A unique choanoflagellate enzyme rhodopsin exhibits lightdependent cyclic nucleotide phosphodiesterase activity

Article abstract of DOI:10.1074/jbc.M117.775569

Photoactivated adenylyl cyclase (PAC) and guanylyl cyclase rhodopsin increase the concentrations of intracellular cyclic nucleotides upon illumination, serving as promising secondgeneration tools in optogenetics. To broaden the arsenal of such tools, it is desirable to have light-activatable enzymes that can decrease cyclic nucleotide concentrations in cells. Here, we report on an unusual microbial rhodopsin that may be able to meet the demand. It is found in the choanoflagellate Salpingoeca rosetta and contains a C-terminal cyclic nucleotide phosphodiesterase (PDE) domain. We examined the enzymatic activity of the protein (named Rh-PDE) both in HEK293 membranes and whole cells. Although Rh-PDE was constitutively active in the dark, illumination increased its hydrolytic activity 1.4-fold toward cGMP and 1.6-fold toward cAMP, as measured in isolated crude membranes. Purified full-length Rh-PDE displayed maximal light absorption at 492 nm and formed the M intermediate with the deprotonated Schiff base upon illumination. The M state decayed to the parent spectral state in 7 s, producing long-lasting activation of the enzyme domain with increased activity. We discuss a possible mechanism of the Rh-PDE activation by light. Furthermore, Rh-PDE decreased cAMP concentration in HEK293 cells in a light-dependent manner and could do so repeatedly without losing activity. Thus, Rh-PDE may hold promise as a potential optogenetic tool for light control of intracellular cyclic nucleotides (e.g. to study cyclic nucleotide-associated signal transduction cascades).

Full text of DOI:10.1074/jbc.M117.775569

JBC Papers in Press. Published on March 16, 2017 as Manuscript M117.775569
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M117.775569
Rhodopsin-PDE
A unique choanoflagellate enzyme rhodopsin with cyclic nucleotide phosphodiesterase activity
1
1,2,3
4
1,3
Kazuho Yoshida , Satoshi P. Tsunoda , Leonid S. Brown , Hideki Kandori
From
1
Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Showa-ku,
Nagoya 466-8555, Japan.
2
JST PRESTO, 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan
3
OptoBioTechnology Research Center, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555,
Japan.
4
Department of Physics, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Running title: Rhodopsin-PDE
To whom correspondence should be addressed: Prof. Satoshi P. Tsunoda & Prof. Hideki Kandori
Address: Department of Life Science and Applied Chemistry, Nagoya Institute of Technology,
Showa-ku, Nagoya 466-8555, Japan.
tel: +81 52 735 5218 & +81 52 735 5207
e-mail: tsunoda.satoshi@nitech.ac.jp & kandori@nitech.ac.jp
Keywords: microbial rhodopsin, phosphodiesterase, optogenetics, biophysics, membrane protein,
photoreceptor, cyclic nucleotide
1
ABSTRACT
promising second-generation tools in
Photoactivated adenylyl cyclase (PAC) and
optogenetics. To broaden arsenal of such tools,
guanylyl cyclase rhodopsin (Rh-GC) increase
it is desirable to have light-activatable
the concentrations of intracellular cyclic
enzymes which can decrease cyclic nucleotide
nucleotides upon illumination, serving as
concentrations in cells. Here, we report on an
1
Copyright 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Rhodopsin-PDE
Rhodopsins, heptahelical membrane proteins
unusual microbial rhodopsin which may be
able to meet the demand. It is found in the
choanoflagellate Salpingoeca rosetta and
contains a C-terminal cyclic nucleotide
phosphodiesterase (PDE) domain. We
examined enzymatic activity of the protein
containing retinal as chromophore, are
subdivided into the animal and microbial
types (1). While animal rhodopsins serve
almost exclusively as G-protein coupled
receptors (2–5), the functions of microbial
rhodopsins are diverse, including light-driven
cation and anion pumps, light-gated cation
and anion channels, positive and negative
phototaxis sensors, photochromic sensors,
and light-activated enzymes (1, 6–9). The
first discovered light-activated enzyme was
an algal histidine kinase (HK) (10), while
fungal light-activated guanylyl cyclase (GC)
was reported recently (11–13). These proteins
are composed of a membrane-embedded
(
named Rh-PDE) both in HEK293 membranes
and whole cells. Even though Rh-PDE was
constitutively active in the dark, illumination
increased its hydrolytic activity 1.4-fold
toward cGMP and 1.6-fold toward cAMP, as
measured in isolated crude membranes.
Purified full-length Rh-PDE displayed
maximal light absorption at 492 nm and
formed the M intermediate with the
deprotonated Schiff base upon illumination.
The M state decayed to the parent spectral
state in 7 seconds, producing long-lasting
activation of the enzyme domain with
increased activity. We discuss possible
mechanism of the Rh-PDE activation by light.
Furthermore, Rh-PDE decreased cAMP
concentration in HEK293 cells in a
rhodopsin domain and
a
C-terminal
cytoplasmic enzyme domain, activated by
light absorption by the all-trans-retinal
chromophore. The molecular mechanisms of
enzyme rhodopsins activation are understood
much less than those of other microbial
rhodopsins, mainly because of the difficulty
of preparing functional full-length proteins.
In fact, the light-dependent enzymatic
activity has never been demonstrated for
Rh-HK, and detailed spectroscopic study of
Rh-GC photochemistry has been performed
only for the transmembrane domain without
the soluble enzyme domain (12).
light-dependent manner and could do so
repeatedly without losing activity. Thus,
Rh-PDE may hold promise as a potential
optogenetic tool for light control of
intracellular cyclic nucleotides, for example,
to study cyclic nucleotide–associated signal
transduction cascades.
Optogenetics has revolutionized life
sciences, due to its ability to control various
2

Rhodopsin-PDE
biological activities by light (14–18). Since
the early stages of rhodopsin-based
optogenetics, light-gated channels and
light-driven ion pumps have been used to
excite and silence neurons (19, 20). This is
highly advantageous for applications in
neuroscience when fast temporal resolution is
required to change transmembrane potential.
In contrast, for optogenetic control of a wider
engineered light-activated cyclic nucleotide
phosphodiesterase
(PDE)
(26)
and
Gi-coupled animal opsin (27) are able to
lower cyclic nucleotides concentration upon
illumination. The former combines GAF
(cGMP-binding PDEs, adenylyl cyclase and
FhlA) domain and PDE domain, which
allows controlling the PDE activity by light
(26). However, because of the photochromic
nature of the GAF domain, its light-induced
enzymatic activity has to be stopped by light
of a different color. Gi-coupled animal opsin
from mosquito (Opn3) decreases cAMP
concentration under illumination, as Gi
inhibits the activity of adenylyl cyclase (AC)
(27). On the other hand, enzymatic activity of
mosquito Opn3 persists for several tens of
minutes (27), being much longer than that of
Rh-GC (12, 13), presumably because of the
AC-related signalling network. In addition,
GPCRs regulate other signalling cascades
through G and arrestin, triggering complex
intracellular events. Therefore, proteins
composed of a light-sensor domain and a
PDE domain, with fast enzymatic turnover
induced by single light excitation, are highly
desirable.
spectrum
of
biological
functions,
light-induced enzyme activation is more
attractive, because intracellular signalling
processes can be manipulated by light. In
particular, optogenetic control of second
2
+
messengers such as Ca
nucleotides is in high demand.
and cyclic
Photoactivated adenylyl cyclase (PAC)
with flavin adenine nucleotide (FAD)
chromophore was discovered in Euglena
gracilis in 2002 (21). It could be used as an
optogenetic
tool,
which
increases
intracellular cAMP concentration under blue
light (22–25). More recently, a microbial
rhodopsin containing guanylyl cyclase (GC)
domain was discovered in Blastocladiella
emersonii (11) and applied to optogenetics
(
12, 13). In contrast to these light-activated
proteins able to increase cAMP and cGMP
concentrations, an efficient native
In this paper, we report on a novel type of
microbial rhodopsin with C-terminal PDE
domain, found in the choanoflagellate
Salpingoeca rosetta, a unicellular and
light-dependent enzyme capable of lowering
cyclic nucleotides concentration has not been
known until now. It should be noted that
colonial
single
flagellate
eukaryote,
3

