(Received for publication, October 23, 1995; and in revised form, January 6, 1996)
From the
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.
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 -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
), (
)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 -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
. 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.
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
CO
,
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 NaCO
, 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).
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 -D-mannopyranoside. Spectra were
recorded in the dark (continuous lines) and after
acidification to pH
1.9 (dashed
lines).
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 NaHPO
, 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).
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.
Figure 6:
Light-dependent activation of G 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
, and 4 µM [
S]GTP
S. The data shown are averages
± standard error from four determinations using pigments from
two separate transfections.
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 NaHPO
, 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.
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 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 (Fig. 6). This is presumably due to disruption of the
cytoplasmic binding site(s) for G
. 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
-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.
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.
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 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.