From the Department of Earth and Space Science,
Graduate School of Science, Osaka University, Osaka 560-0043, Japan
and ¶ Faculty of Pharmaceutical Science, Osaka University,
Suita 565-0043, Japan
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ABSTRACT |
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S-modulin controls rhodopsin phosphorylation in a calcium-dependent manner, and it has been suggested that it modulates the light sensitivity of the photoreceptor cell. S-modulin binds to the ROS membrane at high Ca2+ concentration, and N-terminal myristoylation is necessary for this property (the calcium-myristoyl switch). S-modulin has four EF-hand motifs, of which two (EF-2 and -3) are functional. Here, we report on the roles of EF-2 and -3 in S-modulin function (calcium binding, membrane association, and inhibition of rhodopsin phosphorylation) by site-directed mutants (E85M and E121M). Surprisingly, E121M, which has a mutation in EF-3, neither binds Ca2+ nor inhibits phosphorylation. In contrast, E85M binds one Ca2+ and has the same membrane affinity as wild-type S-modulin, but has lost the ability to inhibit rhodopsin phosphorylation. It is suggested that the binding of Ca2+ to EF-3 is probably required for EF-2 to be a functional Ca2+-binding site and to induce exposure of the myristoyl group; and that the binding of Ca2+ to EF-2 is important for the interaction with rhodopsin kinase.
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INTRODUCTION |
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In vertebrate rod photoreceptors, cGMP-gated cation channels are opened in the dark-adapted state (1, 2). Light activates rhodopsin and triggers the phototransduction cascade, which results in the closure of cation channels in the rod outer segment (ROS)1 and blocks the influx of Ca2+. As intracellular Ca2+ is continuously pumped out by a Na+-K+/Ca2+ exchanger in the outer segment (3, 4), the cytoplasmic Ca2+ concentration decreases in light-adapted photoreceptors. This decrease of Ca2+ concentration is the underlying mechanism of light adaptation of vertebrate photoreceptors (5, 6).
Phosphorylation of rhodopsin plays a role in shutting off the activation of transducin (7, 8), and the efficiency of phosphorylation is regulated, in a Ca2+-dependent manner, by a Ca2+-binding protein, S-modulin, in frogs (9, 10) or recoverin, its bovine homologue (11). At high Ca2+ concentrations (dark-adapted state), S-modulin inhibits phosphorylation of light-activated rhodopsin, but does not interfere at low Ca2+ concentrations (light-adapted state). Therefore, S-modulin and recoverin contributes to increased light sensitivity in the dark-adapted state.
S-modulin and recoverin are Ca2+-binding proteins which contain covalently attached fatty acyl groups at their N terminus (12). The binding of Ca2+ to these proteins induces exposure of the fatty acyl groups, which enables them to associate with ROS membrane (13, 14). This property is the so-called "Ca2+-myristoyl switch" (13). There are four EF-hand motifs in S-modulin and recoverin, but only two of them (EF-2 and -3) are thought to be able to bind Ca2+ (15). Therefore, S-modulin function (inhibition of frog rhodopsin phosphorylation) is mediated by the binding of Ca2+ ions to EF-2 and -3.
We made site-directed mutants of S-modulin that lack Ca2+ binding ability in their EF-2 or -3. The present study describes the Ca2+ binding properties, membrane-association, and inhibitory effects on rhodopsin phosphorylation of wild-type S-modulin and these mutants. The results suggest that EF-3 first binds Ca2+, which enables S-modulin to associate with ROS membrane and to bind Ca2+ at EF-2. Subsequent EF-2 binding of Ca2+ ions probably causes a conformational change permitting interaction of S-modulin with rhodopsin kinase, which inhibits rhodopsin phosphorylation.
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EXPERIMENTAL PROCEDURES |
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Site-directed Mutagenesis-- Oligonucleotides, 5'-GGTCATATGTAGCGCTATCATGTACATCTTAAA-3' and 5'-TTGAATATTGCCGTAATGATTTCAAGCACCATTTTTTT-3' were used as antisense primers to generate site-directed mutants, E85M and E121M, respectively. The S-modulin cDNA fragment (10) and the SMD-NTF primer (16) were used as a template and sense primer, respectively, for the polymerase chain reactions. cDNA fragments encoding E85M and E121M were inserted between the NcoI and XhoI sites of pET-16b (Novagen) plasmid vector, designated pET-E85M and pET-E121M, respectively.
