EUKARYOTIC CELL, Oct. 2005, p. 1605–1612 Vol. 4, No.

10
1535-9778/05/$08.00⫹0 doi:10.1128/EC.4.10.1605–1612.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Photoreceptor for Curling Behavior in Peranema trichophorum and

Evolution of Eukaryotic Rhodopsins
Jureepan Saranak and Kenneth W. Foster*
Physics Department, Syracuse University, 201 Physics Building, Syracuse, New York 13244-1130
Received 11 November 2004/Accepted 18 July 2005

When it is gliding, the unicellular euglenoid Peranema trichophorum uses activation of the photoreceptor
rhodopsin to control the probability of its curling behavior. From the curled state, the cell takes off in a new
direction. In a similar manner, archaea such as Halobacterium use light activation of bacterio- and sensory
rhodopsins to control the probability of reversal of the rotation direction of flagella. Each reversal causes the
cell to change its direction. In neither case does the cell track light, as known for the rhodopsin-dependent
eukaryotic phototaxis of fungi, green algae, cryptomonads, dinoflagellates, and animal larvae. Rhodopsin was
identified in Peranema by its native action spectrum (peak at 2.43 eV or 510 nm) and by the shifted spectrum
(peak at 3.73 eV or 332 nm) upon replacement of the native chromophore with the retinal analog n-hexenal.
The in vivo physiological activity of n-hexenal incorporated to become a chromophore also demonstrates that
charge redistribution of a short asymmetric chromophore is sufficient for receptor activation and that the
following isomerization step is probably not required when the rest of the native chromophore is missing. This
property seems universal among the Euglenozoa, Plant, and Fungus kingdom rhodopsins. The rhodopsins of
animals have yet to be studied in this respect. The photoresponse appears to be mediated by Ca2ⴙ influx.

Since rhodopsins sharing sequence similarities are found in
bacteria, archaea, eukaryotic microorganisms, and animals, it
is possible to gain insight into the evolutionary progression and
diversification leading to the receptor proteins of human visual
photoreceptors as well as other photoreceptors. As part of this
continuing project on the evolution and mechanisms of visual
systems, the light-dependent responses of Peranema have been
studied with respect to behavior, action spectra, the mecha-
nism of activation, and signaling to the cell.
Peranema trichophorum is a colorless eukaryotic phagotroph
of the Euglenophyta that lives in fresh water (8). It does not
have a chloroplast but rapidly deforms in shape and is very
effective at capturing and eating prey (41). Peranema vora-
ciously takes any particles into its body by phagocytosis (1).
This “feeding behavior” is clearly and commonly observed
under the microscope. Its life cycle is not well studied, but from
light microscopic observations Peranema appears to have sev-
eral distinct stages of growth. After the cells are put into fresh
medium, which is initially low on waste products, one sees
small round cells of about 10 to 15 ␮m in diameter. These cells
elongate into oval cells that swim freely in solution and fast in
a helical euglenoid motion. Over 2 weeks, these cells elongate
further to 25 to 70 ␮m in length, remaining 10 ␮m wide, and
are seen to glide forward on a surface pulled by their leading
anterior cilium, which is about the length of the cell body. (We
use the word “cilium” rather than “eukaryotic flagellum” to
describe the leading appendage that enables the cell to glide to
emphasize that this appendage is not the more familiar rotary
motor-driven flagellum of bacteria. The cilium controls linear
motors that slide microtubules relative to each other in the
axenome and is the same as the cilia of Ciliata. This choice of
* Corresponding author. Mailing address: 201 Physics Building, Syr-
acuse University, Syracuse, NY 13244-1130. Phone: (315) 443-9220.
Fax: (315) 443-9103. E-mail: foster@phy.syr.edu.
terminology follows the advice of Irene Manton [24] and Tom
Cavalier-Smith [5], who says “the word flagellum should be
dropped altogether from eukaryotic biology.”) These fully
grown or mature cells are pulled forward along a surface by the
cell surface motility of the proximal five-sixths of the anterior
cilium (32). The cilium is straight except for the waving ante-
rior one-sixth, which presumably senses particles of food and
obstacles. A second thinner cilium runs posteriorly and tightly
appressed to the cell body, lying between the cell body and
gliding surface, and is rarely visible under the light microscope
(41). Apparently, the thin cilium does not participate in driving
the cell forward. The cells are in sufficient contact with the
surface to not rotate as they glide. In order to turn, the cell
body curves and then straightens with its cilium pointing to-
ward its new direction. The cells also curl their bodies into a
ball, with fast beating and waving of their cilia, when they eat.
Under our culture conditions, Peranema remains in this ma-
ture stage for about 3 to 4 weeks. The cells gradually become
wider and denser at the last stage of their life cycle. They glide
slowly, spend more time curled, and finally stay in the
curled-up shape and die from starvation or senescence.
Mature Peranema cells glide, turn, and curl spontaneously in
either light or dark. A movie of the curling can be seen at the
Euglenoid Project website (http://www.plantbiology.msu.edu
/triemer/Euglena/movies.htg/peranem2.mov). As originally
noted by Shettles (35), the frequency of curling behavior is
apparently enhanced by an increase in light, as shown by a
shortening of the time before the next curl. Shettles mapped
the location of photosensitivity to the primary cilium and the
front half of the body. Both light and dark adaptations increase
the sensitivity to light (35). At visible wavelengths, the sensi-
tivity is well related to the light intensity and apparently is
highest at 500 to 505 nm (35), which is typical for the spectral
peak of a rhodopsin. While Shettles did not make any partic-
ular hypothesis about the nature of the receptor, the photore-

