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INTRODUCTION |
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 (1-4). The apoprotein folds into a structure of seven transmembrane (TM)1 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 (5). 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 (6, 7).
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 (8, 9), 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) (9). 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 (8, 9) 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.
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Fig. 1.
A secondary structure model of bovine opsin
showing the positions of polypeptide chain discontinuity. The
seven putative TM helices are numbered I-VII from left to
right, and the membrane-solvent boundaries are shown
approximately by the interrupted horizontal lines. A
bold line indicates the point of polypeptide separation
between Glu-232 and Ala-233, while arrows indicate the other
points of polypeptide chain separation examined in this study. Pro-267
and Trp-265 in the sixth TM are underlined. The
hexasaccharide chains attached to Asn-2 and Asn-15 are shown as
circles, the disulfide bond between Cys-110 and Cys-187 is
shown as a dashed line, and the palmitoyl groups attached to
Cys-322 and Cys-323 as zigzag lines.
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EXPERIMENTAL PROCEDURES |
Materials
Restriction 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 (10-12). 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 (8, 9).
Methods
Construction of Opsin Gene Fragments--
All opsin gene
fragments were constructed by restriction fragment replacement of the
bovine opsin gene in the pMT-3 expression vector (13, 14). 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
(15) 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 (8, 9). The sequences of
the opsin gene fragments were confirmed by the dideoxynucleotide chain
termination method of DNA sequencing (16).
Expression and Purification of Opsin Polypeptide
Fragments--
Procedures for the transient transfection of COS-1
cells with the opsin genes and gene fragments have been described (8, 17). The transfected cells were harvested 55-72 h after addition of
DNA and washed with phosphate-buffered saline (10 mM
NaH2PO4, 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 mM
phenylmethylsulfonyl fluoride and purified on immobilized rho 1D4
antibody or concanavalin A as described (8, 17).
SDS-PAGE Analysis of Opsin Polypeptide Fragments--
Protein
samples were analyzed by nonreducing SDS/Tris-glycine PAGE (18) with a
5% stacking and a 15 or 16% resolving gel and electroblotted onto
poly(vinyldifluoride) membranes (19). In some cases, the proteins were
analyzed by nonreducing SDS/Tris-tricine PAGE (20) 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 Methods--
Enzyme-linked immunosorbent assays were
carried out as described (9). COS-1 cell membranes were prepared by
hypotonic lysis essentially as described (21). Membrane integration of
wild-type opsin or the opsin fragments was determined by incubating the crude membrane preparations with 100 mM
Na2CO3, pH 11.0, for 1 h at 4 °C (22).
Protein determinations were done using the method of Peterson (23) with
bovine serum albumin as the standard.
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RESULTS |
Expression of the Opsin Fragments in COS-1 Cells
The Singly Expressed Carboxyl-terminal Bovine Opsin
Fragments--
Cellular expression of the opsin fragments (Table
I) 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. 2A), 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 (9, 24). 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 (25-27).
Notably, the Pro-267 Leu mutation is associated with autosomal
dominant retinitis pigmentosa (26). 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 (25, 28). 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).
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Fig. 2.
Expression 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.
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The Coexpressed EF(1-232) Bovine Opsin Fragments--
Stable
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 (9), 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 mannose
N-glycosylated doublet (Fig.
3A). 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. 2B and Table
II). 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. 2B). Similarly, removal of the
third cytoplasmic loop and the sixth TM (FG(281-348)) also did not
confer stable and reproducible production (Fig. 2C).
However, the EF(249-312) fragment, which lacks the third cytoplasmic
loop and carboxyl-terminal tail, resulted in significant EF(1-232)
production (Fig. 2C 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).
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Fig. 3.
Effect 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 the
left in kilodaltons.
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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-265Phe mutations. As shown in Fig. 3D, 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. 3E 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. 3F 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 Fragments
The 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 (9). 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. (29), 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 (
max = ~480 nm). This is the
same absorbance maximum for the full-length opsin containing the
Trp-265 Phe mutation (27). 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 (9).
