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CHEMISTRY VoI. 269, No. 31, Issue of August 5 , pp. 19738-19744, 1994
0 1994 by The American Society for Biochemistry and Molecular Biology, Inc Printed in U S A .
(Received for publication, February 17, 1994, and in revised form, April 18, 1994)
We concluded previouslyfrom mutagenesis in the in- illumination, the retinal isomerizes to the all-trans form and
tradiscal domain of bovine rhodopsinthat the formation drives the protein througha series of transient photointerme-
of a tertiary structurecomprisingthe N-terminaltail and diates (7). The intermediate, designated metarhodopsin (Meta-
the three polypeptide loopsis essential to the in uiuo as- 111, binds to and activates the G protein, transducin.A cascade
sembly of the functional rhodopsin. We now report on of biochemical reactions ensues that culminates in the closing
more comprehensive mutagenicstudies in the intradis- of the cation conductance channel in the plasma membrane. A
cal domainto determine more preciselythe requirement neural signal is thus generated(8,9).
for the formation of the above-proposed tertiary struc- In structure-function studies, we have previously introduced
ture. Three large deletions, two consisting of groups of 10 by design a variety of deletions as well as point mutations in a
amino acids each,and the thirdof 34 amino acids, were synthetic rhodopsin gene (1,lO-17). Focusing, in particular,on
carried out in the N-terminal loop. All the threemutant
the intradiscal domain, we observed three main types of phe-
opsins only poorly formed the rhodopsin chromophore.
In the BC loop, wecarried out five 2 amino acid deletions,notypes (14). Type I phenotype resembled the wild-type, in that
2 single amino acid deletions, and three mutations in it folded correctly and formed the normalchromophore with 11-
which short sequences in the loop were reversed. AI1 the cis-retinal. Type I1 mutants did not fold correctly. They re-
resulting mutant opsins had lostthe ability to bind 11- mained in the endoplasmic reticulum andfailed to bind ll-cis-
cis-retinal.In the DE loop, where extensive mutagenesis retinal. Type 111 mutants were also defective in folding. They
had previously beencarried out, we carried out 3 amino showed varying extents of chromophore regeneration with 11-
acid replacements(Am, Thr, ”&I-) at Cys’*’. None ofthese cis-retinal but always less than quantitative.With the aim of
mutants bound ll-cis-retinal. In loopFG, we carried out defining more closely the requirementsfor the formationof the
four 2 amino acid deletions, 1single amino acid deletion,specific structure in the intradiscal domain, we nowreport on a
3 amino acid replacements,and one mutation in which more comprehensive set of mutagenic studies in this domain.
the sequence of the 7 amino acids was reversed. All the The total mutations reported and characterized in this paper are
mutants in FG loop partially formed the rhodopsin chro- shown in Fig. 1and Table I. Thus, we carried out large deletions
mophore. Allthe mutants now described appeared to be in the N-terminal tail. We introduced 1and 2 amino acid dele-
retained in the endoplasmic reticulum: several that were tions in the BC and FG loops and single amino acid replacements
examined in detail were complexed with non-opsinpro- in theFG and DE loops. Further, to test the effect of substantial
teins, the chaperonins. Treatment with ATP-MgCl, re- alterations in amino acid interactions withoutaffecting the sizes
leased the latterfrom the mutant rhodopsins. Our overall of the loops BC and FG, we introduced mutations inwhich se-
conclusion is that the formation of the specific structure quences of short peptide segments in theloops were reversed.
in the intradiscal domain has highly stringent spatial The phenotypes of the different groups of mutations now de-
requirements. scribed mostly paralleled the phenotypes encountered previ-
ously (14). The overall conclusion from the total mutagenesis
performed in the intradiscal domain is that thestereochemical
Rhodopsin, the dim light photoreceptor of the vertebraterod requirements of the intradiscal structure necessaryfor the as-
cell, is anexample par excellance of the receptors that couple to sembly of the functional rhodopsin are highly stringent.