Rhodopsin-PDE
considered to be one of the closest living
relatives of metazoans. We successfully
expressed and purified the full-length
Rh-PDE from mammalian cells, and its
enzymatic functions were studied in cells, in
membranes, and in detergent, both
biochemically and spectroscopically. We
discuss a putative molecular mechanism of
the Rh-PDE activation based on the
presented studies of the full-length protein
and its applicability to optogenetics.
changes to induce (or modulate) the
enzymatic activity.
Fig. 1B shows a phylogenetic tree of
selected microbial rhodopsins, based on the
amino acid sequences of their transmembrane
domains. Rh-PDE clusters with other enzyme
rhodopsins of lower eukaryotes, e.g.,
histidine kinases and guanylate cyclases.
Many common residues of microbial
rhodopsins are conserved in Rh-PDE (Fig.
1C, see Supplementary Fig. S2 for the
complete alignment). Specifically, D85, the
primary counterion of the protonated Schiff
RESULTS
A new microbial rhodopsin containing
a PDE domain. The genome of the
base
in
light-driven
proton
pump
bacteriorhodopsin (BR), is conserved as
E164 in Rh-PDE. The residues constituting
the all-trans-retinal binding pocket of BR
(W86, T89, W182, and Y185) (1) are also
conserved as W165, T168, W264, and F267,
respectively, in Rh-PDE. The PDE domain
contains many conserved residues typical for
the active centers of soluble PDEs (Fig. 1D)
but it is not possible to predict the substrate
specificity of Rh-PDE (cAMP, cGMP, or
both) from the sequence.
choanoflagellate
Salpingoeca
rosetta
(
sequenced by the Broad Institute, NCBI
accession PRJNA37927) contains an unusual
gene (NCBI Gene ID: 16078606) encoding a
rhodopsin fused to a C-terminal PDE domain
(
Rh-PDE). Fig. 1A shows a schematic
drawing of the Rh-PDE architecture, which is
composed of an N-terminal 7 transmembrane
helical rhodopsin domain and a cytoplasmic
PDE domain (see Supplementary Fig. S1 for
the complete amino acid sequence). It is
possible that Rh-PDE exists as a dimer, as
typical for known PDEs (28–30). From the
sequence analysis, we propose that Rh-PDE
Enzymatic activity of Rh-PDE in
mammalian cells. To investigate enzyme
rhodopsins of lower eukaryotes, it is
important to find a suitable expression
system. Previous studies on Rh-GC used
yeast expression, where the full-length
is
a
microbial rhodopsin that binds
all-trans-retinal as a chromophore, where
light absorption triggers protein structural
4

Rhodopsin-PDE
protein could not be obtained (12). In this
study, we first measured enzymatic activity
of Rh-PDE using HEK293 cells, from which
the full-length protein was subsequently
purified and characterized in vitro. To
measure in cell enzymatic activity of
Rh-PDE, we used the GloSensor assay, which
is based on a cyclic nucleotide dependent
luciferase. In this assay, the Rh-PDE
expressing cells were first incubated in the
serum-containing culture medium after
addition of all-trans-retinal in the dark at
room temperature (~27 °C), and the
luminescence intensity was increased by
sodium nitroprusside (SNP) or forskolin,
which activate GC or AC resulting in
elevation of the cytoplasmic cGMP or cAMP
level, respectively.
illumination. Consistent with this idea,
light-independent gradual increase of
luminescence occurred for the D605A mutant
(bottom panel of Fig. 2A), with inactivating
replacement in the active center of the PDE
domain.
Fig. 2B and C show the activity of
Rh-PDE toward cAMP. The luminescence
intensity has gradually increased after
addition of forskolin, and reached a plateau
after 40 min, similar to the cGMP assay (Fig.
2B). At that time point, illumination at 510
-2
nm with 2.28 W mm intensity caused a
decrease of the luminescence in cells
expressing Rh-PDE (red line), but not in
those with the empty vector (blue line) (Fig.
2
B and C). 28 % (±2.3 % SD) reduction in
the luminescence intensity was reproducibly
observed under these experimental conditions
Fig. 2A shows the activity of Rh-PDE
toward cGMP. In the control experiments, the
luminescence intensity has gradually
increased after addition of SNP and reached a
plateau after 40 min for HEK293 cells
expressing GloSensor only (empty vector).
As expected, there was no effect of light on
the level of cGMP in this system (top panel
of Fig. 2A). In contrast, such gradual increase
of luminescence after addition of SNP has not
been observed upon expression of Rh-PDE
(
N=6). Thus, it can be concluded that the
decrease of cAMP concentration must
originate from the light-induced Rh-PDE
activity, which is further supported by the
lack of signal for the catalytically inactive
D605A mutant (yellow line in Fig. 2B and C).
It should be noted that after the light was
switched off, the light-induced luminescence
decrease persisted for a significant amount of
time (~10 sec), and then decayed with 1/e of
(
middle panel of Fig. 2A), which suggests
1
95 s (red line in Fig. 2C). This kinetics
constitutive activity of Rh-PDE toward
cGMP, both in the dark and upon
involves at least two components,
5

Rhodopsin-PDE
deactivation of Rh-PDE, and production of
cAMP by endogenous adenylyl cyclases.
Based on the exponential fitting, we estimate
the life time of the light-activated state of
Rh-PDE as about 71 sec (Fig. S3). Another
notable feature is that the stationary
luminescence intensity in the dark was
smaller for Rh-PDE (middle panel of Fig.
decrease of intracellular cAMP concentration
suggests that Rh-PDE may be useful as an
optogenetic tool. Together with PAC and
Gs-coupled opsins capable of increasing
intracellular cAMP concentration, Rh-PDE
increases the diversity of optogenetics toolkit.
2
6
Unlike the engineered light-activated PDE ,
Rh-PDE is switched off by its photocycle,
without the need for an additional
illumination. On the other hand, Gi-opsin,
which can decrease intracellular cAMP
concentration (27), also switches off in the
dark, warranting a comparison of the
properties of Rh-PDE and Gi opsin.
2
B) than for the empty vector and the D605A
mutant (top and bottom panels of Fig. 2B),
suggesting constitutive dark activity toward
cAMP, although smaller than toward cGMP.
Fig. 2D shows that the light-induced
reduction of luminescence increased with the
applied light intensity, and saturated with an
Thus, we expressed mosquito Opn3 in
HEK cells with addition of all-trans-retinal,
using the same protocol as for Rh-PDE.
Although the native chromophore of
mosquito Opn3 is 11-cis-retinal, it is known
that this opsin binds 13-cis-retinal, which can
be easily formed from all-trans-retinal, and
that the regenerated pigment shows
absorption maximum at 500 nm (27). Upon
application of the 510 nm light, the cells
expressing mosquito Opn3 show strong
decrease in the intracellular cAMP
concentration, which continues to decrease
after the light is switched off reaching a
minimum in ~5 min, and then recovers with
2
EC50 of 0.244 W/mm (Fig. 2E). One
could expect that the intensity increase is a
sigmoidal function if any two-photon
processes were involved, e.g., both protein
molecules in a dimer need to be activated.
Nearly linear increase at lower light
intensities in Fig. 2E suggests that Rh-PDE
activation is a single photon event, and
activation of one molecule is sufficient even
if Rh-PDE likely to form a dimer. In
summary, the in cell assays demonstrated that
Rh-PDE shows strong constitutive activity
toward cGMP, while the activity toward
cAMP is light-dependent.