Expression and Purification of Recombinant Proteins--
The
procedures for expression and purification of recombinants follow
Hisatomi et al. (16). Briefly, the expression vectors, pET-Smd (16), pET-E85M, and pET-E121M were transfected to
Escherichia coli BL21DE3 (Novagen) with (+myr) or without
(myr) pBB131, an expression vector of
N-myristoyltransferase. The recombinant proteins were
expressed by the addition of 1 mM
isopropyl-
-D-thiogalactopyranoside. Recombinant proteins
were solubilized in 8 M urea buffer, refolded by dialysis,
and applied to a DEAE-Sephadex column. The fraction containing
recombinant S-modulins was dialyzed against the buffer containing 5 mM Ca2+ and applied to a phenyl-Sepharose
column. Recombinant S-modulins were eluted with 5 mM
EGTA.
HPLC-- Purified recombinant proteins were injected onto a reverse-phase C-18 column. Recombinant S-modulin was eluted with a linear gradient of 0-80% acetonitrile (1.3%/min) in 0.1% trifluoroacetic acid at a flow rate of 1.5 ml/min, monitoring absorbance of the eluate at 280 nm.
Ca2+ Binding Assay-- Binding of Ca2+ ions to S-modulin or mutant proteins was evaluated by ultrafiltration (17). Purified proteins were extensively dialyzed against 25 mM Tris-HCl (pH 8.0) to remove EGTA and calcium, then 20 µmol of each calcium-free protein in 1 ml of 25 mM Tris-HCl (pH 8.0) were placed in a Centricon-10 concentrator (Amicon). 1 ml of 0.2 mM CaCl2 solution in 25 mM Tris-HCl (pH 8.0) was then added, and the solution was thoroughly mixed. The calcium-protein mixtures were then centrifuged, and the amounts of calcium in the filtrated fractions were measured by atomic absorption (Shimazu AA-660).
Tryptophan Emission Spectrum-- Spectroscopic measurement was carried out as described by Hisatomi et al. (16). Briefly, fluorescence emission spectra were recorded from 300 to 400 nm with a fluorescence spectrophotometer (Hitachi, F-4500) at an excitation wavelength of 290 nm, in a mixture containing 2 µM recombinant protein, 100 mM KCl, 5 mM 2-mercaptoethanol, 1 mM EGTA, and 100 mM HEPES (pH 7.0). The free Ca2+ concentration was adjusted by adding 1 M CaCl2.
Ca2+-dependent Membrane Association of Recombinant Proteins-- Frog ROS were isolated by flotation with 45% sucrose in gluconate buffer (40 mM potassium gluconate, 2.5 mM KCl, 2 mM MgCl2, 1 mM dithiothreitol, 1 mM EGTA, and 10 mM HEPES, pH 7.5), and washed with gluconate buffer containing 4 M urea to eliminate endogenous S-modulin, s26 (cone homologue of S-modulin) and other peripheral proteins. Urea-stripped ROS membranes were mixed with gluconate buffer containing 1% bovine serum albumin to prevent nonspecific binding of the recombinant proteins to the ROS membrane and tube. After washing with gluconate buffer containing various concentrations of Ca2+ (Ca2+ gluconate buffer), the ROS membranes were resuspended in Ca2+ gluconate buffer containing recombinant proteins (120 pmol). The mixtures were incubated at room temperature for 30 min, and the soluble and membrane fractions after centrifugation (37,000 × g for 5 min) were analyzed by SDS-polyacrylamide gel electrophoresis. The integrated densities of Coomassie Brilliant Blue-stained bands of the recombinant proteins were quantified by a two-dimensional densitometer (The Discovery Series, pdi Inc.).
Phosphorylation Assay--
Phosphorylation of rhodopsin was
measured by the methods of Kawamura (9) and Sanada et al.