1605
1606 SARANAK AND FOSTER EUKARYOT. CELL

ceptor rhodopsin has been found in several evolutionary

branches that diverged early from animals, including green
algae (16) and fungi (34). Replotting of Shettles’s data (35, 36)
yielded an action spectrum which fit reasonably well with the
rhodopsin standard curve (12) if one considers that the tech-
nology used did not provide sufficiently narrow-wavelength
bands of light. These overly broad bands of light resulted in a
flattened action spectrum. We are using the traditional (pre-
molecular biology) operational definition of rhodopsin,
namely, a protein that forms a chromophore with a retinal
molecule and that has a light-sensing function. So far, all rho-
dopsins identified with this definition have subsequently
proved to have similar (and probably homologous) amino acid
sequences.
Since the available evidence suggests that rhodopsin might
be the photoreceptor responsible for enhancing curling in Per-
anema, we used our established techniques for the identifica-
tion of photoreceptors (12, 16, 34) to show here that rhodopsin
is indeed the photoreceptor for the curling behavior of Per-
anema. We also confirm unusual features of the light/dark
adaptation and link rhodopsin activation to calcium ions and FIG. 1. Schematic diagram of the experimental apparatus.
channels during curling behavior.
The availability of rhodopsins in the exterior plasma mem-
branes of microorganisms makes it relatively easy to incorpo- For characterization of dark/light adaptation, aliquots of the cell suspension
rate and test for the physiological in vivo activity of rhodopsin were put through different protocols of adaptation as described below. For action
spectrum experiments, the culture jar was kept in the dark 30 min before and
analogs that can help to elucidate the mechanisms of receptor
during the experiments. Due to phagocytosis, Peranema takes up chemicals very
activation. Our report in this paper of a rhodopsin from the rapidly. Therefore, for pharmacological study, the chemicals were incorporated
Euglenozoa kingdom (4) allows us to ascertain the universality with cell samples for only 5 min in the dark.
of the activation properties of eukaryotic rhodopsins found Experimental setup. The experimental setup is depicted in Fig. 1. All exper-
previously in the Fungus and Plant kingdoms. iments were done in a constant-temperature (22°C) dark room. A phase-contrast
microscope (Nikon Labophot-2) was equipped with a near-infrared (near-IR)
This finding also permits some speculation about the origin charge-coupled-device video camera projecting the image of the cells onto a TV
of rhodopsins in eukaryotes and their relationship to the rho- monitor. The cells were illuminated from below with dim near-IR light. A 10⫻
dopsins of bacteria and archaea. Note that an analysis of the dry objective was used both for magnification of the image of the cells and to
structures of bacteriorhodopsin and rhodopsin showed that concentrate the focused beam of stimulating light from above onto the cells.
Except for the light/dark adaptation experiments, a 300-W tungsten lamp (EXR)
they have the same overall topology of their polypeptide folds,
in a Kodak carousel slide projector was used as a stimulating light source.
with helices I to III and also helices VI and VII superimposing Three-cavity interference filters with transmission peaks of 300, 334, 350, 380,
reasonably well (40). The amino acid sequences of helix VI are 420, 460, 500, 546, 600, and 640 nm (10-nm bandwidth) from Microcoating
remarkably similar for all rhodopsins. We also note that a new (Westford, MA) were used to generate different wavelengths of light for stimu-
consensus is emerging that bikonts such as Peranema, the lation. Optical density filters were used to vary the light intensity. The light beam
was collected by three convex lenses onto a 45° dichroic mirror, which reflected
green algae, ciliates, foramins, and cryptomonads diverged wavelengths of ⬍675 nm down to the objective lens to directly stimulate the cells
from unikonts such as the fungi and animals early in eukaryotic in the field of observation while transmitting those above 675 nm from the
divergence (6, 7, 39). The finding of rhodopsin in Peranema near-IR observing light up through to the video camera.
supports the notion of a common eukaryotic ancestor having a For light/dark adaptation studies, the stimulating light was the microscope
illumination light without the near-IR filter. Light adaptation was done by ex-
rhodopsin photoreceptor.
posing the cell suspension to a tungsten lamp at 5.0 mW/cm2 for 30, 60, or 120
min and then keeping it in the dark for 30 min before testing for the response
time. One sample with 30-min light adaptation was tested without being kept in
MATERIALS AND METHODS
the dark. Dark-adapted samples were kept in the dark for the same 30 min and
Culture. P. trichophorum was bought from Carolina Biological Supply Com- then an additional 30 min before being tested.
pany, Burlington, NC. The cells came in a mixed culture in spring water with Test for response. The response time, defined as the time in seconds for a cell
decaying material as food. They were kept in a constant room temperature of to curl (from a straight shape to a ball shape), was recorded manually with a
22°C under fluorescent room light during the day and in the dark at night. The stopwatch. The end point was defined as a tight curl if the cell body became a
culture was used for experiments when there was a sufficient number of elon- balled shape or the cell completed a 360° tight turn. A mature cell was identified
gated cells to observe their curling behavior as they glided. These cells were good and tracked to obtain the response time in the dark two times (geometric mean
for experiments for about 3 to 4 weeks. Every 2 weeks, cells were transferred to ⫽ D), followed by stimulation in the light (L) one time. The geometric mean is

冉 冊
1
about 30 ml of spring water (Spring Brook Springs II) in a culture jar with a grain n n
of wheat and a small piece (1 to 2 mm) of hard-boiled egg yolk to produce the 写 ai for the data sequence 共ai兲in⫽ 1. Cells were only stimulated once because
i⫽1
next batch of active cells. The gliding cells from the original or newly transferred
we found that prior exposure strongly influenced the response. Measuring the
jar were adapted to the dark for at least 30 min before the experiments. After a
spontaneous response twice in the dark improved the estimate of the dark
brief mixing and aeration step, an aliquot of 0.3 ml from the top half of the
response time without extending the experiment too long. The light response, R,
suspension was transferred to a glass microscope slide with a circular depression
was usually defined as follows (except in Table 1, where R ⫽ D/L):
in the middle. The uncovered slide was placed on the microscope’s X-Y trans-

冉 冊
lation stage. Moving the stage allowed us to track a cell during behavioral D⫺L
observation. R⫽
D⫹L
VOL. 4, 2005 RHODOPSIN AND CURLING IN PERANEMA TRICHOPHORUM 1607

TABLE 1. Effect of light and dark adaptation on curling response

Follow-up Response to stimulating lighta (mean ⫾ SEM [n])

Procedure Prestimulus condition
condition Expt 1 Expt 2 Expt 3 Avg

a Dark 30 min of dark 1.32 ⫾ 0.28 (9) 1.41 ⫾ 0.36 (8) 1.10 ⫾ 0.27 (8) 1.28 ⫾ 0.18
b 30 min of light 30 min of dark 2.94 ⫾ 0.43 (4) 2.38 ⫾ 0.29 (5) 2.86 ⫾ 0.41
c 60 min of light 30 min of dark 3.03 ⫾ 0.18 (2) 3.03 ⫾ 0.18
d Over 120 min of light 30 min of dark 1.54 ⫾ 0.40 (5) 1.69 ⫾ 0.54 (7) 1.12 ⫾ 0.34 (4) 1.49 ⫾ 0.25
e 30 min of light None 1.41 ⫾ 0.52 (12) 1.41 ⫾ 0.52
a
Light-stimulated rate of curling (s⫺1)/spontaneous (in the dark) rate of curling (s⫺1).

