the first transmembrane helix were not N-glycosylated and were only partially membrane integrated. One of the singly expressed fragments, which is truncated after the retinal attachment site, bound 11-cis-retinal. Of the coexpressed complementary fragments, only those separated in the second intradiscal and third cytoplasmic regions formed noncovalently linked rhodopsins. Both of these pigments showed reduced transducin activation. Therefore, while many opsin fragments contain enough information to fold and insert into a membrane, only those separated at specific locations assemble to a retinal-binding opsin.


INTRODUCTION

Bovine rhodopsin is composed of the apoprotein opsin, a single polypeptide chain of 348 amino acids, and a covalently linked 11-cis-retinal chromophore(1, 2, 3, 4) . Opsin is posttranslationally acetylated at its amino terminus, and N-glycosylated at Asn-2 and Asn-15(5, 6) . A disulfide bond between Cys-110 and Cys-187, and two palmitic acid chains linked to Cys-322 and Cys-323, are also present(7, 8) . Numerous studies (for reviews, see (9) and (10) ) have suggested that rhodopsin contains seven membrane-embedded alpha-helices connected by solvent-exposed polypeptide segments on the intradiscal and cytoplasmic surfaces (Fig. 1). The seven transmembrane segments form a binding pocket for the retinal. An important function of rhodopsin is to convey information stored in the specific geometry of the chromophore to the surface of the molecule upon light absorption. This involves the exposure of cytoplasmic binding sites for the guanine nucleotide-binding protein transducin (G(T)), (^1)the interface between the receptor and effector molecules involved in visual transduction(9, 10) . At present, only limited information exists about the nature of the structural changes in rhodopsin accompanying this process (11, 12, 13, 14) .


Figure 1: A secondary structure model of bovine opsin showing the positions of polypeptide chain discontinuity. The seven transmembrane alpha-helical segments are lettered A-G, and the membrane-solvent boundaries are shown approximately by the interrupted horizontal lines. The points of polypeptide chain discontinuity examined here and in an earlier study (27) are indicated by arrows. The hexasaccharide chains attached to Asn-2 and Asn-15 are shown as circles, the disulfide bond between Cys-110 and Cys-187 as a dashed line, and the palmitoyl groups attached to Cys-322 and Cys-323 as zigzag lines.



Another aspect of rhodopsin conformation that has eluded a detailed understanding is the folding and assembly process. The first clues into the in vivo folding and assembly of bovine opsin came from a series of mutagenesis studies in the intradiscal region(15, 16) . Short in-frame deletions and/or amino acid substitutions in all of the intradiscal polypeptide segments resulted in opsins that failed to bind 11-cis-retinal or bound it poorly, showed altered cellular processing, and were wholly or partially retained in the endoplasmic reticulum when expressed in cultured cells. Recently, a number of naturally occurring mutations in the human opsin gene have been discovered in patients afflicted with some forms of retinitis pigmentosa (RP), a heterogenous group of inherited retinal diseases whose clinical hallmark is night blindness and, in some cases, progressive degeneration of the retina. Many of these mutations have also been examined after expression of the corresponding human, bovine, or murine opsin genes in cultured cells (17, 18, 19, 20, 21) or transgenic mice (22, 23, 24, 25, 26) . Interestingly, many of the mutations that preclude chromophore formation and result in abnormal cellular processing and transport of opsin in cultured cells or cause degeneration of the rod outer segment in the retina reside in the intradiscal and membrane-embedded regions. Thus, the evidence to date strongly supports the notion that polypeptide segments in both the intradiscal and transmembrane regions are intimately involved in the folding and assembly of opsin.

In an earlier study(27) , we examined whether expressed complementary bovine opsin fragments separated in the second and third cytoplasmic regions (Fig. 1) contain sufficient information to independently fold, insert into a membrane, and assemble into a functional photoreceptor. The five opsin fragments investigated were stably produced upon expression in COS-1 cells and coexpression of two or three complementary fragments formed noncovalently-linked rhodopsins. These results provided the first evidence that the functional assembly of bovine rhodopsin is mediated by the association of multiple folding domains and demonstrated the utility of defined polypeptide fragments for studying the folding and assembly process. We have now focused on further defining the boundaries of these putative folding domains by identifying additional sites where discontinuity of the opsin polypeptide chain is tolerated. For this purpose, 20 additional opsin gene fragments were constructed. The points of separation in these fragments are located in all three topographical regions of the opsin (Fig. 1) and correspond to intron/exon boundaries (Gly-120/Gly-121, Ser-176/Arg-177, Glu-232/Ala-233, and Gln-312/Phe-313, (3) ), permissive areas peptide or protein insertion (Leu-68/Arg-69, Pro-194/His-195, and Gly-280/Ser-281, (28) ), a papain cleavage site (Ser-186/Cys-187, (29) ), and naturally occurring stop codon mutations associated with autosomal dominant or autosomal recessive RP (Gln-64 Stop and Glu-249 Stop; (30) and (31) ). The gene fragments were expressed in COS-1 cells singly or in combination and the polypeptide fragments were examined for stable production and for their ability to form a chromophore and activate G(T). Coupled with our earlier findings, the present results show that while many opsin fragments with discontinuities in the intradiscal, transmembrane, and cytoplasmic regions fold and insert into a membrane, only those separated at key positions in the intradiscal and cytoplasmic regions assemble to form noncovalently linked rhodopsins.