1/e of 16 min (green line in Fig. 3A). These
Suitability of Rh-PDE for optogenetic
data reproduce the ones previously reported
(27), and it is obvious that the luminescence
applications. The observed light-dependent
6

Rhodopsin-PDE
signal is smaller and faster (both in the rise
and the decay) in the case of Rh-PDE under
the same illumination conditions (red line in
Fig. 3A). In the case of mosquito Opn3,
inter-molecular protein-protein interactions
between opsin and Gi, and between Gi and
AC should amplify the signal. Thus, as an
optogenetic tool, Rh-PDE exhibits less
sensitive, but about 5-times faster response
than mosquito Opn3. In addition, Rh-PDE
only acts on cyclic nucleotides, while
additional intracellular effects of mosquito
Opn3 are unclear.
without addition of 11-cis-retinal was
27
reduced more than twofold . In contrast,
Rh-PDE binds naturally abundant
all-trans-retinal, and the active state is
rapidly reverted to the resting state as a result
of the photocycle. This comes along with the
high repeatability, another important property
for optogenetics. Fig. 3C shows that
activation of Rh-PDE can be repeated for
longer than 2 hours, showing the suitability
of Rh-PDE as an optogenetic tool. To define
the usable spectral range of Rh-PDE, we
systematically changed the irradiation
wavelength at the same light intensity (Fig.
3D) and found that the luminescence is
maximally decreased by the blue-green light
(480-510 nm).
Another
important
parameter
for
optogenetic applications is chromophore
availability. We compared the activity of
Rh-PDE toward cAMP with and without
addition of all-trans-retinal in the cell culture
media after transfection (Fig. 3B). Even
though the luminescence decrease is smaller
without addition of all-trans-retinal, it still
retains robust activity at 70 % of the
amplitude upon its addition, which is
explained by the fact that all-trans-retinal is
commonly present in cells. While mosquito
Opn3 11-cis-retinal chromophore is not
abundant in cells, Koyanagi et al. found that
mosquito Opn3 can bind 13-cis-retinal, a
ubiquitously present retinal isomer (27). As
HPLC analysis of the enzymatic
activity
of
Rh-PDE.
We
further
characterized enzymatic activity of Rh-PDE
by the HPLC analysis using crude
membranes of HEK293 cells. In these
experiments, cGMP or cAMP is mixed with
the crude cell membranes expressing
Rh-PDE, and time- and light-dependent
hydrolysis of cyclic nucleotides into 5’GMP
or 5’AMP is monitored by HPLC. Fig. 4A
shows the HPLC profile for cGMP, where
Rh-PDE decreases the amount of cGMP and
concomitantly increases that of 5’GMP.
Importantly, 10 min incubation under the
13-cis- and all-trans- retinals exist in
photoequilibrium, mosquito Opn3 is usable
in general tissues, even though its activity
7

Rhodopsin-PDE
illumination results in the 85 % decrease of
cGMP concentration, compared to only 60 %
in the dark (Fig. 4B). This indicates that
Rh-PDE has light-dependent enzymatic
activity toward cGMP, even though its dark
constitutive activity is significant. The
hydrolysis rates were calculated from the
slopes of the curves (0-10 min) in Fig. 4B as
entirely abolished in the D605A mutant, with
inactivating replacement in the active center
of the PDE domain (data not shown).
Although crude cell membranes contain
many other proteins, the comparison between
the wild-type and the D605A mutant Rh-PDE
indicates that the observed hydrolysis of both
nucleotides originates exclusively from
Rh-PDE, consistent with the in-cell results
(Fig. 2).
353 nmol/mg total protein/min in the dark
and 511 nmol/mg total protein/min under
illumination, giving 142 % activation by
light.
Molecular properties of purified
Fig. 4C shows the HPLC profiles for
cAMP in the dark (top panel) and under the
illumination (bottom panel). Rh-PDE
decreases cAMP concentration in both cases,
but the activity is smaller compared to that
for cGMP. It should be noted that 10 fold
higher amount of the membranes was used
for the cAMP hydrolysis assay (see
full-length Rh-PDE.
The in-cell and
in-membrane studies described above
showed that the enzymatic activity of
Rh-PDE is stimulated by light toward both
cAMP and cGMP. To gain insight into the
molecular mechanism of photoactivation, it is
necessary to purify and characterize the
full-length protein. While expression levels
of rhodopsins are usually much smaller in
HEK293T cells than in yeast and E. coli,
previously, we have been able to conduct
infrared spectroscopic studies on monkey
red- and green-sensitive pigments based on
HEK293 cell expression (31, 32). We thus
applied the sample preparation procedure as
used for monkey red- and green-sensitive
pigments to Rh-PDE, using HEK293T cell
Experimental
Procedures).
A
clear
light-dependent activation can be seen for the
cAMP hydrolysis as was observed in the
cGMP assay above. The hydrolysis rates
were calculated as 32.6 nmol/mg total
protein/min in the dark and 53.6 nmol/mg
total protein/min under illumination, giving
~164% stimulation by light. The activity
toward cGMP is about 10 fold higher than
toward cAMP (Fig. 4), consistent with the
in-cell results.
expression,
solubilisation
by
Dodecyl-β-D-maltopyranoside (DDM), and
Hydrolysis of cGMP and cAMP was
purification by monoclonal antibody column
8

Rhodopsin-PDE
chromatography, with all-trans-retinal being
added into the culture medium. As a result,
we were able to obtain purified full-length
protein (Fig. 5A inset) with the yields
comparable to those of monkey color
pigments (0.4 mg of Rh-PDE vs. 0.3 mg of
monkey color pigments from 300 culture
plates of HEK293 cells with 10 cm diameter
absorption band appears in the 350-420 nm
region. It should be noted that the
measurements shown in Fig. 5A were
performed at 4 °C, as the decay of the
blue-shifted photointermediate was too fast to
record its spectrum at room temperature. Fig.
5C shows the difference spectra of the
photoactivation
and
its
relaxation,
(
31, 32)).
demonstrating bleaching of the dark state at
499 nm and concomitant appearance of the
intermediate state with the maximum at 392
nm. This peak wavelength suggests the
light-induced deprotonation of the retinal
Schiff base, in a state known as the M
intermediate, which decays to the original
spectral state.
We characterized the photoreactions of
the purified full-length Rh-PDE using
UV-Vis spectroscopy. Fig. 5A shows the
absorption spectrum of Rh-PDE in the dark,
with the max located at 492 nm (black). This
absorption spectrum shows good correlation
with the action spectrum obtained from the
GloSensor assays (Fig. 5B) using the
wavelength dependence of the light-induced
Rh-PDE activity toward cAMP (Fig. 3D).
The spectrum possesses a shoulder at about
Fig. 5D represents the M decay and the
parent state recovery kinetics at 4 °C, with

1/e of about 45 sec (43.1 sec at 395 nm and
48.7 sec at 492 nm). It should be noted that
the initial absorption at 492 nm does not fully
recover after the complete decay of the M
intermediate indicating some irreversible
processes. We infer that Rh-PDE in detergent
tends to form aggregates (and/or partially
bleaches) upon continuous illumination,
which does not occur in membranes judging
from the perfect repeatability of the in-cell
Rh-PDE activation (Fig. 3C). To estimate the
M decay kinetics under the conditions of the
enzyme activity experiments (27°C), we
measured its temperature dependence. As
4
60 nm, which resembles that of sensory
rhodopsin II (SRII) (33). The absorption at
80 nm is 4.8 times higher than that at 492
2
nm, which is significantly higher than the
value of ~2 known for most microbial
rhodopsins. Among the possible reasons is
the additional large extramembrane domain,
incomplete chromophore regeneration or
bleaching, and/or co-purified proteins other
than Rh-GC (see Materials and Methods for
details). Upon the irradiation, the absorption
peak at 492 nm decreases and a new
9