Cells that did not show spontaneous curling (in the dark) within 5 min were of EGTA were done using a calcium electrode (ISE25Ca-9; Radiometer Ana-
rejected. For action spectrum experiments, at least seven cells were observed at lytical, France) and a reference electrode (REF 201; Radiometer Analytical,
each intensity. The field of view was changed after one cell was observed to France).
minimize the chance of using cells that were prematurely exposed to light. A new To detect the free calcium in the cells, we used Calcium Green 1 (5 mM in
aliquot of cell suspension was used at each intensity. After testing to find ap- dimethyl sulfoxide; Molecular Probes, Eugene, Oregon) at 1 ␮l/1 ml of Peranema
proximately the right range of intensities to use, at least three intensities near the suspension incorporated on a shaker in the dark for 15 min and then observed
threshold were tested for each wavelength to plot the log-intensity linear-re- with an Olympus BX51W1 microscope with 60⫻ and 100⫻ water immersion
sponse curve. objectives at excitation/emission wavelengths of 500 and 535 nm.
Plot of action spectra. The threshold action spectra for native rhodopsin and Data analysis of delays in spontaneous (dark) and elicited (dark and light
n-hexenal-opsin were plotted as previously described (12, 34). In brief, the adaptation) responses. Because the distribution of response times followed a log
threshold for each stimulating wavelength was obtained by linear extrapolation normal distribution, the following calculations were carried out. For each cell,
of its log-intensity linear-response curve (see Fig. 1 in reference 16 and Fig. 3 and the geometric mean of the delays in spontaneous responses in the dark (D) was
4 in reference 12). The log of sensitivity (reciprocal of threshold) was plotted calculated. The ratio (R) of the rate of curling (reciprocal of delay) in response
against wavelength (nm)/energy (eV) using SigmaPlot. The action spectrum was to a standard light stimulus (1/L) to the spontaneous (in the dark) rate of curling
fitted to the standard rhodopsin curve (33). (1/D) was then calculated (R ⫽ D/L). The mean and the standard error of the
Chromophore removal and analog reconstitution. A nonreversible removal of mean (SEM) for each condition, which consisted of 2 to 12 cells, were also
the anticipated retinal of Peranema was attempted by adding hydroxylamine (to calculated using the PSIPlot computer program, and the data are presented in
trap free retinal) to the cell suspension before exposure to high-intensity light. Table 1. Since the frequency distribution of reaction times was fit by the log
However, hydroxylamine as low as 5 nM was still toxic to the cells. Therefore, normal distribution, the level of significance was calculated from the standard
only strong light was used to bleach the cells and remove the chromophore. The normal distribution using the natural logarithm values of D and L, as follows:
cell suspension (0.3 ml) in the microscope chamber was laterally exposed to 500
nm (32-nm-bandwidth interference filter; Barr Associates, Westford, MA) of
intensity up to 105 mW/cm2 for 2 min to bleach the rhodopsin molecules,
៮
Z ⫽ 关ln ៮
D ⫺ ln L兴 冒冑 s2 ln D s2 ln L
nD
⫹
nL
removing the native chromophore. The bleached suspension was then allowed to
take up 5 pM n-hexenal (Aldrich), which incorporated into rhodopsin, and the where s2ln D is the variance of ln D and nD is the number of measurements in the
cells were tested immediately for their response time as described above. Testing sample.
of the response of each sample was completed within 30 min, the time period in
which the native pigment remained suppressed by photobleaching and the n-
hexenal activity was still effective. RESULTS
It is essential to note that Peranema is enormously more sensitive to added
chemicals than any other microorganism we have tested. The uptake of chemi- Spontaneous and light-stimulated curling. The frequency
cals into the cells is evidently fast and efficient, probably due to the frequent distribution of the response times (times to onset of curling)
phago/endocytosis used for their feeding. Hydroxylamine at ⬎5 nM, n-hexenal at for spontaneous curling under constant conditions fits the log
⬎5 pM, and retinal at ⬎1 ␮M are toxic to the cells. The cells became tightly normal distribution curve (Fig. 2A). This fact guided all our
curled and eventually died upon exposure to these chemicals. We had to omit the
hydroxylamine for irreversible bleaching and the vitamin E (0.25%) as an anti-
analyses. The fit of the log normal distribution to the response
oxidant for retinal and hexenal that we routinely used in our other studies. This
was unfortunate since added antioxidant approximately doubles the period of
activity for hexenal. We also observed that the regeneration of rhodopsin after
bleaching without hydroxylamine took place after 30 min. The effects of added
retinal and hexenal without added vitamin E last for 45 and 30 min, respectively,
in Chlamydomonas (J. Saranak, unpublished). In the absence of added antioxi-
dant after 3 h of incubation, no chromophore is detected in the binding site in
Chlamydomonas and hence no activity is detected (19). Furthermore, the activity
of n-hexenal analogues must be tested before they can be significantly oxidized
into the carboxylic acid form that does not bind opsin and before they have
evaporated. These constraints gave us a mere 30-min window for n-hexenal
experiments. Fortunately, since uptake into the Peranema cell was almost instan-
taneous, we did not have to wait before testing. Since we were able to complete
each test within 30 min, the regeneration of rhodopsin and the oxidation of
retinal or hexenal did not affect our results. The above details are important if
one desires to repeat these experiments.
Calcium experiments. To study the effect of calcium on curling behavior, we
used the calcium ionophore A23187 (40 mM in dimethyl sulfoxide); a calcium
chelator, the free acid form of EGTA; and a calcium channel blocker, nifedipine FIG. 2. Time distribution of curling. Repeated measurements were
(50 mM in ethyl alcohol). Chemicals were purchased from Sigma and dissolved made of the length of time before cell curling (time to curl). The
in spring water (Spring Brook Springs II) unless otherwise indicated. They were figures are histograms of the natural Naperian logarithms of the time,