(18). For the phosphorylation assay, ROS were isolated in
phosphorylation buffer (115 mM potassium gluconate, 2.5 mM KCl, 2 mM MgCl2, 1 mM dithiothreitol, 1 mM EGTA, 10 mM
HEPES, pH 7.5) in complete darkness, and washed with the buffer to
eliminate endogenous S-modulin, s26, and ATP. The reaction was carried
out in 25 µl of the mixture containing 10 µM (final concentration) rhodopsin and various concentrations of S-modulin and/or
its mutants in phosphorylation buffer. The free calcium concentration
in the mixture was adjusted by adding 1 M CaCl2 solution. The reaction mixtures were exposed to light for 2 min, and
the reaction was initiated by addition of a mixture of ATP (0.1 mM final concentration), [-32P]ATP (168 TBq/µmol, 0.25 µM), and GTP (0.5 mM). After
2 min of incubation at room temperature, the reaction was terminated by adding 150 µl of 10% trichloroacetic acid. After centrifugation (10,000 × g for 5 min) of the reaction mixture, the
precipitates were washed with 500 µl of phosphorylation buffer and
subjected to SDS-polyacrylamide gel electrophoresis. The amount of
32P incorporated into rhodopsin was quantified by using an
image analyzer (BAS 2000, Fuji Film).
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RESULTS |
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Myristoylation of Recombinant S-modulin--
It has been
established that the conserved glutamic acid at position 12 of the
Ca2+-binding loop is important for coordinating
Ca2+ (19, 20). To investigate the role of each
Ca2+-binding site, EF-2 or EF-3 was inactivated by
replacing glutamic acid with the hydrophobic amino acid, methionine.
Myristoylated wild-type (+myr) and mutant S-modulins, E85M (+myr) and
E121M (+myr), and unmyristoylated wild-type (myr) were expressed and purified as described in the experimental procedures. Myristoylated recombinant proteins eluted from a C-18 column at almost the same retention time (Fig. 1, b,
c, and d), which is longer than that of wild-type
(
myr) (Fig. 1a) and suggests that these recombinants expressed with N-myrstoyltransferase are in fact
myristoylated.
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Ca2+ Binding of S-modulin and Mutants-- The number of Ca2+ ions bound to each of these proteins was quantified in the presence of 0.1 mM Ca2+ (Table I). As expected, wild-type (+myr) and E85M (+myr) bind two and one Ca2+ per a molecule, respectively. On the other hand, E121M (+myr) can not bind Ca2+. These results indicate that Ca2+ binding to EF-3 is necessary for EF-2 to bind Ca2+ at physiological Ca2+ concentrations. It has been reported that EF-3 has the conformation of a classic EF-hand, but EF-2 is rather different (21). The conformational change induced by Ca2+ binding to EF-3 may be required to raise the Ca2+ affinity of EF-2, in order for EF-2 to become functional.
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Fluorescence Properties of S-modulin and Mutants--
E85M (+myr)
and E121M (+myr) can be purified in the same way as for wild-type
S-modulin, so it seems that the structure of S-modulin is not largely
disrupted by the mutagenesis. Fig. 2 shows the tryptophan emission spectra of the wild-type and mutant S-modulins. Wild-type (+myr), E85M (+myr) and E121M (+myr) showed almost the same spectrum in the presence of 1 nM
Ca2+ (Fig. 2, upper panel), but different from
wild-type (myr). This suggests that E86M (+myr) and E121M (+myr) are
in fact myristoylated in a similar way to the wild-type (+myr), and
that the mutations of glutamic acid to methionine in EF-2 and EF-3 do
not significantly change the environment of the three tryptophan
residues in the Ca2+-free form of these proteins. However,
the emission spectra of mutants were different from that of wild-type
at a concentration of 0.1 mM Ca2+ (Fig. 2,
lower panel). The spectrum of the wild-type is red-shifted by an increasing Ca2+ concentration (16); that of E85M
(+myr) shows a smaller red shift; and that of E121M (+myr) was not
affected by Ca2+ concentration. The red shift observed in
E85M (+myr), which can bind one Ca2+ ion per molecule, is
probably caused by the binding of Ca2+ to EF-3.
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Membrane Association of Wild-type, E85M, and E121M S-modulins-- Each myristoylated recombinant, (wild-type (+myr), E85M (+myr), or E121M (+myr)) was mixed with urea-stripped ROS membranes at various Ca2+ concentrations and separated by centrifugation into membrane and soluble fractions. The soluble fraction (containing proteins free from ROS membranes) and the membrane fractions (containing proteins bound to ROS membranes) were subjected to SDS-polyacrylamide gel electrophoresis. The densities of Coomassie Brilliant Blue-stained bands of the wild-type and mutant S-modulins were analyzed quantitatively, and the ratio of membrane-bound protein, (membrane fraction)/(membrane + soluble fraction), was plotted against Ca2+ concentration (Fig. 3). This shows that E85M (+myr) has almost the same membrane affinity as the wild-type. As exposure of the myristoyl group is essential for membrane binding (13, 14), our results suggest that binding of Ca2+ to EF-3 induces exposure of the myristoyl group. It has been reported that ejection of the myristoyl group is required for rotation at Gly-42, unclamping of the myristoyl group, and melting of part of the N-terminal helix (21). Ca2+ binding to EF-3 may induce these changes until the level necessary for membrane association.
