Aug 29, 1995 - Thus, the abundant 30 kDa protein of S3 membranes was identified as .... contrast (blue) and the anti-opsin fluorescence (green-white). Bar = 8 ...
The EMBO Journal vol.14 no.23 pp.5849-5858, 1995
Chiamyrhodopsin represents photoreceptor
a new
Werner Deininger, Petra Kroger, Utah Hegemann, Friedrich Lottspeichl and Peter Hegemann2 Institut fur Biochemie I, Universitat Regensburg, 93040 Regensburg and 'Max-Planck-Institut fiir Biochemie, 82152 Martinsried, Germany 2Corresponding author
In order to find optimal light conditions for photosynthetic growth, the green alga Chlamydomonas uses a visual system. An optical device, a rhodopsin photoreceptor and an electrical signal transduction chain that mediates between photoreceptor and flagella comprise this system. Here we present an improved strategy for the preparation of eyespot membranes. These membranes contain a retinal binding protein, which has been proposed to be the apoprotein of the phototaxis receptor. The retinal binding protein, which we named chlamyopsin, was purified and opsin-specific antibodies were raised. Using these antibodies, the opsin was localized in the eyespot region of whole cells during growth and cell devision. The opsin cDNA was purified and sequenced. The sequence reveals that chlamyopsin is not a typical seven helix receptor. It shows some homology to invertebrate opsins but not to opsins from halobacteria. It contains many polar and charged residues and might function as a light-gated ion channel complex. It is likely that this lower plant rhodopsin diverged from animal opsins early in opsin evolution. Keywords: calcium channel/green algae/photoreceptor/plant
rhodopsin/phototaxis
Introduction To orient in its light environment, Chlamydomonas developed a visual system which enables it not only to measure the light intensity but also to detect the direction from which the light is coming. Direction of light incidence is recognized with the help of the eyespot, which looks like an orange spot having a diameter of 1-1.5 ,um in an almost equatorial position of the cell. Though the function of the eyespot has been the subject of scientific attention for many years, only Foster and Smyth (1980) have come up with an elegant interpretation which states that the layers of carotenoid-containing vesicles operate as an interference reflector (quarter wave stack) and thereby perfectly accomplish the role of an optical device. The eyespot produces a front to back contrast of factor 8 (Harz et al., 1992). This is >5 times higher than the value realized by absorption and scattering, and by unisotropic chromophore orientation. Due to these optical properties of the eyespot, the eyespot overlaying part of the plasmalemma has been regarded as the most probable Oxford University Press
type of
sensory
location of the photoreceptor. Yoshimura (1994) measured photocurrents upon stimulation with polarized light. His results confirm the residence of the receptor in the eyespot, and his data suggest that the chromophore is oriented parallel to the eyespot membrane. The nature of the photoreceptor was first investigated by action spectroscopy at low light levels, resulting in threshold action spectra. The action spectra for phototaxis, like those for flash-induced phobic responses, are rhodopsinlike, having maxima close to 500 nm (Foster et al., 1984; Uhl and Hegemann, 1990). Moreover, in blind retinaldeficient cells, movement responses can be restored by incubating the cells with all-trans retinal or distinct retinal analogues. All behavioural light responses are restored by the same set of analogues (Hegemann et al., 1991; Lawson et al., 1991; Zacks et al., 1993). All-trans retinal and alltrans retinol are by far the most dominant retinoids in whole cells and in putative eyespot membranes. In general, the data suggest that the photoreceptor for both behavioural responses is the same rhodopsin with an all-trans retinal chromophore which is activated by an all-trans to 13-cis isomerization (Kroger and Hegemann, 1994; Zacks and Spudich, 1994). Despite the quite detailed information that has been gained about the chlamyrhodopsin chromophore purely from behavioural studies, little is known about the protein itself. A retinal binding protein with a molecular weight of 30 kDa was identified initially in putative eyespot membranes. In the same membrane preparation, a rhodopsinlike absorption was recorded by absorption difference spectroscopy (Beckmann and Hegemann, 1991). A detailed spectroscopic analysis has been precluded by the huge amount of carotenoids present even in the final eyespot membrane preparation. Since the [3H]retinal label was weak and the rhodopsin absorption was only a few milli-optical density units (mOD), the presence of other retinal proteins could not be excluded. Therefore, the labelled 30 kDa protein had not been generally accepted as the photoreceptor. Later, retinal-deficient cells were reconstituted with [3H]retinal. Also, in these cells, the 30 kDa protein had been identified as the only retinal protein. This substantiated the two conclusions that the 30 kDa protein is the photoreceptor and that this receptor mediates all movement responses (Kroger and Hegemann, 1994). As concluded from electrical measurements with suction electrodes, chlamyrhodopsin triggers a sequence of photoreceptor and flagellar currents, which appear in a time range of 100 ms after a flash. The photoreceptor current located within the eyespot region appears with a delay of
