The Distinct Signaling Mechanisms of Microbial Sensory Rhodopsins in Archaea, Eubacteria and Eukarya

Authors

  • Kwang-Hwan Jung

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    1. Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University, Shinsu-Dong, Mapo-Gu, Seoul, Korea
      *email: kjung@sogang.ac.kr (Kwang-Hwan Jung)
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  • This invited paper is part of the Symposium-in-Print: Photobiology in Asia.

*email: kjung@sogang.ac.kr (Kwang-Hwan Jung)

Abstract

Most of the known archaeal-type microbial rhodopsins are retinal-binding ion transporters, such as bacteriorhodopsin (BR) and proteorhodopsin (PR). Their identification is the result of extensive studies of their photochemical and biophysical properties. The cells containing these pigments, however, use other microbial rhodopsins as photosensors to monitor environmental light signals. From the early studies of sensory rhodopsin I (HsSRI) in Halobacterium salinarum and sensory rhodopsin II (NpSRII) in Natronomonas pharaonis, we now know that several microbial sensory rhodopsins in the other major domain of life relay information on light intensity and quality to the cell. Three of the most studied photosensory transduction mechanisms of these microbial rhodopsins are dealt with in this review. We discuss recent progress in the understanding of genomic organization, photochemical properties and photosignaling mechanisms with respect to biological function.

Introduction

Diversity of microbial rhodopsins: transport and sensory rhodopsins

Microorganisms have evolved into efficient photo-energy converters (1–3) and sensitive photodetectors that monitor temporal variations of light intensity and light quality. How then do many living organisms sense light? One of the most widely used photosensitive proteins is rhodopsin, which involves a relatively well-studied signal transduction process (4–8). Rhodopsins can be divided into two types or classes and each of these types has been characterized (4,7). Type I rhodopsins function as light-driven ion transporters and sensory transducers and have been found in microbes, while type II rhodopsins are found in the photoreceptor cells of animal eyes, and control the activation of hetero-trimeric G-proteins leading to visual reception (6,7).

Type I microbial rhodopsins are photoactive 7-transmembrane helix proteins that use all-trans or 13-cis retinal as their chromophore. The type I microbial rhodopsins were observed initially in Halobacterium salinarum in the Archaea domain and appeared to grow in extreme halophilic conditions. Genome sequencing, cDNA library searches and the environmental genomics of ocean water have revealed many archaeal-type rhodopsins in the other two major domains of life, namely Bacteria and Eukarya over the past six years (Fig. 1) (7,9,10). Most of the newly found microbial rhodopsins are in the proteorhodopsin (PR) families (2,3) where they are involved in converting the energy of a light quantum to a proton motive force by proton pumping through the plasma membrane of the cell (11). There are several sensory rhodopsins (12,13). In general, the difference between transport and sensory rhodopsins seem to be characterized by the rate of photocycle and the amino acid residue at the proton donor position (Asp96 in BR). If the photocycle is more than 300 ms and the proton donor group is neither aspartate nor glutamate, the rhodopsin may serve a sensory role rather than that of pumping protons. However, there are some exceptions. For example, the photocycle of BPR (blue absorbing proteorhodopsin) is not fast (14) unlike GPR (green absorbing proteorhodopsin) and the decay rate of M state (a deprotonated Schiff base) for ASR (Anabaena sensory rhodopsin) is not slow like other sensory rhodopsins (15). The focus of this review will be a discussion of the three most studied microbial sensory rhodopsins (Fig. 2) (13).

Figure 1.

 Primary sequence comparisons of 13 microbial rhodopsins. We selected several opsin genes from each domain of life. Archaea- BR: Halobacterium salinarum bacteriorhodopsin, SRI: H. salinarum sensory rhodopsin I, NpSRII: Natronomonas pharaonis sensory rhodopsin II; Eubacteria- GPR: γ-proteobacterium (BAC31A8) proteorhodopsin, BPR: γ-proteobacterium (HOT75m4) proteorhodopsin, GR: microbial rhodopsin from Gloeobacter violaceus PCC 7421, ASR: sensory rhodopsin from Anabaena (Nostoc) sp. PCC7120; Eukarya- Fungi- NR: rhodopsin from Neurospora crassa, LR: rhodopsin from Leptosphaeria maculans, and CR: rhodopsin from Cryptococcus neoformans, Algae- CSRA & CSRB: Chlamydomonas reinhardtii sensory rhodopsins A and B (N-terminal portions), Aceta I: rhodopsin from Acetabularia acetabulum I. Conserved residues are marked with black background and the 22 residues in the retinal-binding pocket are marked with asterisks. The position of BR at Arg82, Asp85 and Asp96 in helix C and Glu194 and Glu 204 are marked.

Figure 2.

 Three different signal transduction pathways of microbial sensory rhodopsin. The drawing for the seven transmembrane helices of Anabaena sensory rhodopsin and halobacterial sensory rhodopsin are based on X-ray crystal structure, and the domains of CSRA are based on secondary structure predictions. The retinal chromophore is in red and the colors of each rhodopsin mimic the color of the pigments. Transducer proteins and domains are shown in green. The amino acid differences at the corresponding positions of proton acceptor and donor in BR are shown in helix C for each microbial sensory rhodopsin.