EXPERIMENTAL PROCEDURES

Materials

Endoglycosidase H and PNGase F were from New England Biolabs. Immunolon 4 microtiter plates were from Dynatech Laboratories, and the 3,3`,5,5`-tetramethylbenzidine peroxidase substrate was from Kirkegaard and Perry Laboratories. The rho 4D2 and rho 1D4 antibodies, which are specific for amino acid sequences 2-39 and 341-348 of opsin, respectively, have been described previously(32, 33) . The B6-30N antibody, which recognizes the amino acid sequence 3-14, has also been described previously(34) . Bovine retinae were from J. A. & W. L. Lawson Co. (Lincoln, NE), and 11-cis-retinal was a gift of R. Crouch (Medical University of South Carolina and the National Eye Institute). The sources of other materials used in this investigation have been reported(27) .

Methods

Construction of Opsin Gene Fragments

All opsin gene fragments were constructed by restriction fragment replacement of the synthetic bovine opsin gene in the pMT-3 expression vector(35, 36) . Oligonucleotides were synthesized on an Applied Biosystems 380B DNA synthesizer and purified by high performance liquid chromatography. The AB(1-63), AB(1-68), HC(1-120), DE(1-176), DE(1-186), DE(1-194), EF(1-232), EF(1-248), FG(1-280), and HG(1-312) gene fragments (Table 1) were prepared by replacement of the appropriate restriction fragment with a synthetic oligonucleotide duplex containing a termination codon (TAA) after the last encoded amino acid. The AB(64-348), AB(69-348), HC(121-348), DE(177-348), DE(187-348), DE(195-348), EF(233-348), EF(249-348), FG(281-348), and HG(313-348) gene fragments (Table 2) were constructed by replacing the appropriate restriction fragment with a synthetic oligonucleotide duplex containing a CCACC consensus sequence (37) and a Met codon (ATG) to provide a translation initiation site. The sequences of the gene fragments were confirmed by the dideoxynucleotide chain-termination method of DNA sequencing(38) .





Expression and Purification of Opsin Polypeptide Fragments

Procedures for the transient transfection of COS-1 cells with the opsin gene and gene fragments have been described previously(27) . The transfected cells were harvested 55-72 h after addition of DNA, washed with phosphate-buffered saline (PBS), pH 7.0, and either solubilized with 1% (w/v) DM in PBS, 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 PBS, 0.1 mM phenylmethylsulfonyl fluoride and purified on immobilized concanavalin A or rho 1D4 antibody as described previously (27) .

SDS-PAGE Analysis of Opsin Polypeptide Fragments

Protein samples were analyzed by nonreducing SDS/Tris-glycine PAGE (39) with a 5% stacking and a 15 or 16% resolving gel and electroblotted onto poly(vinyldifluoride) membranes(40) . In some cases, the proteins were analyzed by nonreducing SDS/Tris-Tricine PAGE (41) with a 4% stacking and a 10 or 12% resolving gel. Immunoreactive protein was detected using the rho 4D2, rho 1D4, or B6-30N primary antibodies and horseradish peroxidase-conjugated goat anti-mouse IgG as the second antibody. The protein bands were visualized by chemiluminescence.

Cleavage of Oligosaccharide Chains with Endoglycosidase H and PNGase F

Whole cell detergent extracts were incubated with endoglycosidase H or PNGase F (500 units) in 20 mM Tris-HCl, pH 8.0, 0.5% (w/v) SDS, 1% (v/v) Nonidet P-40, 5 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride for 30 min at 20 °C. The glycosidase treated proteins were analyzed by SDS/Tris-glycine or SDS/Tris-Tricine PAGE and immunoblotting.