Rhodopsin-PDE
expected, the M decay becomes faster at
activation mechanism is unknown and
higher temperatures (Fig. 5E), and the
intriguing. Above, we presented the data
temperature
dependence
follows
the
showing
that
Rh-PDE
exhibits
Arrhenius behaviour well (Fig. 5F), with the
activation energy (Ea) of 57.8 kJ/mol, and
activation enthalpy (H) and entropy (S) of
light-dependent hydrolysis activity both
toward cGMP and cAMP while retaining
some levels of constitutive activity in the
dark. The data in Figure 4 shows that
Rh-PDE has higher activity toward cGMP
than toward cAMP, both in the dark and in
the light-activated form. It is known that a
substrate specificity differs among PDE
families as shown in figure 1D (28). The
observed dark activity was not anticipated, as
the light-sensitive rhodopsin domain was
expected to contribute to the tight regulation
of the enzymatic activity. In fact, the Rh-GC
exhibits a very tight light-dependent cyclase
activity, in which the enzyme has only 1 %
basal activity in the dark (12). Whether or not
the high constitutive activity of Rh-PDE is
derived from the background activity of the
PDE domain, or from a specific conformation
of the rhodopsin part, or from instability of
the oligomer in the heterologous expression
system will have to be addressed in the future
studies. At this point, we believe that it is
logical to suggest that the membrane and
cellular environments differ between
Salpingoeca rosetta and HEK293 cells,
preventing hydrolytic activity in the dark in
the former. For example, it is possible that
the PDE domain is post-translationally
5
5.3 kJ/mol and -76.5 J/mol•K, respectively.
We estimated the M decay time constant at
7 °C to be 6.78 sec, which suggests that the
2
enzymatic activity persists well after its
decay.
We co-expressed the 1D4-tagged Rh-PDE
and its eGFP-tagged version in HEK293 cells
followed by solubilisation and purification by
1D4 agarose resin. The eluate contained not
only the 1D4-tagged protein but also the
eGFP-tagged version, judging from the
fluorescence spectrum and SDS-PAGE (Fig.
S4). This result indicates that Rh-PDE forms
stable oligomers which survive solubilisation.
DISCUSSION
We report on a novel type of microbial
rhodopsin
from
the
choanoflagellate
Salpingoeca rosetta containing a C-terminal
PDE domain. Animal rhodopsins and sensory
microbial rhodopsins have their respective
transducers (G proteins, Htr membrane
transducers, and others) as separate proteins
(
1–8). In contrast, enzyme rhodopsins such as
Rh-HK, Rh-GC, and Rh-PDE contain
covalently bound enzymatic domains, whose
1
0

Rhodopsin-PDE
modified
(phosphorylated)
or
other
PDE-2A, where the elongated C-terminal
regulatory protein(s) associate with it and
keep Rh-PDE inactive in Salpingoeca rosetta
in the dark.
helix of the GAF domain directly connects to
the catalytic region of the PDE domain (30)
may offer some insights into the architecture
of Rh-PDE. In Rh-PDE, the linker region
between the end of transmembrane helix 7 of
the rhodopsin domain and the beginning of
the catalytic region contains 50-60 residues
(Supplementary Fig. S1), which corresponds
to 7-8 nm in length assuming a single helix.
On the other hand, it is well known that
HAMP (Histidine kinases, Adenylyl cyclases,
Methyl-accepting chemotaxis proteins, and
Phosphatases) domain, which connects
transmembrane and cytosolic signaling
domains in many sensory proteins, forms a
coiled-coil bundle of two amphipathic helices
(34, 35). Indeed, such two helical
amphipathic sequences can be seen in the
linker region of Rh-PDE (Supplementary Fig.
During the in-cell cyclic nucleotides
concentration measurements by Glo-sensor
assay, the light-dependent cAMP decrease
was observed (Figure 2), consistent with the
results obtained with crude membranes
shown in Fig. 4. In contrast, we did not
observe any light-dependent change of the
intracellular cGMP level in Glo-sensor assay
in Figure 2A, but observed an enhancement
of the Rh-PDE cGMP activity in crude
membranes by light. This can be explained
taking into the account that cGMP hydrolysis
rate by Rh-PDE is about 10 fold faster than
that for cAMP (Fig. 4C and F). Thus, the
dark activity toward cGMP is high enough to
completely
degrade
cellular
cGMP,
S1).
Therefore,
we
suggest
that
preventing observation of the effects of light.
In addition, concentration of endogenous
cGMP is about 10 fold lower than that of
cAMP, which is another plausible reason for
our observations.
transmembrane helix 7 of the Rh domain
elongates and forms the HAMP-like domain,
which connects to the catalytic region of the
PDE domain (Fig. 6). Dimer formation of the
PDE domain makes the catalytic region
inaccessible to the cytoplasmic bulk, so that
the PDE activity remains shut off in the dark
(the condition which may not be fully
reproduced in our mammalian cells).
Based on the presented results, we
propose the activation mechanism of Rh-PDE
as shown in Fig. 6. We suggest that Rh-PDE
exists in a putative dimeric form, consistent
with the reported PDE structures (21, 22).
The crystal structure of the full-length
The resting state of Rh-PDE shows the
max at 492 nm, which coincides well with
11

Rhodopsin-PDE
the action spectrum of the light-induced
enzymatic activity (Fig. 5B). The absorption
in the visible region indicates that the
chromophore Schiff base is protonated in
Rh-PDE. In light-driven proton pump
bacteriorhodopsin (BR), the protonated
Schiff base, R82, D85, and D212 constitute a
quadrupole inside the protein, where D85
acts as a primary counterion of the Schiff
base (1). Similarly, the Schiff base, R161,
E164, and D292 probably form a quadrupole
in Rh-PDE (Fig. 1C). The absorption
spectrum is considerably blue-shifted
compared to other microbial rhodopsins, and
is close to those of SRII and
channelrhodopsin-2 (ChR2). The position
corresponding to A215 in BR is T in SRII,
whose side chain O-H group contributes to
the blue spectral shift (36, 37). ChR2 has O-H
bearing S at the homologous position as well,
but Rh-PDE has A (Fig. 1C), so that the
mechanism of its blue-shifted absorption is
not clear.
changes at the cytoplasmic surface of the
rhodopsin domain, which results in the
light-induced increase in PDE activity. The
observed increase in the in-cell enzymatic
activity toward cAMP for ~10 sec after the
light was switched off (Fig. 2C) coincides
with the timescale of the M decay (7 sec; Fig.
5F), suggesting that the enzymatic activity is
not yet induced in the PDE-domain in the M
intermediate. In other words, structural
changes of the Rh-domain in the M
intermediate lead to those of the PDE-domain
at the later times after the photoactivation,
presumably altering the structure of helix 7
and the HAMP-like domain (Fig. 6). Linear
light intensity dependence of the activity
toward cAMP (Fig. 2E) suggests that
activation of just one Rh-domain in a putative
dimer is sufficient to trigger an increase in
the enzymatic activity. An alternative (even
though less plausible) explanation for the
linear intensity dependence is that Rh-PDE is
monomeric in the mammalian membranes,
which could also explain its significant dark
activity, even though this contradicts our
results with eGFP tagged Rh-PDE, which
suggest oligomerization (Fig. S4).
After the photoexcitation, the
M
intermediate with deprotonated retinal Schiff
base forms in the Rh-PDE photocycle (Fig.
5
A and C), similar to many other microbial
and animal rhodopsins. In many cases, proton
transfer from the Schiff base induces large
structural changes, leading to the respective
functional processes (1). In Rh-PDE, the M
formation probably triggers protein structural
The recovery of the original spectral state
occurs parallel to the M decay (Fig. 5D),
suggesting that the photocycle completes in
~7 sec at 27 °C (~45 sec at 4 °C). This fact
appears to contradict the long-lived
1
2