added to the cell suspension 5 min before testing. Subsequently, measurements in seconds. (A) Dark conditions; (B) immediately following light stim-
of free Ca2⫹ in cell suspensions and spring water before and after the addition ulation at 500 nm at 26 ␮W/cm2.
1608 SARANAK AND FOSTER
time histogram for light stimulation (Fig. 2B) shows an about
threefold decrease in geometric mean response time compared
to that for spontaneous curling. (The geometric mean is the
appropriate metric for log normal distributions, just as the
mean is appropriate for normal distributions.) The geometric
mean response time for light-stimulated curling was 11 ⫹4/⫺3
s (value ⫾ SEM; nL ⫽ 21) (Fig. 2B) versus 36 ⫹6/⫺5 s (nD ⫽
42) (Fig. 2A) for spontaneous curling of this set of cells. (Note
that the SEM of a log normal distribution is symmetric on a
logarithmic scale but skewed on a linear scale.) For these data,
the probability that there is not a response to light is ⬍0.0001
(level of significance). While possibly not significant, given the
very limited amount of data, the light-stimulated log normal
distribution shows an extra two cells responding earlier than
expected. If significant, this would suggest that there could
transiently be a higher probability of a response immediately
following a step change in light than later. Such a transient
increase in response is common in biological signal transduc-
tion networks and occurs whenever signal processing involves a
differentiating function which leads to a faster response.
Light and dark adaptation was studied briefly here primarily
to gain the experience of the light-dependent behavior of Per-
anema and to choose optimal parameters for action spectrum
experiments. Our results on light/dark adaptation were similar
to those of Shettles (35) in that the cells had the fastest re-
sponse after 30 to 60 min of light followed by 30 min of dark
(Table 1, compare first four rows). Longer light exposures
(experiment d) reduced the response. Having a dark adapta-
tion period after the light period also increased the response
(compare experiments e and b). Thus, we have confirmed the
finding that unlike the case in animals, both light and dark
adaptation increased the response to light. This unique adap-
tation process is still not well understood.
Action spectra. To establish the native and n-hexenal chro-
mophore action spectra, intensity-response curves for six wave-
lengths of light over the range of 300 to 640 nm were deter-
mined. Straight-line extrapolation of the intensity response to
the log-intensity axis yielded the threshold for each wave-
length, which varied ⬎4 orders of magnitude. The reciprocal of
the threshold, the sensitivity, is plotted as a function of photon
energy in Fig. 3, resulting in the action spectrum of curling
behavior.
The spectrum of the native chromophore fits well with the
Chlamydomonas rhodopsin standard curve (33), with a peak of
2.43 ⫾ 0.04 eV (510 nm) and a half-spectral width at a half-
maximum of 0.21 eV. Bleaching of the chromophore with
high-intensity (up to 105 mW/cm2) 500-nm light abolished the
response to light. Light responses (R; see Materials and Meth-
ods) on a scale ranging from approximately zero to one were
0.33 and ⫺0.13 for unbleached and bleached samples, respec-
tively. As expected for rhodopsin, the light response could be
restored by adding retinal (1 ␮M), which brought the response
to 0.54. Bleaching causes rhodopsin to break down to free
retinal and opsin. This set of results indicates that the loss of
light response after bleaching is due to the loss of native chro-
mophore. We have shown with Chlamydomonas (14, 15, 16)
and Allomyces (34) that retinal and many retinal analogs that
reach and incorporate into the retinal-binding site of opsin,
including n-hexenal, can restore phototaxis. Because of n-hex-
enal’s volatility and potential for relatively rapid oxidation,
EUKARYOT. CELL
FIG. 3. Action spectra of curling behavior. (A) Action spectrum of
native untreated cells. (B) Action spectrum of cells whose chro-
mophores have been bleached and then replaced by 5 pM n-hexenal.
The spectra are shown on the same relative sensitivity scale, and the
scales are in a standard proportion, where a 1-eV shift is the same
length as 2 log sensitivity units (12).
special care must be taken in its use (see Materials and Meth-
ods). For this study, we chose to use this small retinal analog,
which has only two conjugated double bonds in the resulting
chromophore, to substitute for the native chromophore. n-
Hexenal is easy to incorporate with a rapid onset, and most
importantly, has the bluest absorption, the largest spectral shift
in action spectrum that we can measure; hence, it is the most
obviously shifted among the available retinal analogs. As ex-
pected, n-hexenal at 5 pM restores the light-stimulated curling
behavior in Peranema as soon as it is added and lasts for at
least 30 min. Since we always measure threshold action spectra
very near the threshold, as expected and intended, the level of
significance for being different from behavior in the dark was
low. For example, at 460 nm with n-hexenal incorporated, the
point nearest the threshold had a level of significance of 0.35.
Doubling the intensity, however, gave a response time (geo-
metric mean, 19 ⫹6/⫺5 s) different from that in the dark (34
⫹6/⫺5 s), with a level of significance or P value of 0.04. Thirty-
five cells were used to obtain the 460-nm point, with 28 cells on
average at each wavelength.
Since the action spectrum has the typical rhodopsin shape
and the spectral peak anticipated for opsin with hexenal incor-
porated, clearly Peranema recovers its light response due to the
presence of n-hexenal in the retinal-binding site. Since retinal
was removed prior to the addition of the exogenous analog,
activation of that analog could not transfer its signal to a native
rhodopsin molecule via a retinal chromophore. The analog
must activate the opsin directly by being incorporated into the
available binding site. As a short chromophore with only two
conjugated double bonds, the analog has an action spectrum
(Fig. 3B) shifted to a higher energy (3.73 ⫾ 0.04 eV [332 nm])
than that of the native pigment. This peak is similar to 3.66 ⫾
0.35 eV (339 nm), obtained from Allomyces zoospore photo-
taxis (34), and 3.50 eV (354 nm), obtained from Chlamydomo-
VOL. 4, 2005 RHODOPSIN AND CURLING IN PERANEMA TRICHOPHORUM 1609