ELISA Assays

Whole cell detergent extracts containing wild-type opsin or opsin fragments were serially diluted in detergent extracts obtained from cells transfected with the pMT-3 vector. After drying the samples for 12-16 h at 37 °C in microtiter plates, the unbound proteins were washed away with PBS, 0.1% (w/v) bovine serum albumin, 0.05% (v/v) Tween 20, and the wells were blocked with 10 mM KH(2)PO(4), pH 7.4, 2.5% (w/v) bovine serum albumin, 0.5% (w/v) gelatin, 0.02% (w/v) NaN(3) for 6 h at 20 °C. Immunoreactive protein was detected by incubating the samples with the rho 4D2, rho 1D4, or B6-30N primary antibodies in blocking buffer for 16 h at 4 °C. After washing away excess primary antibody, horseradish peroxidase-conjugated goat anti-mouse IgG was added as the second antibody for 1 h at 20 °C. Excess peroxidase-conjugated IgG was washed away before adding the 3,3`,5,5`-tetramethylbenzidine peroxidase substrate. Reactions were monitored using an automated ELISA reader equipped with a 650-nm filter (Dynatech Laboratories). A known amount of photobleached rod outer segment rhodopsin (3.2-400 ng) solubilized in 1% DM was used as the standard for quantitation. The absorbance at 650 nm was linear over this range of protein concentration.

Other Methods

Spectral characterization of the opsin fragments and G(T) activation assays were performed as described previously(27) . COS-1 cell membranes were prepared by hypotonic lysis essentially as described previously(42) . Membrane integration of wild-type opsin or the opsin fragments was determined by incubating the crude membrane preparations with 100 mM Na(2)CO(3), pH 11.0, for 1 h at 4 °C(43) . Protein determinations were done using the method of Peterson (44) with bovine serum albumin as the standard.


RESULTS

Expression of the Opsin Fragments in COS-1 Cells

The Amino-terminal Opsin Fragments

Cellular expression of the amino-terminal opsin fragments (Table 1) was examined by immunoblotting of whole cell detergent extracts with the rho 4D2 or B6-30N antibodies. In contrast to wild-type opsin, the AB(1-63) and AB(1-68) fragments were not detected with the rho 4D2 antibody (Fig. 2A). This finding suggested that these opsin fragments had not folded to a conformation that was resistant to cellular proteolysis. However, fragments of the appropriate size (7-8 kDa) were detected with the B6-30N antibody (Fig. 2B), indicating that the rho 4D2 antibody was unable to recognize these particular fragments. Expression of the EF(1-248) fragment showed the presence of two distinct species of 30 and 27 kDa, which were detected with the rho 4D2 antibody (Fig. 2A). Both the HC(1-120) and DE(1-176) opsin fragments showed three distinct polypeptides (Fig. 2C), while expression of the EF(1-232) opsin fragment resulted in two faint polypeptides that were observed sporadically (Fig. 2C). However, coexpression of this opsin fragment with its complementary partner, EF(233-348), resulted in stable and reproducible production (Fig. 2D). Similar to the EF(1-248) polypeptide, expression of the DE(1-186), DE(1-194), FG(1-280), and HG(1-312) fragments also showed two prominent polypeptides (Fig. 2, C and E). The levels of amino-terminal opsin fragment production relative to wild-type opsin were estimated from ELISA assays. All of the opsin fragments showed a lower amount of polypeptide(s) when compared with wild-type opsin (Table 1).


Figure 2: Expression of bovine opsin fragments in COS-1 cells. Cells expressing the indicated opsin fragments were solubilized in DM (A-G, and I), and equivalent amounts of protein (25 µg) were analyzed by immunoblotting following nonreducing SDS/Tris-glycine (A, C-F, and I) or SDS/Tris-Tricine (B and G) PAGE. In F and G, the whole-cell detergent extracts were incubated with endoglycosidase H prior to electrophoretic analysis. In H, cells transfected with the wild-type, AB(1-63), and AB(1-68) genes were subjected to hypotonic lysis, and the crude membranes were recovered by centrifugation. Equivalent amounts of protein (25 µg) from the cell cytosol (lanes 1, 4, and 7), from membranes extracted with 100 mM Na(2)CO(3), pH 11.0 (lanes 2, 5, and 8), or from DM solubilized membranes (lanes 3, 6, and 9) were analyzed by nonreducing SDS/Tris-Tricine PAGE. Opsin and opsin fragments were detected with the rho 4D2 (A, C-F), B6-30N (B, G, and H), or rho 1D4 (I) antibodies, and visualized by chemiluminescence. Detergent extracts prepared from cells transfected with the pMT-3 expression vector served as control. Positions of molecular size standards are shown at the left in kilodaltons.