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Fig. 4.
UV-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.
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DISCUSSION |
The 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 (8, 9, 30, 31). 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 mutations in the
TH(241-348) fragment result in the formation of a noncovalent complex
with EF(1-232) that is capable of forming a 500-nm chromophore with
11-cis-retinal (Fig. 4). These findings suggest that
replacement of this highly conserved proline residue has a deleterious
effect on a portion of the third cytoplasmic loop (amino acids
233-240) without dramatically altering the conformation and/or
orientation of the sixth TM, a critical component of the retinal
binding pocket. This is consistent with previous studies suggesting
that Pro-267 mutations impair the kinetics of transducin activation by
affecting the structure of the G-protein interaction site on the mutant protein (25, 27).
Site-directed spin labeling studies on cysteine substitution mutants of
the sequence 232-249 indicate that this portion of the third
cytoplasmic loop consists of solvent-exposed, regular secondary
structure (32). Further, NMR studies on a synthetic peptide
encompassing amino acids 231-252 suggests a turn-helix-turn motif for
this segment of rhodopsin (33). Other models based on the available
experimental data suggest that the 233-237 segment is in all
probability a continuation of helical structure from the fifth TM,
whereas residues 238-240 comprise a connecting turn to the sixth TM.
Thus, it is not unreasonable to expect that addition of the
"remainder" of the sequence, when in the appropriate conformation (i.e. in the absence of the Pro-267 mutations), helps
stabilize the cytoplasmic end of the fifth TM. This conclusion is
consistent with earlier work from Engelman and co-workers (34) on
bacteriorhodopsin showing that a reduction in the length of the TM
flanking sequences below a critical limit destabilizes the TM helix,
thereby rendering it more susceptible to proteolysis. Despite this
wealth of accumulated experimental and theoretical data on bovine opsin
and related receptors, it is difficult to rationalize how the Pro-267
mutations exert their effects on the 233-240 sequence in EF(233-348)
to preclude a meaningful interaction with EF(1-232). Perhaps this will
become more evident as high resolution structural data on rhodopsin
becomes available. An equally intriguing matter is whether the effects
of the Pro-267 mutations are confined to the 233-240 sequence or have
an impact on other regions of opsin as well.
Stabilization of one fragment by a complementary fragment(s) is not
unique to bovine opsin. Bibi and Kaback (35) reported that the
amino-terminal six TM segments of lactose permease are required for
stable membrane insertion of the complementary carboxyl-terminal six TM
segments. Further, this association appears to be specific for
fragments from lactose permease because analogous fragments from the
structurally related tetracycline and sucrose transporters are unable
to direct insertion of the carboxyl-terminal six TM segments (36). We
have also examined whether fragments analogous to EF(233-348) from
octopus rhodopsin and bacteriorhodopsin would also complement and
confer stable EF(1-232) production. Although both fragments were
stably expressed in COS-1 cells, only the octopus opsin fragment (amino
acids 235-322 plus amino acids 312-348 from bovine opsin) afforded a
modest degree of EF(1-232)
expression.2 This was not
entirely unexpected because there is considerable amino acid
conservation and homology among the sixth and seventh TMs of bovine and
octopus opsins.
The results reported here highlight a key residue in the sixth TM
(Pro-267) that appears to exert long range effects on the conformation
of the third cytoplasmic loop. Although the third cytoplasmic loop is
not critical for stabilization of the EF(1-232) fragment, when
present, its conformation is important for establishing the proper
contacts required for opsin folding, membrane insertion, assembly,
and/or function. Future studies will focus on whether other
site-directed or naturally occurring mutations in the TM helices also
affect the conformation of the solvent exposed loop regions. For this
purpose, the TOXCAT system recently developed by Russ and Engelman (37)
to examine helix-helix interactions in biological membranes may prove useful.
,
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed. Tel.: 301-738-6218;
Fax: 301-738-6255; E-mail: ridge@carb.nist.gov.
-D-maltoside;
PAGE, polyacrylamide gel electrophoresis.
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