Rhodopsin-PDE
enzymatic activity (71 sec) of PDE in cells
Fig. 2C and Fig. S3). As the light absorption
half of helix F in SRII, which causes
(
rotational
motion
of
the
second
originates from the retinal chromophore in
the membrane core of the Rh domain, it may
be not sensitive to the conformation of the
PDE domain, which probably switches on
upon the transition from the M intermediate
to the initial spectral state in the Rh domain.
Structural changes of the Rh-domain,
particularly in helix 7 (and ensuing changes
in the HAMP-like domain), possibly
dissociate the PDE dimer, leading to the two
catalytic domains being exposed to the
aqueous phase (Fig. 6). The photocycles of
microbial rhodopsins are generally too short
to keep sustained enzymatic activity, so that a
special mechanism is needed to achieve this.
The presence of the two domains, Rh and
PDE, connected through the HAMP-like
linker could allow for 10 times longer
enzymatic activity than the photocycle of the
Rh domain alone.
transmembrane helix of the transducer.
Outward motion of helix F on the
cytoplasmic side is common for activation of
microbial and animal rhodopsins, and also
causes motion of helix G. Presently, there is
no structural information on Rh-PDE, and
further studies will be needed to reveal the
activation mechanism of the PDE domain by
light absorption in the Rh domain.
Light control of second messengers is in
high demand in optogenetics, and PAC
(22–25) and Rh-GC (12, 13) are promising
tools
for
increasing
intracellular
concentration of cyclic nucleotides by light.
In addition to the engineered light-activated
PDE (26) and mosquito Opn3 (27), Rh-PDE
may become a promising optogenetics tool,
but all of them have their specific drawbacks.
The enzymatic activity of the engineered
light-activated PDE has to be stopped by
additional irradiation of different color due to
the photochromic nature of the GAF domain.
Although the chromophore of mosquito Opn3
is 11-cis retinal, not abundant in mammalian
cells, it can be used for optogenetics because
it can bind 13-cis retinal. Mosquito Opn3 is
more sensitive and has longer activation time
than Rh-PDE, because of the amplification
processes. In addition, mosquito Opn3
regulates other signalling cascades through
Molecular
mechanism
of
the
light-activated sensory function has been
extensively studied for SRII, which forms a
2
:2 complex with two transmembrane helical
Htr transducer (38, 39). The linker region of
the transducer contains a HAMP-like domain.
The M intermediate structure of the
receptor-transducer complex (40) and
numerous EPR studies (41, 42) revealed
significant outward tilting of the cytoplasmic
1
3

Rhodopsin-PDE
G
 and arrestin, triggering complex
synthesized
after
a
human
codon
intracellular events. In contrast, Rh-PDE
regulates only catalysis of cyclic nucleotides
hydrolysis. While Rh-PDE lowers cAMP
concentration by light, its constitutive
activity would be problematic for optogenetic
applications, as Rh-PDE maintains very low
intracellular cGMP levels. This requires
further efforts to make it suitable for
optogenetic applications, and additional
engineering of the active center of the PDE
domain is possibly needed.
optimization (Gen Script) and fused to the
cDNA encoding a venus fluorescent protein
and cloned into pCDNA3.1 vector between
HindIII and XbaI sites. All constructs were
verified by the DNA sequencing. Expression
of Rh-PDE with 1D4 epitope tag was
confirmed by Western blotting.
Assay of the enzymatic activity of
Rh-PDE in mammalian cells. HEK293 cells
were purchased from JCRB Cell Bank and
cultured in E-MEM media with L-Glutamine
and Phenol Red (Wako) containing 10 %
(vol/vol) FBS and penicillin-streptomycin.
The cells were co-transfected with the PDE
EXPERIMENTAL PROCEDURES
Molecular biology. A full-length gene
encoding Rh-PDE (NCBI Gene ID:
or
MosOpn3
plasmid
and
the
1
6078606) was synthesized after a human
pGloSensor-42F cGMP or pGloSensor-22F
cAMP vector (Promega) by using
Lipofectamine 2000 (Invitrogen). Changes in
codon optimization (Gen Script) and cloned
into peGFP-N1 vector between HindIII and
BamHI sites. We amplified Rh-PDE gene by
PCR with the forward primer encoding the
first 20 bp and the reverse primer encoding
the last 15 bp of Rh-PDE gene followed by
the rho 1D4 epitope-tag (TETSQVAPA)
sequence. The amplified fragment was
inserted into pCDNA3.1 vector (Invitrogen)
between HindIII and XbaI sites. The plasmid
for D605A Rh-PDE was generated by
QuikChange Site-Directed Mutagenesis Kit
the
intracellular
cGMP
or
cAMP
concentration of the HEK293 cells were
measured by using the GloSensor assay
(Promega). The transfected cells were
incubated with or without 0.5 M
all-trans-retinal
(Toronto
Research
Chemicals). Before measurements, the
culture medium was replaced with a
2
CO -independent medium containing 10 %
(vol/vol) FBS and 2 % (vol/vol) GloSensor
cAMP or cGMP stock solution (Promega).
Cells were then incubated for 2 hours at room
(
Agilent Technologies). MosOpn3 gene that
lacks 99 C-terminal amino acids (27) was
1
4

Rhodopsin-PDE
temperature in the dark. Intracellular cGMP
or cAMP level was observed by monitoring
luminescence by using a microplate reader
buffer
A
and
homogenized
using
Potter-Elehjem Grinder (Wheaton) and a
syringe with a 27G needle. The syringe was
filled and drained 5 times while stirring the
homogenate. Protein amount was determined
by BCA protein assay (Thermo Fisher
Scientific). Samples were kept in the dark
before measurement for at least 2 hours.
Catalytic activity was measured at room
temperature in 100 L buffer A with 1.6-1.8
g (in the case of cGMP) or 16-18 g (in the
case of cAMP) of total protein in a 1.5 ml
sample tube. The sample was illuminated
with a xenon lamp (MAX-303, Asahi Spectra
Co., Ltd., Japan) through Y52 filter (7 mW
(
Corona Electric) at 27 °C. The cells were
treated with 100 M sodium nitroprusside
SNP) (Wako), direct activator of
(
a
endogenous guanylyl cyclase, to elevate
intracellular cGMP level. Alternatively, the
cells were treated with 3.5 M forskolin
(Wako), a direct activator of adenylyl cyclase,
to elevate intracellular cAMP level. The cells
were illuminated with a xenon lamp
(
LAX-103, Asahi Spectra Co., Ltd., Japan)
through interference filter (420, 464, 487,
10 and 550 nm). The light intensity was
5
-
2
mm ). Reaction was initiated by adding
cyclic nucleotides (final concentration 100
adjusted to desired values by a variable ND
filter mounted in the LAX-103. The light
intensity was measured by a power meter
LP1 (Sanwa Electric Instruments Co., Ltd.,
Japan).