Treatment of the HC(1-120), DE(1-176), DE(1-186), DE(1-194), EF(1-232), EF(1-248), FG(1-280), and HG(1-312) fragments with endoglycosidase H all resulted in a single faster migrating polypeptide (Fig. 2F). As previously noted for the CH(1-146) and TH(1-240) opsin fragments(27) , these results indicate high mannose N-glycosylation at Asn-2, Asn-15, or both. However, neither the AB(1-63) or AB(1-68) fragments showed a shift in migration after similar treatment (Fig. 2G), suggesting that they lacked high mannose N-glycosylation. Similarly, incubating these fragments with PNGase F, which cleaves both high mannose and complex N-linked oligosaccharides, also caused no shift in migration (data not shown). To determine whether these fragments had inserted into a membrane, cells transfected with the AB(1-63) and AB(1-68) genes were subjected to hypotonic lysis, and the crude membranes were extracted with 100 mM Na(2)CO(3), pH 11.0, prior to detergent solubilization. Like wild-type opsin, neither of these fragments was found in the cell cytosol (Fig. 2H, lanes 1, 4, and 7). However, both fragments were partially extracted with alkali (Fig. 2H, lanes 5 and 8), indicating that a portion of the fragments had not integrated into a membrane. The remainder of the fragments, as well as wild-type opsin, were extracted from membranes with DM detergent (Fig. 2H, lanes 3, 6, and 9).

The Carboxyl-terminal Opsin Fragments

Cellular expression of the carboxyl-terminal opsin fragments (Table 2) was examined by immunoblotting of whole cell detergent extracts with the rho 1D4 antibody (Fig. 2I). The AB(64-348), AB(69-348), and EF(249-348) fragments each showed one prominent band of 30, 29, and 17-18 kDa, respectively. Although a single prominent band of the appropriate size was noted for the HC(121-348), DE(177-348), and EF(233-348) fragments, numerous higher molecular weight species, presumably aggregates, were also present. The HG(313-348) opsin fragment was also stably produced to yield a single species. However, on this and other immunoblots, the HG(313-348) fragment migrated between the 6.4- and 16.5-kDa markers, which is considerably higher than its expected molecular weight (4 kDa). (^2)Both the DE(187-348) and DE(195-348) fragments each showed a prominent band 19-20 kDa and numerous aggregated products. In contrast, the FG(281-348) opsin fragment showed only a faint single polypeptide of 9-10 kDa. ELISA assays showed that with the exception of the FG(281-348) and HG(313-348) fragments, the carboxyl-terminal fragments were present at equivalent or higher levels than wild-type opsin (Table 2). Whether this difference reflects a higher level of expression or greater stability of many of the carboxyl-terminal opsin fragments or is due to more efficient recognition by the rho 1D4 antibody remains to be determined.

Spectral Characterization of the Expressed Opsin Fragments

The Singly Expressed Opsin Fragments

Of the amino- and carboxyl-terminal opsin fragments, only the HG(1-312) fragment (Fig. 3) formed a chromophore with 11-cis-retinal. This result was not unexpected since the HG(1-312) polypeptide chain is terminated after the retinal attachment site (Lys-296) in helix G (Fig. 1). However, in contrast to the wild-type protein purified under the same conditions (Fig. 3), the HG(1-312) pigment showed a shorter wavelength species ((max) 360 nm) in addition to the 500-nm chromophore. A portion of the 360-nm species appeared to be covalently linked as acidification of the pigment resulted in the formation of a higher absorbing peak ((max) 420 nm). Similar treatment of wild-type rhodopsin produced only the 440-nm protonated retinyl-Schiff base (Fig. 3). Based on the absorbance at 500 nm and assuming a similar chromophore extinction (40,600 Mbulletcm), the yield of the HG(1-312) pigment was 25-33% of wild-type rhodopsin.


Figure 3: UV-visible absorption spectra of wild-type and HG(1-312) rhodopsins. Cells expressing the wild-type or HG(1-312) opsins were incubated with 11-cis-retinal and solubilized in DM, and the proteins were purified on immobilized concanavalin A. The bound proteins were eluted in PBS, 0.1% DM, 0.3 M methyl alpha-D-mannopyranoside. Spectra were recorded in the dark (continuous lines) and after acidification to pH 1.9 (dashed lines).