theguanine nucleotide-binding regulatoryproteins (G pro-
teins) in signal transduction (2). Structural models propose EXPERIMENTALPROCEDURES
seven transmembranehelical segments as a common motif for
Materials
this class of membrane receptors (3). The primary structureof
rhodopsin has been determined both by protein (4,5) and DNA Dodecylmaltoside (DM)’ wasfromAnatrace.[35SlMethionine,de-
oxyadenosine 5’-[~x-~~S]th1otriphosphatewere from Dupont-New Eng-
sequencing (6). The protein containsa single polypeptidechain land Nuclear. Goat anti-mouse IgG conjugatedalkaline
to phosphatase,
of 348 amino acidsfolded approximately toform the secondary fluorescein-conjugated goat anti-mouse IgG, and rhodamine-conjugated
structure pattern shown Fig. in 1. Amolecule of ll-cis retinal is goat anti-mouse IgG were from Boehringer Mannheim. The protease
linked to theopsin by a protonated Schiff base at LysZg6.Upon inhibitors, aprotinin, benzamidine-HC1, leupeptin and pepstatin, and
ATP-agarosewerefromSigma.Sequenase(version 2.0) wasfrom
United States Biochemicals Corp. ll-cis-Retinal was a gift of Dr. P.
* This work was supported by National Institutes of Health Grant Sorter (Hoffman-La Roche). Sepharose 4B was purchased from Phar-
GM28289 (to H. G. K.). This is Paper 8 in the series “Structure and
Function in Rhodopsin.” Ref. 1 is Paper 7 in this series. The costs of
publication of this article were defrayed inpart by the payment of page
charges. This article must therefore be hereby marked “aduertisement” The abbreviations used are: DM, n-dodecyl p-D-maltoside; IgG, im-
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. munoglobulin G; rho-1D4, an antirhodopsin monoclonal antibody spe-
t Recipient of Government of India (Department of Biotechnology) cific for the C-terminal tail of rhodopsin; PBS, phosphate-bufferedsa-
fellowship and an additionalfellowship from Flight for Sight funded in line; TBS, “is-buffered saline; PMSF, phenylmethylsulfonyl fluoride;
memory ofAlexander P. and Mary E. Hirsch. Permanent address: Dept. BiP, immunoglobulin heavy chain binding protein; Grp, glucose-rep-
of Biophysics, School of Biological Sciences, MaduraiKamaraj Univer- lated protein; Hsp,heat shock protein; PAGE, polyacrylamide gel elec-
sity, Madurai 625 021, India. trophoresis.
19738
Tertiary
Rhodopsin
Structure
Intradiscal
in Domain 19739
macia LKB Biotechnology Inc.and rho-1D4-Sepharose4B was prepared Immunoblotting
as described (18). Anti-BiP antibody was a gift from Dr. David Bole The opsins after SDS-PAGE werevisualized by immunoblottingwith
(University of Michigan)while the antibodies to the chaperonins Hsp6O the monoclonal antibody rho-1D4. For immunoblotting with anti-chap-
and Grp94 (19) were from Stress Genes, British Columbia. eronin antibodies, the total eluates from the rho-1D4 Sepharose 4B
were concentrated using Millipore ultra filters (10,000 normal molecu-
lar weight limit filters) and subjected to gel electrophoresis.
Construction of the Rhodopsin Gene Mutants
The desired mutations in the synthetic rhodopsin gene (18) were Expression of Rhodopsin Mutants in the Presence of
introduced by the restriction fragment replacement method (20) by P5SlMethionine
replacement of appropriate restriction fragments by synthetic DNA
Rhodopsin mutant genes weretransiently expressed in COS cells for
duplexes containing the required changed codons. All the oligonucleo-
48 h. [35SlMethionine,final concentration, 65 mCi/ml in the medium,
tides corresponding to the DNA fragments were synthesized on an Ap-
was then added for 30min. The cells wereharvested and solubilized in
plied Biosystems 380B DNA synthesizer and purified as previously
the solubilizationbuffer in thepresence of protease inhibitors aprotinin,
described (20). The amino acid replacements, the correspondingrestric- benzamidine-HCI, leupeptin and pepstatin (10 pg/ml of each), and 1mM
tion fragments, and their positions in thegene are shown in Table I. All PMSF and 10 unitdml of apyrase. The insoluble fraction was removed
constructions were checked by sequencing the appropriate regions cor- by centrifugation and rhodopsin was purified from the fraction by ab-
responding to insertions of the fragments containing the modified sorption to rho-1D4 Sepharose 4B.