nas phototaxis (15). n-Hexenal–opsin was as active as rhodop-

sin as a photoreceptor for curling behavior, as shown by its
action spectrum (Fig. 3B). The red shift of the n-hexenal–opsin
action spectrum from the free n-hexenal absorption spectrum
(265-nm peak in methanol) indicates that hexenal forms a
chromophore with opsin. The blue shift of its action spectrum
from the native one indicates that hexenal-opsin has a physi-
ological function as a photoreceptor for the curling behavior of
Peranema. Together, the results form conclusive evidence that
the euglenoid Peranema uses a rhodopsin as a photoreceptor.
As anticipated for such a short chromophore, the low-energy
cutoff slope of the hexenal-opsin action spectrum is less than
that of the native chromophore.
Calcium effects. The motion observed when the cell curls
could be described as a contraction of the cell body on one
side. Shettles (37) found that the reaction time to light de-
creased with more calcium and was the shortest with calcium
relative to that with magnesium, potassium, or sodium. Since
this seemed similar to skeletal muscle contraction and since
rhodopsins typically couple with calcium channels (2), we de-
cided to test the hypothesis of calcium involvement in curling.
The response is strongly dependent on the external Ca2⫹ con-
centration. Decreasing the external free Ca2⫹ concentration by
EGTA (50 to 500 ␮M) inhibited the curling behavior in a
dose-dependent manner (Fig. 4A). At these concentrations of
EGTA, motility is unaffected. Note, however, that much higher
concentrations of EGTA are toxic, causing the cell to perma-
nently curl. Table 2 shows the free Ca2⫹ concentration as well
as the pH of a Peranema cell suspension before and after the
addition of EGTA. The values for spring water only are in-
cluded for comparison. Note that the cells removed 85% of the
free calcium in the suspension by binding or by sequestering it
in concentrated vesicles. The vesicles were observed by label-
ing with Calcium Green 1, a dye that is fluorescent if it binds FIG. 4. (A) Dose-response effect of EGTA on curling behavior.
free calcium. Consequently, one cannot accurately predict the The response, R, was measured in seven cells for each data point after
free calcium levels from consideration of the medium (spring stimulation at 500 nm and 26 ␮W/cm2. (B) Effect of light and Ca2⫹
water in this case) alone. Under our conditions, the calcium- ionophore on curling rate. F, dark;
, light at 500 nm and 22 ␮W/cm2;
■, light at 500 nm and 342 ␮W/cm2.
chelating effect of EGTA very significantly lowered the free
calcium level in the Peranema sample. Free Ca2⫹ was reduced
from 2.7 ⫻ 10⫺4 M without EGTA to ⬍10⫺8 M with 500 ␮M
EGTA. As shown in Table 2, EGTA specifically reduced free flux through Ca2⫹ channels in the cell membrane. Rhodopsin
Ca2⫹ up to 4 to 5 orders of magnitude but only reduced the pH might, in addition, act as a channel. For the same level of light
from 7.7 to 6.4 at the highest dose. According to Shettles (37), stimulation and batch of cells, the response, R, was 0.62, but
this drop in pH corresponds to an increase in the average with 100 ␮M nifedipine, which blocks L-type calcium channels,
reaction time (n ⫽ 10), from 16.5 to 18.8 s. Shettles (37) also R was 0.21, and with 300 ␮M nifedipine, R was 0.15. This
found that the response to light was quite normal from pH 5.5 suggests a significant role of calcium channels apart from a
to 8.5, and our conditions were well within that range. It is direct role of rhodopsin. As anticipated if light induces calcium
likely that the EGTA effect on curling behavior is predomi-
nantly due to a reduction of extracellular free Ca2⫹. To further
elucidate the roles of calcium in curling behavior, the effects of TABLE 2. Free Ca2⫹ concentration and pH of Peranema cell
suspension and spring water with and without EGTA
a calcium ionophore (A23187) and a calcium channel blocker
(nifedipine) were studied. A23187 at 20, 40, and 80 ␮M in- Value for Peranema as Value for spring water
creased the spontaneous response rate, as shown in Fig. 4B. received from Carolina only (Spring Brook
EGTA (␮M) Biological Supply Springs II)
The effect is similar to that of light stimulation. Both the
calcium ionophore and light stimulation result in dose-depen- Free Ca2⫹ (M) pH Free Ca2⫹ (M) pH
dent increases in curling probability. In the presence of the 0 2.7 ⫻ 10 ⫺4
7.77 1.8 ⫻ 10 ⫺3
7.72
calcium ionophore, light does not have much, if any, further 50 1.5 ⫻ 10⫺4 7.32 1.65 ⫻ 10⫺3 7.62
effect on the response time. This implies that when the cell 200 7.5 ⫻ 10⫺7 6.50 1.0 ⫻ 10⫺3 7.35
membrane is made permeable to Ca2⫹, light does not have a 500 ⬍⬍10⫺8a 6.43 2.2 ⫻ 10⫺4 6.98
significant additional effect. Light probably causes a Ca2⫹ in- a
The concentration is much less than the Ca electrode sensitivity.
1610 SARANAK AND FOSTER
influx and intracellular calcium induces curling, reducing the
entry of calcium will reduce the curling response. These results
suggest that the curling behavior of Peranema is calcium de-
pendent and that light induces a calcium influx.
DISCUSSION
Rhodopsin is the photoreceptor for curling in Peranema. As
we have done previously with the green alga Chlamydomonas
(16) and the fungus Allomyces (34), we have shown that retinal
analogs incorporated into cells that have been depleted of their
native pigment shift the action spectrum of a photoreceptor in
a microorganism. Furthermore, we have found a native action
spectrum consistent with a rhodopsin pigment. In both the case
of Chlamydomonas and that of Allomyces, subsequent work by
others found rhodopsin-like amino acid sequences in Chlamyd-
omonas and other fungi such as Neurospora. Hence, Peranema
has a rhodopsin photoreceptor which has yet to be proven to
be of the same family as all other rhodopsins found so far in all
domains of nature. This finding has many implications for the
evolution of vision in eukaryotes.
Phylogenic significance of and sequence similarities in the
rhodopsin family. Peranema rhodopsin joins the growing list of
nonanimal eukaryotic rhodopsins, including those identified in
dinoflagellates by the evaluation of protein sequences (30) and
action spectral analysis (11, 12, 18), in cryptophytes by action
spectra (9, 12) and protein sequence evaluation (22), in fungi
by shifted action spectra (34) and protein sequence evaluation
(3), and in green algae by shifted action spectra (16) and
protein sequence evaluation (20, 38). Two other probable
groups with rhodopsins suggested by action spectra are the