The Coexpressed Opsin Fragments

Of the 10 complementary fragment combinations examined, only the DE(1-194) + DE(195-348) and EF(1-248) + EF(249-348) fragments formed rhodopsin-like pigments, which were amenable to affinity purification on immobilized rho 1D4 (Fig. 4). These rhodopsins appear to have folded and assembled to a correct ground-state structure as shown by their 500 nm (max), similar chromophore bandwidth, and molar absorption coefficient (40,200-40,700 Mbulletcm). Furthermore, these rhodopsins, like the wild-type pigment, showed little reactivity toward hydroxylamine in the dark over a 6-h period (data not shown). They also showed the characteristic shift in (max) to 380 nm upon illumination and formed the 440-nm protonated retinyl-Schiff base after acidification (Fig. 4). The yield of the DE(1-194) + DE(195-348) rhodopsin relative to wild-type rhodopsin was only 10-12%, while that for the EF(1-248) + EF(249-348) pigment was 60-70%. Notably, coexpression of the HG(1-312) and HG(313-348) fragments did not allow the formation of a complex that could be purified on immobilized rho 1D4. Furthermore, coexpression had no effect on the amount of chromophore obtained after purification of the HG(1-312) fragment on immobilized concanavalin A (data not shown).


Figure 4: UV-vis absorption spectra of wild-type, DE(1-194) + DE(195-348), and EF(1-248) + EF(249-348) rhodopsins. Cells expressing wild-type opsin or the opsin fragments were incubated with 11-cis-retinal, solubilized in DM, and purified on immobilized rho 1D4. The bound proteins were eluted in 2 mM NaH(2)PO(4), pH 6.0, 0.1% DM, 35 µM c'-1-9 peptide. Spectra were recorded in the dark (continuous lines), after illumination (>495 nm) for 10 s (dashed lines), and after acidification to pH 1.9 (dotted lines).



Properties of the Rhodopsin Fragment Complexes

Nature of the Rhodopsin Fragment Complexes

The disposition of the polypeptide fragments in the DE(1-194) + DE(195-348) and EF(1-248) + EF(249-348) rhodopsins were examined by immunoblotting with the rho 4D2 and rho 1D4 antibodies. Like the previously described CH(1-146) + CH(147-348) and TH(1-240) + TH(241-348) rhodopsins (27) , both the DE(1-194) + DE(195-348) and EF(1-248) + EF(249-348) rhodopsins showed two opsin polypeptide fragments in which the amino-terminal fragment now migrated as an elongated band with a sharp leading edge and a trailing smear (Fig. 5). This results from further processing to the complex carbohydrate form(12) .


Figure 5: Immunoblot analysis of purified wild-type, DE(1-194) + DE(195-348), and EF(1-248) + EF(249-348) rhodopsins. The purified pigments were analyzed by nonreducing SDS/Tris-Tricine PAGE, detected with the rho 4D2 and rho 1D4 antibodies, and visualized by chemiluminescence. Positions of molecular size standards are shown at the left in kilodaltons.



G(T) Activation by the Rhodopsin Fragment Complexes

To test whether the DE(1-194) + DE(195-348) and EF(1-248) + EF(249-348) rhodopsins activated G(T), in vitro GTPS binding assays were performed. While the DE(1-194) + DE(195-348) rhodopsin catalyzed light-dependent GTPS binding by G(T) to about 45% of the level of wild-type rhodopsin, the EF(1-248) + EF(249-348) rhodopsin showed only 12% the level of activity after a 1-min reaction (Fig. 6).


Figure 6: Light-dependent activation of G(T) by wild-type rhodopsin and rhodopsin fragment complexes. Time course for the reaction catalyzed by wild-type (circles), DE(1-194) + DE(195-348) (squares), and EF(1-248) + EF(249-348) (triangles) rhodopsins. The assay mixture contained 2 nM rhodopsin, 2 µM G(T), and 4 µM [S]GTPS. The data shown are averages ± standard error from four determinations using pigments from two separate transfections.