codons (21). The two N-terminal tail mutants, A 1 and A2, were cloned
between the restriction sites EcoRI and FspI and A3 was cloned be-
Opsin-bound Chaperonins and Deatmentsfor Their Dissociation
tween the restriction sites EcoRI and BclI in the vector pMT4. The BC
loop mutants were cloned first in the cloning vector pOP3 (22) and Urea Wash-Mutant rhodopsins and opsins were prepared from COS
subsequently the opsin gene (EcoRI-Not1 fragment, 1060 base pairs) cells as above and adsorbed to rho-1D4-Sepharose4B. The resin was
was transferred to the expression vector pMT4 (Table I). The mutant A4 washed with 50 resin bed volumes of PBS containing 1M urea and then
was cloned between the restriction sites BgZII and NcoI while A5-A10 with 200 volumes of the wash buffer (10 mM Tris-HC1, pH 7.0, 150 mM
were cloned betweenthe restriction sites NcoI (nucleotidenumber 302) NaC1, 0.1% (wiv) DM). The proteins were then eluted with the antibody-
antipeptide as described previously (18).
andXhoI (nucleotidenumber 339) (Table I) (20).The DE loop mutants,
Removal ofNon-opsinProteins by ATP + MgCZ,-Protocol 1: the total
involving single amino acid replacements, wereclonedbetween the
proteins (opsins and non-opsin proteins) were boundto rho-1D4-Sepha-
restriction sites XbaI (nucleotide number 531) and CZaI (nucleotide
rose. The resin was washed at room temperature with 200 volumes of
number 571) in the vector pMT4. The FG loop deletion mutants ( A l l -
the wash buffer, pH6 (see above) containing 1mMATP and 3mM MgCl,.
A151 (Table I) and the triple amino acid replacement mutant, S281Tl The non-opsin proteins thus eluted were examined by SDS-PAGE. The
D282E/F283Y, were cloned between the NdeI and ApaI sites. The se- opsins were then eluted with the antibody-antipeptide in the wash
quence reversal mutants, R-1 to R-3, in the BCloop (Table I) were buffer, pH 7.0. Protocol 2: the COS cells were harvested 3 days after
cloned between the restriction sites NcoI and XhoI in the vector pMT4, transfection, the chromophorewas generated with 11-cis-retinalfor 3 h,
while the mutantR-4 (FG loop) was cloned between the restriction sites and the cells weretreated with the solubilizationbuffer, pH 6, contain-
NdeI and ApaI. ing 5 mM ATP + 5 mM MgC1, at room temperature for 45 min to disso-
ciate the non-opsin proteins from the mutant rhodopsins. The latter
Expression, Chromophore Formation by the Expressed Opsins, were purified by immunoadsorption as described. Protocol 3:COS cells
a n d Purification of Rhodopsin Mutants were harvested 3 days after transfection. They weresuspended in PBS,
and 11-cis-retinal was added for 3 h. The medium was supplemented
The wild-type rhodopsin gene and the rhodopsin mutant genes in the
with DM (0.1% final) and PMSF (0.1 mM final). After45 min, the
pMT4 vector were transiently expressed in COS-1 cells as described insoluble fraction was removed by centrifugation, and the supernatant
previously (18). COS cells were routinely harvested 3 days after trans- fraction was treated with ATP-agarose for2 hat 4 "C. The supernatant
fection and were treated inone of the following different ways to recon- solution was used to purify themutant rhodopsins by immuno-
stitute the opsins with 11-cis-retinal. absorption.
Deatment-They were treated in PBS (10 mM NaH,PO,, pH 7, 150
m~ NaCl) buffer with 11-cis-retinal(3.5 p ~for ) 3 h in thedark. The cells
Spectral Characterization of Mutant Rhodopsins
were pelleted and treated with the solubilization buffer 1%(w/v) DM
and PBS and 0.1 mM PMSF in the dark at 4 "C for 45 min to solubilize UV-Vis absorption spectra of the wild-type and mutant rhodopsins
the rhodopsin and free opsin. The latter were then purified by immu- were measured in the dark with a Perkin-Elmer A-7 spectrophotometer
noabsorption on rho-1D4-Sepharose4B (18). at 20 "C. Samples were in TBS (10 mM Tris-HC1, pH 7.0, 150 mM NaC1)
Chromophore Formation at Difjrent Temperatures-The cells were containing 0.1% (w/vi DM. Irradiation of the samples was with a 300
watt fiber opticlight source with a <495 nm cut-off filter at 20 "C forthe
incubated with 11-cis-retinal as above, but at 4, 20, or 37 "C. Solubili-
specified times. For absorption spectra at acidic pH, the samples were
zation and purification of the resulting rhodopsin was as above.