M). Aliquots were taken out at different
time points and the reactions were
immediately terminated by adding 100 L of
0
.1 N HCl and frozen in liquid nitrogen.
HPLC analysis for in vitro assay of the
enzymatic activity of Rh-PDE. HEK293T
cells were transfected with plasmid
pCDNA3.1-Rh-PDE by calcium phosphate
method. The DMEM/F-12 media contained
After thawing, the samples were centrifuged
to remove the membranes and denatured
proteins. Nucleotides (20 L of aliquot) were
separated by HPLC (Shimadzu systems) with
C18 reversed-phase column (Waters) and 100
mM potassium phosphate (pH 5.9), 4 mM
tetrabutylammonium iodide, and 10 %
(vol/vol) methanol as eluent (12, 26).
Nucleotides were monitored at 254 nm. Data
0
.5 M all-trans-retinal and penicillin and
streptomycin. The cells were harvested after
4 hours and washed in buffer A (140 mM
NaCl, 3 mM MgCl , 50 mM HEPES-NaOH,
pH 6.5). The cells were resuspended with
2
2
were
evaluated
with
LabSolutions
1
5

Rhodopsin-PDE
(
Shimadzu). Peak areas were integrated and
Estimation of chromophore regeneration.
assigned to the educt cyclic nucleotide based
on retention times of corresponding standard
compound.
The extinction coefficient of the full-length
Rh-PDE protein was determined as 106,340
-1
-1
M cm based on the protein sequence by
using ProtParam tool of Expasy (43). The
absorption change, representing the bleaching
of a purified Rh-PDE by hydroxylamine
Purification of Rh-PDE. HEK293T cells
expressing Rh-PDE, described above, were
harvested after 24 hours and homogenized by
dounce tissue grinder (Wheaton). Membrane
proteins were then extracted with 2%
(HA), was measured with
a
UV-vis
spectrometer (Shimadzu, Japan) equipped
with an integrating sphere after the addition
of HA to a final concentration of 500 mM.
The molecular extinction coefficient of the
chromophore in Rh-PDE () was determined
(
(
weight/vol) Dodecyl-β-D-maltopyranoside
DDM) in buffer A, followed by
o
centrifugation (6,500 g, 1 hour, 4 C) to
remove unsolubilized fractions. The extract
was mixed with 1D4-agarose resin and
incubated at room temperature for 15-30 min.
After washing the resin with buffer A
containing 0.02% (weight/vol) DDM (buffer
B), proteins were eluted with buffer B
containing 0.11-0.18 mg/mL 1D4 peptide.
Protein fractions were concentrated with
Amicon Ultra 30K centrifugal devices
-1
-1
as 33,710 M cm from the ratio between the
absorbance of Rh-PDE at 492 nm and retinal
-1
-1
oxime ( = 33,600 M cm at 360 nm (44))
produced by the reaction between retinal
Schiff base and HA. From the values above
and the absorptions at 280 nm and 492 nm in
Figure 5A (black spectrum), the chromophore
regeneration extent was estimated.
(
Millipore).
Phylogenetic analysis of rhodopsin
genes. The amino acid sequences of
Spectroscopy.
illuminated with 1 kW tungsten-halogen
projector lamp (Master HILUX-HR,
The samples were
transmembrane
domains
of
selected
rhodopsins (corresponding to residues 9-234
of bacteriorhodopsin) have been aligned
using CLUSTALO (45) and the resulting
phylogenetic trees were visualized with
TreeView (46).
Rikagaku) through Y52 filter (>500 nm) and
the absorption spectra of Rh-PDE were
recorded at various temperatures by using
JASCO V-650 spectrometer.
1
6

Rhodopsin-PDE
Acknowledgements
We thank Dr. Shoko Hososhima, Mizuna Tanaka and Shunta Nakamura for helpful discussions.
Coflict of interest
The authors declare that they have no conflicts of interest with the contents of this article.
Author contributions
S.P.T., L.S.B. and H.K. contributed to the study design. S.P.T. designed the construct. K.Y.
conducted all measurements. S.P.T., L.S.B and H.K wrote the paper. All authors discussed and
commented on the manuscript.
1
7

Rhodopsin-PDE
REFERENCES
1
.
Ernst, O. P., Lodowski, D. T., Elstner, M., Hegemann, P., Brown, L. S., and Kandori, H. (2014)
Microbial and animal rhodopsins: Structures, functions, and molecular mechanisms. Chem. Rev.
1
14, 126–163
2
3
.
.
Palczewski, K. (2006) G Protein – Coupled Receptor Rhodopsin. 75, 743–767
Shichida, Y., and Morizumi, T. (2007) Mechanism of G-protein activation by rhodopsin.
Photochem. Photobiol. 83, 70–5
4
.
Hofmann, K. P., Scheerer, P., Hildebrand, P. W., Choe, H. W., Park, J. H., Heck, M., and Ernst,
O. P. (2009) A G protein-coupled receptor at work: the rhodopsin model. Trends Biochem. Sci.
3
4, 540–552
5
6
7
.
.
.
Koyanagi, M., and Terakita, A. (2014) Diversity of animal opsin-based pigments and their
optogenetic potential. Biochim. Biophys. Acta - Bioenerg. 1837, 710–716
Jung, K. H., Trivedi, V. D., and Spudich, J. L. (2003) Demonstration of a sensory rhodopsin in
eubacteria. Mol. Microbiol. 47, 1513–1522
Grote, M., Engelhard, M., and Hegemann, P. (2014) Of ion pumps, sensors and channels -
Perspectives on microbial rhodopsins between science and history. Biochim. Biophys. Acta -
Bioenerg. 1837, 533–545
8
9
1
.
.
Brown, L. S. (2014) Eubacterial rhodopsins - Unique photosensors and diverse ion pumps.
Biochim. Biophys. Acta - Bioenerg. 1837, 553–561
Inoue, K., Kato, Y., and Kandori, H. (2015) Light-driven ion-translocating rhodopsins in marine
bacteria. Trends Microbiol. 23, 91–98
0. Luck, M., Mathes, T., Bruun, S., Fudim, R., Hagedorn, R., Tran Nguyen, T. M., Kateriya, S.,
Kennis, J. T. M., Hildebrandt, P., and Hegemann, P. (2012) A photochromic histidine kinase
rhodopsin (HKR1) that is bimodally switched by ultraviolet and blue light. J. Biol. Chem. 287,
4
0083–40090
1. Avelar, G. M., Schumacher, R. I., Zaini, P. A., Leonard, G., Richards, T. A., and Gomes, S. L.
2014) A Rhodopsin-Guanylyl cyclase gene fusion functions in visual perception in a fungus.
1
1
1
(
Curr. Biol. 24, 1234–1240
2. Scheib, U., Stehfest, K., Gee, C. E., Körschen, H. G., Fudim, R., Oertner, T. G., and Hegemann,
P. (2015) The rhodopsin – guanylyl cyclase of the aquatic fungus Blastocladiella emersonii
enables fast optical control of cGMP signaling. Sci. Signal. 8, 1–9
3. Gao, S., Nagpal, J., Schneider, M. W., Kozjak-Pavlovic, V., Nagel, G., and Gottschalk, A.
(
2015) Optogenetic manipulation of cGMP in cells and animals by the tightly light-regulated
guanylyl-cyclase opsin CyclOp. Nat. Commun. 6, 8046
14. Deisseroth, K. (2011) Optogenetics. Nat. Methods. 8, 26–29
15. Yizhar, O., Fenno, L. E., Davidson, T. J., Mogri, M., and Deisseroth, K. (2011) Optogenetics in
1
8