Expression of the AB(1-63) Opsin Fragment in the Presence of Wild-type Opsin

Patients afflicted with RP are often heterozygous for the mutant allele, i.e. they have one copy of the mutant gene and one copy of the wild-type gene. Since the Gln-64 Stop mutation is associated with autosomal dominant RP, we attempted to mimic the in vivo genotype by examining the effects, if any, of AB(1-63) fragment expression on the folding and assembly of wild-type opsin. For this purpose, COS-1 cells were cotransfected with both the wild-type and AB(1-63) opsin genes. Since the AB(1-63) fragment is not recognized by the rho 1D4 antibody, the effect of this opsin fragment on wild-type folding and assembly could be discerned after purification on immobilzed rho 1D4. Both wild-type opsin and the AB(1-63) fragment were stably expressed in the cotransfected cells (Fig. 7A, lane 2). After addition of 11-cis-retinal and affinity purification, wild-type opsin expressed in the presence of the AB(1-63) fragment showed virtually the same amount of protein and 500 nm chromophore as the singly expressed wild-type (Fig. 7B). As expected, immunoblot analysis showed only the wild-type protein in the antibody column eluate (Fig. 7A, lane 3).


Figure 7: Effect of AB(1-63) fragment expression on the folding of wild-type opsin. A, immunoblot analysis of wild-type opsin expressed in the absence and presence of the AB(1-63) fragment. Whole-cell detergent extracts (lanes 1 and 2) or immobilized rho 1D4 purified pigment (lane 3) prepared from cells expressing the indicated opsin genes were analyzed by nonreducing SDS/Tris-Tricine PAGE and detected using the B6-30N antibody and chemiluminescence. Positions of molecular size standards are shown at the left in kilodaltons. B, UV-visible absorption spectra of wild-type rhodopsin expressed in the absence and presence of the AB(1-63) fragment. The expressed opsins were incubated with 11-cis-retinal, solubilized in DM, and the wild-type protein was purified on immobilized rho 1D4. The bound proteins were eluted in 2 mM NaH(2)PO(4), pH 6.0, 150 mM NaCl, 0.1% DM, 35 µM c`-1-9 peptide. The spectra shown were recorded in the dark and correspond to the singly expressed (continuous line) and coexpressed (dashed line) pigments.




DISCUSSION

Previously, we showed that coexpression of two or three complementary bovine opsin fragments separated in the second and third cytoplasmic regions allowed the formation of rhodopsins with spectral characteristics similar to the native pigment(27) . As indicated above, these results provided the first evidence that the functional assembly of rhodopsin is mediated by the association of multiple folding domains. In the present study, we focused on further defining the boundaries of these putative domains by identifying additional sites where discontinuity of the opsin polypeptide chain is tolerated. For this purpose, 20 additional opsin gene fragments were constructed ( Table 1and Table 2), where the points of separation occur in all three topographical regions of opsin.

The Cytoplasmic Region

Although the AB(1-63) and AB(1-68) fragments appear to be stably expressed as judged by immunoblot analysis with the B6-30N antibody (Fig. 2A), they both lack high mannose (Fig. 2G) or complex N-glycosylation. These findings suggest that these fragments may not have been properly translocated and/or appropriately inserted into the endoplasmic reticulum membrane. This conclusion is further supported by the observation that they can be partially extracted from membranes with alkali (Fig. 2H), a commonly used criterion for establishing membrane integration(43) . Although earlier studies indicated that the first transmembrane segment (and adjacent residues) contains both a signal and stop-transfer sequence(45, 46) , the properties of the AB(1-63) and AB(1-68) fragments may be due to the fact that they are simply too short. It has been estimated that the ribosome sequesters 30-40 amino acids of the nascent polypeptide chain and that the signal sequence is presumably available for interaction with the signal recognition particle only when exposed on the surface of the ribosome(47, 48) . Therefore, it is possible that not enough of the signal sequence in these fragments is available for a productive interaction with the signal recognition particle. Alternatively, the disruption or lack of an effective stop-transfer sequence may result in their translocation across the endoplasmic reticulum membrane. In the case of the AB(1-63) fragment, this is a reasonable consideration since it lacks the necessary charged residues in the carboxyl-terminal region, which serve to anchor the helix in the membrane(49) . However, the AB(1-68) fragment contains additional basic, presumably positively charged residues in this region, which could function as a stop-transfer sequence. Notably, these same residues would also result in a net positive charge in this region of the fragment and thereby conform to the ``positive inside'' rule, which specifies correct membrane toplogy(50) . As concerns those fragments that appear to be stably membrane integrated, other factors, presumably structural in nature, may be influencing their ability to adopt the correct membrane orientation or, in the case of those that have attained the correct topology, to interact with the lumenal glycosylation machinery. This is suggested by the observation that both the AB(1-63) and AB(1-68) fragments are not recognized by the rho 4D2 antibody (Fig. 2A and Table 1), whose affinity for rhodopsin is known to exhibit considerable conformational sensitivity(32) . Recently, Wess and co-workers (51) reported that an expressed m(3) muscarinic acetylcholine receptor fragment, which encodes the amino terminus and first transmembrane helix, was localized to the cytoplasm, whereas the complementary six-helix fragment was in the plasma membrane. Although the AB(64-348) and AB(69-348) opsin fragments are stably produced (Fig. 2I), it is not evident from the present work if these fragments are transported to the cell surface and/or adopt the correct membrane topology. Since the AB(1-63) fragment corresponds to a mutant opsin gene associated with autosomal dominant RP(30) , we examined whether expression of this fragment had a deleterious effect on the folding and assembly of wild-type opsin. Interestingly, the level of wild-type expression and 500 nm chromophore formed were essentially the same regardless of whether the AB(1-63) polypeptide was present or not (Fig. 7). Coupled with the aforementioned properties of this opsin fragment, these findings suggest that the negative dominant effect(s) of the AB(1-63) fragment in the rod cell may lie outside or beyond the opsin biosynthetic pathway.