ciliates (12, 25) and foramins (12). The rhodopsins of fungi and
green algae show the greatest similarity to each other and then
to archaeal sensory rhodopsin IIs compared to cyanobacterial
rhodopsins. This association with archaeal rhodopsins and the
ubiquity of these rhodopsins in different eukaryotic groups,
both bikont and unikont, suggest that their last common an-
cestor probably had a rhodopsin photoreceptor. Mitchell (26)
suggested for the origin of cilia that a phagocytic cell ancestor
possibly developed a protocilium, which rather than bending,
glided on a substrate and later evolved into a beating cilium.
The phagocytic Peranema organism seems to have retained this
capability, and its photosensitivity, which we show here de-
pends on rhodopsin, is found in its gliding cilium (35). A
possible single exception to this ancestral origin may be a
dinoflagellate whose rhodopsin sequence shows high similarity
to that found in cyanobacteria (28), implying perhaps that this
rhodopsin may have come recently from a lateral or horizontal
transfer from a cyanobacterium.
Spectral trends in evolution. Interestingly, as one proceeds
along the evolutionary rhodopsin line from bacteria and ar-
chaea toward animals, there is a significant broadening of the
spectral widths of absorption and action spectra. As previously
noted (33), the archaeal pigment bacteriorhodopsin has a half-
width at a half-maximum of 0.16 eV, bikont eukaryotic micro-
organisms such as dinoflagellates, cryptomonads, and green
algae have wider spectra, with a half-maximum at 0.21 eV, and
unikont eukaryotes such as fungi (Allomyces [34]) and animals
have spectra that are wider still, at 0.31 eV. Peranema fits with
these other bikont microeukaryotes. The consensus that uni-
EUKARYOT. CELL
konts and bikonts evolved from a common eukaryote is con-
sistent with a single transition from 0.16 to 0.21 eV occurring
between archaea/bacteria and eukaryotes. A second transition
to 0.31 eV on the unikont line occurred after the bikont-
unikont split and before the animal-fungal branching. The
molecular basis for these spectral changes in evolution has not
yet been reported.
Signal processing and behavioral trends in evolution. In
bacteria and archaea, the rhodopsins frequently serve as pro-
ton or other ion pumps and simultaneously as sensory recep-
tors, which control the probability of a reversal of the direction
of rotation of the flagella changing the direction of the organ-
ism. These organisms do not have localized receptors, and the
cell responds only to the transition of swimming into or out of
a light beam. The ability to move in the direction of light is
weak. In some nonmotile eukaryotes in which there is no visual
function, such as the fungus Leptoshaeria, the proton pumping
function is maintained (42). On the other hand, in animals
rhodopsins are typically associated with localized eyes as visual
receptors. Peranema’s lack of a localized eye and its curling
behavior, which contributes to weak photo avoidance, are
more like characteristics of the prokaryotes than of the eu-
karyotes and suggest that the last ancestor of unikonts and
bikonts may have had similar primitive behavior. If this is true,
then the tracking type of phototaxis behavior evolved sepa-
rately in the bikont and unikont lines. This would mean that all
the physical principles of central location of the eye, looking
orthogonal to the swimming axis, and using constructive inter-
ference in the eyes are the results of convergent evolution (18).
All phototactic microeukaryotes with rhodopsin photorecep-
tors, apart from Peranema, have localized eyespots, which fa-
cilitate true tracking of light and true phototaxis.
Among bikonts, cryptomonads, green algae, and Peranema
appear to use rhodopsin photoreceptors, while flavins are used
for stramenopile and euglenoid phototaxis. An example of a
flavin photoreceptor for phototaxis is an adenylyl cyclase acti-
vated by a flavin chromophore in Euglena (21). The last com-
mon ancestor of bikonts and unikonts probably had flavin
photoreceptors, an alternative type of receptor available for
visual systems. Why rhodopsins came to dominate for photo-
taxis and then vision in some kingdoms and flavin receptors
became dominant in others is not clear. An additional ances-
tral function of rhodopsin may be its control of gene expres-
sion. This was observed for a rhodopsin in the green alga
Chlamydomonas (17) and in rats (43). In the nonmotile fungus
Neurospora, rhodopsin probably (3) controls gene expression.
Rhodopsin- and calcium-dependent curling. Our results
suggest that Ca2⫹ plays a role in curling behavior in Peranema.
Light stimulates the rhodopsin receptors, which through a
presently unknown mechanism open Ca2⫹ channels, increasing
Ca2⫹ influx to trigger curling. Calcium influx into the cell
appears to mimic the effect of light in stimulating the proba-
bility of curling. In view of the demonstration that several of
the rhodopsins of Chlamydomonas are light-gated channels
(27), a reasonable hypothesis is that this is true for Peranema as
well. This association probably extends to the common eu-
karyote ancestor. Certainly, an association between rhodopsin
and calcium is common in many organisms (2) and may reflect
a common origin.
VOL. 4, 2005 RHODOPSIN AND CURLING IN PERANEMA TRICHOPHORUM
Evolution of the rhodopsin activation mechanism and im-
plications of n-hexenal activity in microeukaryotes for in vivo
activation of rhodopsins. It is of interest to know the proper-
ties of the ancestral rhodopsins of unikonts and bikonts and
how those properties have evolved since. Two types of rho-
dopsins probably existed side by side in these ancestors, in-
cluding those which performed an energy conversion function
like that of bacteriorhodopsin and those with a sensory func-
tion. For a strictly sensory function, no specific bond, such as
the 11-12 bond in the human retinal chromophore, needs to be
isomerized to activate a rhodopsin. Isomerization about any
bond would appear to be sufficient. In vivo evidence was shown
with the bikont Chlamydomonas rhodopsin (13, 14), for which
active analogs were found with each bond individually blocked.
More recently, this has also been found in unikont animal
kingdom rhodopsins (10, 23). Hence, rhodopsins are not re-
quired during activation to sense a localized motion about a
particular point of the chromophore, making it unlikely that
such a motion is critical for its activation.
The observed activity of the truncated chromophore n-hex-