Rhodopsin-PDE
Neural Systems. Neuron. 71, 9–34
6. Gero Miesenböck (2011) Optogenetic control of cells and circuits. Annu. Rev. Cell Dev. Biol. 27,
31–758
1
1
7
7. Zhang, F., Vierock, J., Yizhar, O., Fenno, L. E., Tsunoda, S., Kianianmomeni, A., Prigge, M.,
Berndt, A., Cushman, J., Polle, J., Magnuson, J., Hegemann, P., and Deisseroth, K. (2011) The
microbial opsin family of optogenetic tools. Cell. 147, 1446–1457
18. Dugué, G. P., Akemann, W., and Knöpfel, T. (2012) A comprehensive concept of optogenetics.
Prog. Brain Res. 196, 1–28
19. Boyden, E. S., Zhang, F., Bamberg, E., Nagel, G., and Deisseroth, K. (2005)
Millisecond-timescale, genetically targeted optical control of neural activity. Nat. Neurosci. 8,
1
263–8
2
2
2
2
2
0. Zhang, F., Wang, L.-P., Brauner, M., Liewald, J. F., Kay, K., Watzke, N., Wood, P. G., Bamberg,
E., Nagel, G., Gottschalk, A., and Deisseroth, K. (2007) Multimodal fast optical interrogation of
neural circuitry. Nature. 446, 633–639
1. Iseki, M., Matsunaga, S., Murakami, A., Ohno, K., Shiga, K., Yoshida, K., Sugai, M., Takahashi,
T., Hori, T., and Watanabe, M. (2002) A blue-light-activated adenylyl cyclase mediates
photoavoidance in Euglena gracilis. Nature. 415, 1047–1051
2. Schröder-Lang, S., Schwärzel, M., Seifert, R., Strünker, T., Kateriya, S., Looser, J., Watanabe,
M., Kaupp, U. B., Hegemann, P., and Nagel, G. (2007) Fast manipulation of cellular cAMP level
by light in vivo. Nat. Methods. 4, 39–42
3. Ryu, M. H., Moskvin, O. V., Siltberg-Liberles, J., and Gomelsky, M. (2010) Natural and
engineered photoactivated nucleotidyl cyclases for optogenetic applications. J. Biol. Chem. 285,
4
1501–41508
4. Stierl, M., Stumpf, P., Udwari, D., Gueta, R., Hagedorn, R., Losi, A., Gärtner, W., Petereit, L.,
Efetova, M., Schwarzel, M., Oertner, T. G., Nagel, G., and Hegemann, P. (2011) Light
modulation of cellular cAMP by a small bacterial photoactivated adenylyl cyclase, bPAC, of the
soil bacterium Beggiatoa. J. Biol. Chem. 286, 1181–1188
2
5. Costa, W. S., Liewald, J., and Gottschalk, A. (2014) Photoactivated adenylyl cyclases as
optogenetic modulators of neuronal activity. Methods Mol. Biol. 1148, 161–175
6. Gasser, C., Taiber, S., Yeh, C.-M., Wittig, C. H., Hegemann, P., Ryu, S., Wunder, F., and
Moglich, A. (2014) Engineering of a red-light-activated human cAMP/cGMP-specific
phosphodiesterase. Proc. Natl. Acad. Sci. U. S. A. 111, 8803–8808
2
2
7. Koyanagi, M., Takada, E., Nagata, T., Tsukamoto, H., and Terakita, A. (2013) Homologs of
vertebrate Opn3 potentially serve as a light sensor in nonphotoreceptive tissue. Proc. Natl. Acad.
Sci. U. S. A. 110, 4998–5003
28. Bender, A. T., and Beavo, J. A. (2006) Cyclic nucleotide phosphodiesterases: molecular
1
9

Rhodopsin-PDE
regulation to clinical use. Pharmacol. Rev. 58, 488–520
29. Francis, S. H., Blount, M. a, and Corbin, J. D. (2011) Mammalian cyclic nucleotide
phosphodiesterases: molecular mechanisms and physiological functions. Physiol. Rev. 91,
51–690
6
3
0. Pandit, J., Forman, M. D., Fennell, K. F., Dillman, K. S., and Menniti, F. S. (2009) Mechanism
for the allosteric regulation of phosphodiesterase 2A deduced from the X-ray structure of a near
full-length construct. Proc. Natl. Acad. Sci. U. S. A. 106, 18225–30
3
3
3
1. Katayama, K., Furutani, Y., Imai, H., and Kandori, H. (2010) An FTIR study of monkey green-
And red-sensitive visual pigments. Angew. Chemie - Int. Ed. 49, 891–894
2. Katayama, K., Furutani, Y., Imai, H., and Kandori, H. (2012) Protein-bound water molecules in
primate red- and green-sensitive visual pigments. Biochemistry. 51, 1126–1133
3. Shimono, K., Ikeura, Y., Sudo, Y., Iwamoto, M., and Kamo, N. (2001) Environment around the
chromophore in pharaonis phoborhodopsin: Mutation analysis of the retinal binding site.
Biochim. Biophys. Acta - Biomembr. 1515, 92–100
3
3
3
3
3
4. Bordignon, E., Klare, J. P., Doebber, M., Wegener, A. A., Martell, S., Engelhard, M., and
Steinhoff, H. J. (2005) Structural analysis of a HAMP domain: The linker region of the
phototransducer in complex with sensory rhodopsin II. J. Biol. Chem. 280, 38767–38775
5. Hulko, M., Berndt, F., Gruber, M., Linder, J. U., Truffault, V., Schultz, A., Martin, J., Schultz, J.
E., Lupas, A. N., and Coles, M. (2006) The HAMP Domain Structure Implies Helix Rotation in
Transmembrane Signaling. Cell. 126, 929–940
6. Shimono, K., Iwamoto, M., Sumi, M., and Kamo, N. (2000) Effects of three characteristic amino
acid residues of pharaonis phoborhodopsin on the absorption maximum. Photochem. Photobiol.
7
2, 141–5
7. Shimono, K., Hayashi, T., Ikeura, Y., Sudo, Y., Iwamoto, M., and Kamo, N. (2003) Importance
of the broad regional interaction for spectral tuning in Natronobacterium pharaonis
phoborhodopsin (sensory rhodopsin II). J. Biol. Chem. 278, 23882–9
8. Gordeliy, V. I., Labahn, J., Moukhametzianov, R., Efremov, R., Granzin, J., Schlesinger, R.,
Büldt, G., Savopol, T., Scheidig, A. J., Klare, J. P., and Engelhard, M. (2002) Molecular basis of
transmembrane signalling by sensory rhodopsin II-transducer complex. Nature. 419, 484–487
9. Klare, J. P., Bordignon, E., Engelhard, M., and Steinhoff, H.-J. (2004) Sensory rhodopsin II and
bacteriorhodopsin: light activated helix F movement. Photochem. Photobiol. Sci. 3, 543–547
0. Moukhametzianov, R., Klare, J. P., Efremov, R., Baeken, C., Goppner, A., Labahn, J., Engelhard,
M., Buldt, G., and Gordeliy, V. I. (2006) Development of the signal in sensory rhodopsin and its
transfer to the cognate transducer. Nature. 440, 115–119
3
4
41. Wegener, A. A., Chizhov, I., Engelhard, M., and Steinhoff, H. J. (2000) Time-resolved detection
of transient movement of helix F in spin-labelled pharaonis sensory rhodopsin II. J Mol Biol.
2
0