The EF(1-248) fragment, which is composed of the first five transmembrane helices, corresponds to a mutant opsin gene associated with autosomal recessive RP(31) . Coexpression of this fragment with its complementary partner, EF(249-348), resulted in a noncovalently-linked rhodopsin (Fig. 4). However, like the TH(1-240) + TH(241-348) and CH(1-146) + CT(147-240) + TH(241-348) rhodopsins (27) , this fragment complex also showed a significantly reduced capacity to activate G(T) (Fig. 6). This is presumably due to disruption of the cytoplasmic binding site(s) for G(T). Nevertheless, it is clear that the EF(1-248) and EF(249-348) fragments have the capacity to fold, insert into the membrane, and self-associate. Based on a model for the structural organization of the alpha-helices in bovine rhodopsin(52) , it is reasonable to consider that these fragments simply associate through side-to-side interactions and that the amino acids at positions 241-248 are not critical for this process. Of the naturally occurring stop codon mutations identified in the human opsin gene so far, all have been localized to the cytoplasmic region. However, dominant rhodopsin mutations in the Drosophila ninaE gene that result in a premature termination codon have been identified in both the extracellular (equivalent to intradiscal) and cytoplasmic regions(53, 54) . In the case of ninaE, the mutation results in Gln-251 Stop, which is located in the same region of the opsin as the human Glu-249 Stop. It would be of interest to see whether this polypeptide fragment can be functionally complemented in Drosophila photoreceptor cells as well.

The Transmembrane Region

The HC(1-120), HC(121-348), DE(1-176), DE(177-348), EF(1-232), EF(233-348), HG(1-312), and HG(313-348) opsin fragments ( Table 1and Table 2) encode one to four exons, and the point of polypeptide chain discontinuity occurs well within the transmembrane region or near the membrane/solvent intersections (Fig. 1). Although there are a number of examples where individual exons encode stable folding domains, the only case where the resulting polypeptide fragments have been shown to complement in vivo is with triose-phosphate isomerase(55) . Nevertheless, it was anticipated that these fragments would provide clues about the boundaries of the different folding domains and the location of topological determinants.

With the exception of the EF(1-232) fragment, all of the singly expressed fragments separated at intron/exon boundaries were stably produced (Fig. 2). When compared with the TH(1-240) and EF(1-248) fragments, the finding that the EF(1-232) polypeptide is not stably expressed highlights the importance of location for polypeptide chain discontinuity in this region of opsin. Based on these results, it is likely that the boundary that separates two of the putative folding domains is located between Glu-232 and Ser-240. Equally intriguing is the observation that this fragment is stably produced when coexpressed with its complementary fragment. A similar finding has been observed upon coexpression of some lactose permease fragments(56, 57, 58) . Although the EF(1-232) and EF(233-348) fragments do not assemble to a retinal-binding opsin, which is amenable to purification, they clearly interact during some stage of their biosynthesis. Whether this occurs before, during, or after the polypeptide fragments have inserted into the membrane deserves further study. Coexpression of the HC(1-120) and the DE(1-176) fragments with their complementary partners also did not permit a chromophore containing pigment to be isolated. However, the likelihood that these fragments would associate to form noncovalent complexes was somewhat remote since it is energetically unfavorable to bury juxtaposed carboxyl and amino groups in the hydrophobic transmembrane region(59) .