acidified with 2 N H,SO, to pH 1.9. Stability of the chromophore toward
Chromophore Formation from the Lysed Cells-The cells were lysed
hydroxylamine in the dark was examined by adding a solution of neu-
in the hypotonic buffer (15 mM Tris-HC1, pH 7.5, 2 mM MgC1, + 1 mM
tral hydroxylamine hydrochloride (final concentration, 100 mM).
dithiothreitol) in the presence of the protease inhibitors (10 pg/mleach
of aprotinin, benzamidine-HC1, leupeptin and pepstatin, and 0.1 mM
PMSF). Lysis was monitored by phase contrast microscopy, and the Immunofluorescence
suspension was passed through a 25 gauge needle five times to homog- cos cells were grown on glass coverslips in 10-cm plates for 16 h,
enize the lysedcell suspension. Formation of the chromophore and transfected as described, and furthergrown for 72h. Cells were washed
purification of the rhodopsin formed were as above. with cold PBS at 4 "C three times, permeabilized in methanol (-20 "C)
Chromophore Formation fromtheMembraneFraction-Thecells for 5 min, and then fixed in acetone (-20 "C) for 5 min. After air-drying
were lysedas described above. The suspension was pelleted by centrifu- in the hood, the cells were rehydrated with PBS + 0.02%NaN,. After 1
gation, and the pellet was homogenized and layered on a 20%/50% h of incubation with the antibody rho-1D4 (10 pg/ml), cells were washed
sucrose gradient. The membrane band at the gradient interface was three times with PBS and thenincubated with anti-mouse goat IgG (10
resuspended in PBS, and chromophore formation was carried out with pg/ml) conjugated with either fluorescein or rhodamine for 1h. The cells
11-cis-retinal for 3 h. After solubilization, rhodopsin and opsin were were washed with PBS and mounted on microscope glass slides. The
purified as described above. stained cells were observed by confocal laser scanning microscope and
Urea Wash ofthe Membranes-The membrane fraction was prepared by immunofluorescence microscope. The stained cells were scanned at
from transfected COS cells as above. The membrane pellet was resus- 0.5-pm thickness using Bio-Rad MRC6OO confocal laser scanning mi-
pended in the PBS buffer containing 5 M urea and the suspension croscope, and the collected images were integrated.
incubated for 10 min at 4 "C. The pellet was collectedby centrifugation,
and the stepwas repeated once and then the pellet was washed in PBS RESULTS
buffer twice. The membrane pellet was resuspended in PBS, and the
chromophore was generated with 11-cis-retinal as describedabove. Mutants in the N-terminal Tail
Solubilization and immunopurification of the mutant rhodopsins were Previously, Doi et al. (14)reported four deletions in the N-
as above. terminal tail. These eliminated, respectively, the amino acids
19740 Tertiary Structure in Rhodopsin
Intradiscal
Domain
A B C D E F G Thus, in R1 (Table I) the sequence of 7 amino acids was re-
versed while in R2 and R3 only 2 amino acids were reversed.
None of the three mutants with the reversed amino acid se-
quences formed opsins that were able to bind ll-cis-retinal.
BC loop: deletions
A4 98-99 BglII-NCOI 251-302
A5 102-103 NCOI-XhoI 302339
A6 104-105 NcoI-XhoI 302-339
A7 106-107 NcoI-XhoI 302-339
A8 108-109 NCOI-XhoI 302-339
A9 104 NCOI-XhoI 302-339
A10 105 NCOI-XhoI 302-339
C187 Y -
DE loop: amino acid replacements
XbaI-ClaI 531-571
C187 + N
C187 T - XbaI-ClaI
XbaI-ClaI
531-571
531-571
29 -
a, 0.0451 \ I - kDo
FIG. 5. Visualization of the location of the mutant opsins in
COS cells by immunofluorescence.Shown are thefluorescence pat-
terns of wild-type, A4, and A l l opsins. The arrows point to the opsin
cn locations as shown by fluorescence. Details of the procedure are intext.
n
L!!&AsY
300 400 500 600
B
Wavelength (nm)
FIG.4. A, UV-Vis absorption spectra of opsins from mutantsA4 and - 97
A l l after chromophore generation with 114s retinal. The conditions for - 60 97 kd ”“
r
0
A l l (B)
a
0.02