enal (Fig. 3) puts a limit on how much chromophore is needed
to activate rhodopsin. n-Hexenal was also active with rhodop-
sin in the bikont Chlamydomonas (13, 14, 15) and in the zoo-
spore of the unikont chytridiomycete fungus Allomyces (34). In
Chlamydomonas, n-hexenal recovered the sensitivity of photo-
taxis at 354 nm to 2.9 orders of magnitude above the back-
ground, with no loss of sensitivity relative to the native chro-
mophore and with an action spectrum consistent for
n-hexenal–opsin amply demonstrating its activity (29).
Whether this property has evolved to be different in animals
has not been adequately tested. A chromophore strain may not
be needed for activation, since n-hexenal lacks the ␤-ionone
ring and a significant part of the normal conjugated chain
found in retinal. As evident from the n-hexenal structure, as-
suming isomerization about the only bond available for cis-
trans isomerization, the only structural change that would oc-
cur is that the short saturated chain would flop the other way.
Such a small change in such a weak-kneed molecule (only two
conjugated double bonds in the chromophore) is very unlikely
to produce significant strain on the site to result in activation of
the photoreceptor. The opsin protein is specifically sensitive to
what happens at the site near the lysine N to which the chro-
mophore is attached, as this is the only area that neighbors the
n-hexenal chromophore.
Without a need for the strain of the chromophore acting on
the protein, something else must be driving the protein con-
formation change. Rousso et al. (31) used atomic force micros-
copy to show that bacteriorhodopsin’s conformation changed
with isomerization-locked chromophores (no strain on the re-
ceptor site) and that an electrically asymmetric chromophore
was required for activation, presumably to establish charge
separation in the excited state. This point was confirmed with
analogs showing that this charge redistribution or displace-
ment is necessary for initiating the bacteriorhodopsin photo-
cycle (44). Hence, it would seem reasonable for the sensory
role of rhodopsins in Peranema, Chlamydomonas, and Allomy-
ces (13, 14, 15, 29), for which the n-hexenal is an active chro-
mophore, that a similar charge displacement in the photore-
ceptor protein is sufficient to drive a conformation change and
activation.
1611
Peranema as a new model system. This work provides an-
other unicellular model for the comparative study of rho-
dopsins. The model is convenient and inexpensive for studying
rhodopsin activation and its transduction pathway. A particu-
larly noteworthy advantage is that molecules of all sizes and
charges can enter this organism simply by being placed in
solution. We observed a whole Chlamydomonas cell being
trapped inside Peranema by phagocytosis in seconds. While
substances in the food vacuoles inside the cell are not guaran-
teed access to the intracellular compartment, chemicals that
rapidly show toxicity must have gone to their sites of action.
There is no sexual cycle or photosynthesis to interfere with
behavioral studies. Saito et al. (32) have used Peranema as a
model system for gliding motility. Furthermore, they have de-
veloped a monoxenic culture system so that it is possible to
obtain sufficient bacterium-free cells for biochemical and mo-
lecular biological studies. The development of further bio-
chemical and genomic analysis tools would seem appropriate.
Conclusions. Rhodopsin is the receptor that controls curling
in Peranema. This response involves calcium signaling. The
study of this curling photoresponse has yielded new insight into
the evolution of eukaryotic vision and better predictions of the
properties of the ancestral eukaryotic rhodopsin. This ances-
tral sensory rhodopsin does not appear to require isomeriza-
tion of its chromophore for activation. Rather, its activation
may be best described by an essential charge redistribution
near the lysine nitrogen, followed by isomerization if necessary
to move the ␤-ionone ring of the native chromophore out of
the way. Both unikont and bikont rhodopsin receptors are
probably predominantly derived from an ancestral eukaryote
with a characteristic spectral width of more than that of bac-
teriorhodopsins. The spectral width of Peranema’s rhodopsin is
consistent with that of all other bikonts measured and is dis-
tinct from the greater spectral width of all measured unikont
rhodopsins. Peranema has possibly maintained several other
traits postulated for the ancestral eukaryote, namely, phagocy-
tosis, gliding behavior, and an association of rhodopsin with
the cilium. This makes it an interesting organism for further
study.
ACKNOWLEDGMENTS
Nisa Foster and Narie Foster started this work as their middle and
high school science fair projects, and later Jeanne Hansen and Nich-
olas Peruzzi continued it as their high school summer projects. Their
scientific curiosity and enthusiasm combined with industrious data
collection contributed to this finding.
REFERENCES
1. Allen, J. R., J. J. Lee, S. H. Hutner, and J. Storm. 1966. Prolonged culture
of the voracious flagellate Peranema in antioxidant-containing media. Pro-
tozoology 13:103–108.
2. Baehr, W., and K. Palczewski. 2002. Photoreceptors and calcium, p. 632.
Springer, Berlin, Germany.
3. Bieszke, J. A., E. L. Braun, L. E. Bean, S. Kang, D. O. Natvig, and K. A.
Borkovich. 1999. The nop-1 gene of Neurospora crassa encodes a seven
transmembrane helix retinal-binding protein homologous to archaeal rho-
dopsins. Proc. Natl. Acad. Sci. USA 96:8034–8039.
4. Cavalier-Smith, T. 1981. Eukaryote kingdoms: seven or nine? BioSystems
14:461–481.
5. Cavalier-Smith, T. 1986. Cilia versus undulipodia. Bioscience 36:293.
6. Cavalier-Smith, T. 2004. Only six kingdoms of life. Proc. R. Soc. London Ser.
B 271:1251–1262.
7. Douzery, E. J. P., E. A. Snell, E. Bapteste, F. Delsuc, and H. Philippe. 2004.
The timing of eukaryotic evolution: does a relaxed molecular clock reconcile
proteins and fossils? Proc. Natl. Acad. Sci. USA 101:15386–15391.
1612 SARANAK AND FOSTER
8. Ehrenberg, C. G. 1838. Die infusionsthierchen als vollkommene organismen.
Verlag von Leopold Voss, Leipzig, Germany.
9. Erata, M., M. Kubota, T. Takahashi, I. Inouye, and M. Watanabe. 1995.
Ultrastructure and phototactic action spectra of two genera of cryptophyte
flagellate algae, Cryptomonas and Chroomonas. Protoplasma 188:258–266.
10. Fan, G., F. Siebert, M. Sheves, and R. Vogel. 2002. Rhodopsin with 11-cis
locked chromophore is capable of forming an active state photoproduct.
J. Biol. Chem. 277:40229–40234.