It is noteworthy that the high mannose N-glycosylated HG(1-312) fragment formed a chromophore with 11-cis-retinal (Fig. 3). In an earlier study examining carboxyl-terminal truncation mutants of bovine opsin(60) , it was found that terminating the polypeptide chain after Asn-310 resulted in an opsin that failed to form the rhodopsin chromophore. This was ascribed, in part, to disruption of the putative stop-transfer sequence in the distal portion of the seventh transmembrane helix. If this is indeed the case, then the two-amino acid difference in the HG(1-312) fragment may be sufficient for preventing translocation of this segment into the endoplasmic reticulum lumen, thereby allowing correct formation of the retinal binding pocket. However, the presence of a covalently attached near-UV absorbing species in this pigment indicates that some of the chromophore may be linked as an unprotonated Schiff base (Fig. 3). This would suggest that not all of the expressed fragment has adopted the correct structure.

The Intradiscal Region

Of the opsin fragments separated in this region, only the DE(1-194) + DE(195-348) polypeptides formed a noncovalently-linked rhodopsin (Fig. 4). Yu et al.(61) also observed that coexpression of these same two fragments formed a rhodopsin-like complex. Although it is evident that the polypeptide fragments in this rhodopsin have the capacity to fold, insert into a membrane, and stably associate, the yield of the complex after affinity purification was very poor when compared with those rhodopsins separated in the cytoplasmic region. There are several potential reasons for this observation. First, this may be due to the lack of a signal sequence in the fifth transmembrane helix(45, 46) , which would preclude efficient translocation of the DE(195-348) fragment. Second, on the basis of energetic considerations, it has been suggested that membrane insertion of all the alpha-helices subsequent to the most amino-terminal one occurs in the form of a helical hairpin(59) , a motif to which neither the DE(1-194) or DE(195-348) fragments, unlike the cytoplasmically separated fragments, conform. However, the finding that a portion of these complementary fragments do indeed insert and productively associate suggests that, in principle, these thermodynamic barriers can be overcome. Finally, the lack of chain connectivity in the DE loop region may preclude the establishment of specific interactions between adjacent intradiscal polypeptide segments, a step that has been postulated to be of considerable importance during the assembly process(15, 16) .

Similar to the other rhodopsin fragment complexes, the DE(1-194) + DE(195-348) rhodopsin also showed reduced signaling capacity (Fig. 6). However, since the point of polypeptide chain discontinuity occurs on the intradiscal surface, the lower level of G(T) activation is presumably due to something other than disruption of the cytoplasmic binding site(s). In a study examining a rhodopsin mutant in which only the intradiscal cysteines were retained, it was shown that N-ethylmaleimide alkylation of the correctly folded pigment occurred only after illumination(13) . These results strongly argue for a conformational change on the intradiscal surface during photoactivation, a process that may also be compromised in the DE(1-194) + DE(195-348) rhodopsin due to the lack of chain connectivity in this region.

Conclusions

We have examined the contributions of various segments of the opsin polypeptide chain to the mechanism of folding and assembly by expressing and characterizing opsin fragments separated in the intradiscal, membrane-embedded, and cytoplasmic regions. The results reported here and in our earlier study suggest that while many opsin polypeptide fragments contain sufficient information to fold and insert into a membrane, only those separated at specific positions in the cytoplasmic and intradiscal regions assemble to a retinal-binding opsin.


FOOTNOTES

*
This work was supported by the National Institute of Standards and Technology and by Grant EY11112 from the National Eye Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Center for Advanced Research in Biotechnology, National Institute of Standards and Technology and the University of Maryland Biotechnology Institute, 9600 Gudelsky Dr., Rockville, MD 20850. Tel.: 301-738-6218; Fax: 301-738-6255; ridge{at}indigo2.carb.nist.gov.

(^1)
The abbreviations used are: G(T), transducin; RP, retinitis pigmentosa; PBS, phosphate-buffered saline; DM, n-dodecyl beta-D-maltoside; PAGE, polyacrylamide gel electrophoresis; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; ELISA, enzyme-linked immunosorbent assay; GTPS, guanosine 5`-[-thio]triphosphate.

(^2)
Recent results indicate that the singly expressed HG(313-348) fragment is palmitoylated, a finding that most probably accounts for its anomalous migration on SDS-PAGE (K. D. Ridge, N. G. Abdulaev, and S. S. J. Lee, unpublished work).


ACKNOWLEDGEMENTS

We thank Drs. Robert S. Molday and Paul A. Hargrave for anti-rhodopsin antibodies; Drs. Prasad Reddy, Frederick P. Schwarz, and Gary L. Gilliland for critical reading of the manuscript; Drs. Xun Liu, Zhijian Lu, Cheng Zhang, and Barry E. Knox for discussions; and Joel Hoskins and Zhanglin Lin for technical assistance.


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