Rhodopsin-PDE
3
01, 881–891
4
4
4
4
2. Wegener, A. A., Klare, J. P., Engelhard, M., and Steinhoff, H. J. (2001) Structural insights into
the early steps of receptor-transducer signal transfer in archaeal phototaxis. EMBO J. 20,
5
312–5319
3. Gasteiger, E., Gattiker, A., Hoogland, C., Ivanyi, I., Appel, R. D., and Bairoch, A. (2003)
ExPASy: The proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res.
3
1, 3784–3788
4. Scharf, B., Pevec, B., Hess, B., and Engelhard, M. (1992) Biochemical and photochemical
properties of the photophobic receptors from Halobacterium halobium and Natronobacterium
pharaonis. Eur. J. Biochem. 206, 359–366
5. Sievers, F., Wilm, A., Dineen, D., Gibson, T. J., Karplus, K., Li, W., Lopez, R., McWilliam, H.,
Remmert, M., Söding, J., Thompson, J. D., and Higgins, D. G. (2011) Fast, scalable generation
of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7,
5
39
46. Page, R. D. M. (2002) Visualizing phylogenetic trees using TreeView. Curr. Protoc.
Bioinformatics. Chapter 6, Unit 6.2
FOOTNOTES
This work was financially supported by grants from the Japanese Ministry of Education, Culture,
Sports, Science and Technology to H.K. (25104009, 15H02391) and by PRESTO, Japan Science and
Technology Agency to SPT.
FIGURE LEGENDS
FIGURE 1 Architecture and amino acid composition of Rh-PDE. A, Rh-PDE is composed of the
7
transmembrane helical rhodopsin domain and the cytoplasmic PDE domain. B, Phylogenetic tree of
selected microbial rhodopsins based on the sequences of the transmembrane domains. C, Conservation
of important residues of the Rh domain. Red and blue colors represent acidic and basic amino acids,
respectively. Residue numbers in Rh-PDE and bacteriorhodopsin (BR) are shown. D, Conservation of
important residues of the PDE domain. Residue numbers in Rh-PDE and PDE10A are shown,
2
1

Rhodopsin-PDE
sequences are grouped according to the substrate specificity.
FIGURE 2 Enzymatic activity of Rh-PDE in HEK293 cells. A, Luminescence signals
representing cGMP levels, as observed in HEK293 cells with the empty vector (top panel), Rh-PDE
(
middle panel), and the PDE-inactive D605A mutant (bottom panel). HEK293 cells were
pre-incubated in the culture medium with all-trans-retinal in the dark. The arrows and vertical
-2
light-blue lines indicate SNP treatments and irradiations at 510 nm with 2.28 W mm intensity,
respectively, and the luminescence signals of non-irradiated cells are shown as a control (black lines).
B, Luminescence signals representing cAMP level, as observed in HEK293 cells with the empty
vector (top panel), Rh-PDE (middle panel), and the PDE-inactive D605A mutant (bottom panel).
HEK293 cells were pre-incubated in the culture medium with all-trans-retinal in the dark. The arrows
and vertical light-blue lines indicate forskolin treatments and irradiations at 510 nm with 2.28 W
-2
mm intensity, respectively, and the luminescence signals of non-irradiated cells are shown as a
control (black lines). Only HEK293 cells expressing Rh-PDE (red line in the middle panel) exhibited
decrease of the luminescence signal, indicating that cAMP concentration is lowered by light. C,
Changes of luminescence signals upon 2 min 510 nm irradiation (light-blue line) of HEK293 cells
with the empty vector (top panel), Rh-PDE (middle panel), the PDE-inactive D605A mutant (middle),
expanded from B. Colored and black lines represent the data with and without irradiation, respectively.
D, Intensity dependence of the light-induced cAMP concentration decrease by Rh-PDE (cells were
-2
irradiated for 2 min with 510 nm light with 0-2.28 W mm intensities). E, Peak amplitudes of
luminescence decrease plotted against the light intensity (N ≧ 5 cells). All error bars are given as SD.
-2
The half maximal light intensity (EC50) was determined to be 0.244 W mm by a single exponential
decay fit (red dashed line).
2
2

Rhodopsin-PDE
FIGURE 3 Rh-PDE as a potential optogenetic tool. A, Comparison of in-cell hydrolytic
activities of Rh-PDE (top) and mosquito Opn3 (bottom) toward cAMP. The top panel is reproduced
from Fig. 2c, mosquito Opn3 was expressed in HEK293 cells similar to Rh-PDE. During the time
-
2
period shown in light blue, 510 nm light with 2.28 W mm intensity was applied to the cells. B,
Effect of addition of all-trans-retinal on the Rh-PDE activity. C, Repeatability of the in-cell Rh-PDE
-2
activation. 2 min irradiation at 510 nm with 2.28 W mm intensity was repeated every 15 min, other
conditions are as in Fig. 2C. D, Irradiation wavelength dependence of the in-cell activity of Rh-PDE
-
2
toward cAMP. All the data were obtained from experiments at the same light intensity (0.4 W mm ).
FIGURE 4 HPLC analysis of the enzymatic activity of Rh-PDE. A, HEK293 cell membranes
expressing Rh-PDE are incubated with 100 M cGMP for 10 sec (purple), 30 sec (light blue), 1 min
(green), and 2 min (red) in the dark (top) and under the illumination (bottom), and the reaction
products are analyzed by HPLC. Black dotted line represents the profile for 100 M cGMP without
Rh-PDE. B, Time course of the decrease of cGMP concentration in the dark (black) and under the
illumination (blue) (N=3). C, cGMP hydrolysis activity in the dark and under the illumination
calculated from the panel B. D, HEK293 cell membranes expressing Rh-PDE are incubated with 100
M cAMP for 2 min (purple), 5 min (light blue), 10 min (green), and 20 min (red) in the dark (top)
and under the illumination (bottom), and the reaction products are analyzed by HPLC. Black dotted
line represents the profile for 100 M cAMP without Rh-PDE. E, Time course of the decrease of
cAMP concentration in the dark (black) and under the illumination (red) (N=3). F, cAMP hydrolysis
activity in the dark and under the illumination calculated from the panel E. The error bars represent
SD.
2
3

Rhodopsin-PDE
FIGURE 5 Molecular properties of purified full-length Rh-PDE. A, Absorption spectra of
full-length Rh-PDE in the dark (black), and after irradiation with 510 nm light (red) at 4 °C. Inset;
CBB-stained purified Rh-PDE on 12 % SDS-PAGE. The arrow corresponds to about 70 kDa. B,
Absorption spectrum of purified full-length Rh-PDE (dotted line; reproduced from the black line in
Fig. 5A) superimposed with the action spectrum of the light-induced enzymatic activity toward cAMP
in HEK293 cells (the peak amplitudes in Fig. 3D are plotted versus irradiation wavelength with 0.4
-2

W mm intensity). C, Difference absorption spectra of the photoactivation. D, The M decay and the
parent state recovery kinetics at 4 °C. E, Temperature dependence of the M decay. F, Arrhenius plot
for the M decay kinetics. From the linear fit (dotted line), the decay time constant at 27 °C is
estimated to be 6.78 sec (red point). The error bars are given as SD.
FIGURE 6 Schematic representation of the Rh-PDE activation mechanism. The model shows
a putative Rh-PDE dimer. In the dark, Rh-PDE exhibits constitutive activity toward cGMP and cAMP,
suggesting that the catalytic region is somewhat exposed to the cytoplasm in mammalian cells. Upon
absorption of blue-green light by the Rh-domain, photoisomerization of the retinal chromophore from
the all-trans (ATR) to 13-cis form produces the M intermediate with deprotonated Schiff base
(
Rh-domain Switch-ON), but the PDE-domain has not changed yet (PDE-domain Switch-OFF). Next,
the M intermediate decays to the parent spectral state in 7 sec (Rh-domain Switch-OFF), accompanied
by the structural changes in the PDE domain (PDE-domain Switch-ON). The resulting increased
enzymatic activity is turned off within ~70 sec (PDE-domain Switch-OFF).
2
4

Figure 1
A
B
C
D
Yoshida et al.

Figure 2
A
B
C
D
E
Yoshida et al.

Figure 3
A
C
B
D
Yoshida et al.

A
D
B
E
C
F
Yoshida et al.

Figure 5
A
D
B
E
C
F
Yoshida et al.

Figure 6
Yoshida et al.