11. Forward, R. B., Jr. 1974. Phototaxis by the dinoflagellate Gymnodinium
splendens Lebour. J. Protozool. 21:312–315.
12. Foster, K. W. 2001. Action spectroscopy of photomovement, p. 51–115. In
D.-P. Häder and M. Lebert (ed.), Photomovement. Comprehensive series in
the photosciences, vol. 1. Elsevier, Amsterdam, The Netherlands.
13. Foster, K. W., J. Saranak, F. Derguini, R. V. Jayathirtha, G. R. Zarrilli, M.
Okabe, J.-M. Fang, N. Shimizu, and K. Nakanishi. 1988. Rhodopsin activa-
tion: a novel view suggested by in vivo Chlamydomonas experiments. J. Am.
Chem. Soc. 110:6588–6589.
14. Foster, K. W., J. Saranak, F. Derguini, G. R. Zarrilli, R. Johnson, M. Okabe,
and K. Nakanishi. 1989. Activation of Chlamydomonas rhodopsin in vivo
does not require isomerization of retinal. Biochemistry 28:819–824.
15. Foster, K. W., J. Saranak, and P. A. Dowben. 1991. Spectral sensitivity,
structure, and activation of eukaryotic rhodopsins: activation spectroscopy of
rhodopsin analogs in Chlamydomonas. J. Photochem. Photobiol. B 8:385–
408.
16. Foster, K. W., J. Saranak, N. Patel, G. Zarrilli, M. Okabe, T. Kline, and K.
Nakanishi. 1984. A rhodopsin is the functioning photoreceptor for photo-
taxis in the unicellular eukaryote Chlamydomonas. Nature 311:756–759.
17. Foster, K. W., J. Saranak, and G. Zarrilli. 1988. Autoregulation of rhodop-
sin synthesis in Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. USA
85:6379–6383.
18. Foster, K. W., and R. D. Smyth. 1980. Light antennas in phototactic algae.
Microbiol. Rev. 44:572–630.
19. Hegemann, P., W. Gärtner, and R. Uhl. 1991. All-trans retinal constitutes the
functional chromophore in Chlamydomonas rhodopsin. Biophys. J. 60:1477–
1489.
20. Hegemann, P., M. Fuhrmann, and S. Kateriya. 2001. Sensory algal photo-
receptors. J. Phycol. 37:6668–6676.
21. Iseki, M., S. Matsunaga, A. Murakami, K. Ohno, K. Shiga, K. Yoshida, M.
Sugai, T. Takahashi, T. Hori, and M. Watanabe. 2002. A blue-light-activated
adenylyl cyclase mediates photoavoidance in Euglena gracilis. Nature 415:
1047–1051.
22. Jung, K.-H., and J. L. Spudich. 2004. Microbial rhodopsins: transport and
sensory proteins throughout the three domains of life, p. 1–12. In W. M.
Horspool et al. (ed.), CRC handbook of organic photochemistry and pho-
tobiology. CRC Press, Boca Raton, Fla.
23. Kuksa, V., F. Bartl, T. Maeda, G.-F. Jang, E. Ritter, M. Heck, J. P. Van
Hooser, Y. Liang, S. Filipek, M. H. Gelb, K. P. Hofmann, and K. Palczewski.
2002. Biochemical and physiological properties of rhodopsin regenerated
with 11-cis-6-ring and -7-ring retinals. J. Biol. Chem. 277:42315–42324.
24. Manton, I. 1965. Some phyletic implications of flagellar structure in plants.
Adv. Bot. Res. 2:1–34.
25. Marangoni, R., S. Puntoni, L. Favati, and G. Colombetti. 1994. Phototaxis in
Fabrea salina. I. Action spectrum determination. J. Photochem. Photobiol. B
23:149–154.
26. Mitchell, D. R. 2004. Speculations on the evolution of 9⫹2 organelles and
the role of central pair microtubules. Biol. Cell 96:691–696.
EUKARYOT. CELL
27. Nagel, G., T. Szellas, W. Huhn, S. Kateriya, N. Adeishvili, P. Berthold, D.
Ollig, P. Hegemann, and E. Bamberg. 2003. Channelrhodopsin-2, a directly
light-gated cation-selective membrane channel. Proc. Natl. Acad. Sci. USA
100:13940–13945.
28. Nakamura, Y., T. Kaneko, S. Sato, M. Mimuro, H. Miyashita, T. Tsuchiya,
S. Sasamoto, A. Watanabe, K. Kawashima, Y. Kishida, C. Kiyokawa, M.
Kohara, M. Matsumoto, A. Matsuno, N. Nakazaki, S. Shimpo, C. Takeuchi,
M. Yamada, and S. Tabata. 2003. Complete genome structure of
Gloeobacter violaceus PCC 7421, a cyanobacterium that lacks thylakoids.
DNA Res. 10(Suppl.):181–201.
29. Nakanishi, K., F. Derguini, V. J. Rao, G. Zarrilli, M. Okabe, T. Lien, R.
Johnson, K. W. Foster, and J. Saranak. 1989. Theory of rhodopsin activa-
tion: probable charge redistribution of excited state chromophore. Pure
Appl. Chem. 61:361–364.
30. Okamoto, O. K., and J. W. Hastings. 2003. Novel dinoflagellate clock-related
genes identified through microarray analysis. J. Phycol. 39:519–526.
31. Rousso, I., E. Khachatryan, Y. Gat, I. Brodsky, M. Ottolenghi, M. Sheves,
and A. Lewis. 1997. Microsecond atomic force sensing of protein conforma-
tional dynamics: implications for the primary light-induced events in bacte-
riorhodopsin. Proc. Natl. Acad. Sci. USA 94:7937–7941.
32. Saito, A., Y. Suetomo, M. Arikawa, G. Omura, S. M. M. K. Khan, S. Kakuta,
E. Suzaki, K. Kataoka, and T. Suzaki. 2003. Gliding movement in Peranema
tricophorum is powered by flagellar surface motility. Cell Motil. Cytoskel.
55:244–253.
33. Saranak, J., and K. W. Foster. 1994. The in vivo cleavage of carotenoids into
retinoids in Chlamydomonas reinhardtii. J. Exp. Bot. 45:505–511.
34. Saranak, J., and K. W. Foster. 1997. Rhodopsin guides fungal phototaxis.
Nature 387:465–466.
35. Shettles, L. B. 1937. The response to light in Peranema trichophorum with
special reference to dark-adaptation and light-adaptation. J. Exp. Zool.
77:215–249.
36. Shettles, L. B. 1938. Effect of ultraviolet light and X-rays on Peranema
trichophorum. J. Cell Comp. Physiol. 12:263–272.
37. Shettles, L. B. 1939. The relation between salts, hydrogen-ion concentration
and sensitivity to light in Peranema trichophorum. J. Exp. Zool. 81:473–477.
38. Sineshchekov, O. A., K. H. Jung, and J. L. Spudich. 2002. Two rhodopsins
mediate phototaxis to low- and high-intensity light in Chlamydomonas rein-
hardtii. Proc. Natl. Acad. Sci. USA 99:8689–8694.
39. Stechmann, A., and T. Cavalier-Smith. 2002. Rooting the eukaryote tree by
using a derived gene fusion. Science 297:89–91.
40. Teller, D. C., T. Okada, C. A. Behnke, K. Palczewski, and R. E. Stenkamp.
2001. Advances in determination of a high-resolution three-dimensional
structure of rhodopsin, a model of G-protein-coupled receptors (GPCRs).
Biochemistry 40:7761–7772.
41. Triemer, R. E. 1997. Feeding in Peranema trichophorum revisited. J. Phycol.
33:649–654.
42. Waschuk, S. A., A. G. Bezerra, Jr., L. Shi, and L. S. Brown. 2005. Lepto-
sphaeria rhodopsin: bacteriorhodopsin-like proton pump from a eukaryote.
Proc. Natl. Acad. Sci. USA 102:6879–6883.
43. Williams, T. P., A. Squitieri, R. P. Henderson, and J. P. P. Webbers. 1999.
Reciprocity between light intensity and rhodopsin concentration across the
rat retina. J. Physiol. (London) 516:869–874.
44. Zadok, U., A. Khatchatouriants, A. Lewis, M. Ottolenghi, and M. Sheves.
2002. Light-induced charge redistribution in the retinal chromophore is
required for initiating the bacteriorhodopsin photocycle. J. Am. Chem. Soc.
124:11844–11845.