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Inhibition of Rhodopsin Phosphorylation by Wild-type or Mutant S-modulins-- Fig. 4 shows the incorporation of 32P-labeled phosphatic acid into rhodopsin in the presence of various concentrations of wild-type (+myr), E85M (+myr), or E121M (+myr) S-modulins. Wild-type (+myr) inhibits rhodopsin phosphorylation at a high (0.1 mM) Ca2+ concentration, but neither E85M (+myr) nor E121M (+myr) can inhibit rhodopsin phosphorylation even at high Ca2+ concentrations. This suggests that Ca2+ binding to EF-2 is important for the inhibitory activity of rhodopsin phosphorylation.
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DISCUSSION |
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Ca2+ Binding Cooperativity-- Ames et al. (23) reported that unmyristoylated recoverin exhibits heterogeneous and uncooperative binding of two Ca2+ ions, but these two Ca2+ ions bind cooperatively to myristoylated recoverin with a Hill coefficient of 1.75. One explanation of this difference between myristoylated and unmyristoylated recoverins is that the myristoyl group accommodated within the protein moiety may reduce the Ca2+ affinity of EF-3 to a level lower than that of the EF-2 of unmyrisotylated S-modulin. When Ca2+ concentration is high, EF-3 binds Ca2+, which causes the myristoyl group to be exposed and EF-2 to be functional, upon which it binds Ca2+.
The Hydrophobic Region Exposed by Ca2+ Binding--
As
with myristoylated wild-type (+myr), unmyristoylated wild-type (myr)
binds to a phenyl-Sepharose column in a
Ca2+-dependent manner. This suggests that a
hydrophobic region of the protein moiety, in addition to the myristoyl
group, is exposed by binding of Ca2+ (13). In our
preliminary experiments, both unmyrstoylated E85M (
myr) and E121M
(
myr) lose affinity for phenyl-Sepharose. We conclude that exposure
of the hydrophobic region is probably caused by Ca2+
binding at EF-2.
Structural Changes of S-modulin Induced by Ca2+ Binding-- The upper part of Fig. 6 illustrates the conformational changes of S-modulin deduced from our present analysis. The lower part of Fig. 6 represents the corresponding three-dimensional structure of bovine recoverin during Ca2+ binding (15, 21, 22). The structure of N-terminal region in the single Ca2+-bound form is different from that in the Ca2+-free form but similar to the double Ca2+-bound form. It is consistent with our model that the conformational change induced by the Ca2+ binding to the EF-3 (shown in blue) may expose the N-terminal myristoyl group. The structure of EF-2 (shown in red) in the single Ca2+-bound form is also largely different from that in the Ca2+-free form. This difference is probably important for EF-2 to be a functional Ca2+-binding site.
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ACKNOWLEDGEMENTS |
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We thank Prof. Satoru Kawamura, Prof. Mikio Kataoka, and Kumiko Nanda for helpful discussions and technical suggestions. We also thank Dr. Ian G. Gleadall for comments on the manuscript.
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FOOTNOTES |
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* This work was supported in part by a Grant-in-Aid from the Japanese Ministry of Education, Science and Culture of Japan, and by SUNBOR.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Research Fellow of Japan Society for the Promotion of Science. Present address: Laboratory for Memory and Learning, Riken Brain Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0106, Japan.
To whom correspondence should be addressed: Dept. of Earth and
Space Science, Graduate School of Science, Osaka University, Machikaneyama-chyo 1-1, Toyonaka City, Osaka 560, Japan. Tel.: 81-6-850-5499; Fax: 81-6-850-5480; E-mail:
tokunaga{at}ess.sci.osaka-u.ac.jp.
The abbreviations used are:
ROS, rod outer
segment(s); HPLC, high performance liquid chromatography; (+myr), myristoylated recombinant; (myr), unmyristoylated recombinant.
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REFERENCES |
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