The first example is from H. salinarum, which contains a family of four structurally similar type I rhodopsins which capture light energy to power electrogenic ion transport (pump) or to control flagellar rotation for motility (phototaxis) (7). When oxygen is plentiful, the cells grow chemoheterotrophically and synthesize a repellent photoreceptor, sensory rhodopsin II (HsSRII), as their only rhodopsin. When oxygen tension decreases in response to the high density of cells and high intensity of solar energy, the cells produce bacteriorhodopsin (HsBR) and halorhodopsin (HsHR) that function as light-driven proton and chloride pumps, respectively (16,17). The proton motive force generated by BR and HR is used for ATP synthesis. Sensory rhodopsin I (SRI) influences the cells to swim toward favorable light intensities and utilizes the color of the light optimal for the operation of BR and HR, simultaneously avoiding damage by UV radiation (18). An example of a type I sensory rhodopsin is phoborhodopsin (also called NpSRII), which mediates a photophobic response through its transducer in Natronomonas pharaonis (4,19).

The second examples are two functional type I rhodopsins from the green eukaryotic alga Chlamydomonas reinhardtii which were recently identified and extensively characterized and shown to mediate the ability of photons to control cellular motility (20–24). Unlike halobacterial phototaxis in which cells sense the intensity of the light by a two component regulatory system, Chlamydomonas reinhardtii cells respond to the direction (phototaxis) and intensity (photophobic response) of light (7). The Chlamydomonas Sensory Opsin A and B genes (CSOA and CSOB) encode 712 and 737 amino acid proteins, respectively, and the N-terminal regions of Chlamydomonas sensory rhodopsins have similarities to the seven transmembrane helices of archaeal-type rhodopsin along with more than 60% of the retinal-binding pocket being conserved (20,21,23). The physiological roles of Chlamydomonas sensory rhodopsin A (CSRA) and Chlamydomonas sensory rhodopsin B (CSRB) were assigned utilizing RNAi suppression of the gene expression (23). The CSRA and CSRB mediate both phototaxis (orientation) and photophobic (stop) reactions to high- and low-intensity light, respectively (24). The action spectrum of the cell with each RNAi construct was demonstrated using the analysis of electrical currents and the cell’s motility responses. The cell with enriched CSRA and suppressed CSRB has an action spectrum maximum near 510 nm and this pigment mediates a fast photoreceptor current that saturates at high light intensity (20,23). In contrast, CSRB enriched cells show an action spectrum maximum in the blue region at 470 nm and they generate a slow current saturating at low light intensity (21,23). The N-terminal 300 residues of CSRA (20,25) and CSRB (21,25) which has significant homology to archaeal rhodopsins, have been shown to exhibit light-induced proton or cation-channel activity in Xenopus oocytes. The relationship of these channel activities to their control of motility-regulating Ca2+ currents in C. reinhardtii is not clear (26). Three additional microbial rhodopsins, CSRC, CSRD and CSRE, also termed cop5, cop6 and cop7 (27) were predicted from Chlamydomonas genome sequencing but it remains to be determined as to how the proteins are expressed and just what their functions are in cells.

The third example is a sensory rhodopsin gene in Anabaena (Nostoc) sp. PCC7120 (cyanobacteria) which was found in the life domain of Eubacteria (15,28). This opsin gene could be expressed in Escherichia coli, and bound all-trans retinal to form a pink pigment (λmax 549 nm: dark-adapted form) with a photochemical reaction cycle containing K, L, M, N (at low pH) and O photo-intermediates (15,29,30). A typical photochromic equilibrium of bound retinal in ASR as a result of the 13 cis light-adapted form was observed (31,32). The signaling state of ASR has not been determined; it may be the M or O states of the pigment or the ratio of all trans and 13-cis forms at initial state of ASR (30,31). We speculate that the physiological role of ASR is to control the ratio between phycocyanin and phycocyanoerythrin biosynthesis (33,34) (Fig. 2).

Genomic organization: operon vs exon/intron

A major difference in their genome structures is that in prokaryotes there is a clustering of several functionally related genes controlled by one promoter, an operon, but in eukaryotes there is a monocistronic arrangement of the genes. The BR and PR transport rhodopsin genes in prokaryotes (Archaea and Eubacteria) are usually located alone in the genome (Fig. 3) except for the PR gene, which is in an operon of the beta-carotene biosynthesis genes (ctrE, I, B, Y) and a retinal synthase gene (blh) for the retinal production (35,36). In contrast, the microbial sensory rhodopsin genes are always assembled together with the transducer gene adjacent to the opsin gene (Fig. 3) (37). In the HtrI/SRI operon of H. salinarum, the opsin gene of SRI shares one base pair with the termination codon of the HtrI gene and in the case of the NpHtrII/SRII operon, only two base pairs are between the NpHtrII and SRII genes. ASR and the 14 kDa transducer gene are adjacent to each other and separated by 16 base pairs under the control of the same promoter.

Figure 3.

 The prokaryotic and eukaryotic opsin genes are schematically presented for comparison. The transport and sensory opsin operons for each prokaryote are shown. Introns are indicated at the position of each eukaryotic opsin gene. The red colored region is for the opsin gene and the number indicates the length of the rhodopsin protein.

The characteristic eukaryotic genome structure is an exon/intron sequence in the gene and yet is monocistronic. To date, we are aware of only four genomic sequences of microbial rhodopsins in the fungi and green algae. The Neurospora opsin gene contains two introns and the Cryptococcus opsin gene has four; the locus of each intron differs from each other. Chlamydomonas opsins A and B contain 14 and 19 introns, respectively (23) and each of 10 and 13 introns are localized at the seven transmembrane region of these genes. Based on the different locations of the introns of similar eukaryotic microbial opsin genes, we suggest that this supports the concept of “intron late” than “intron early” to exon shuffling.

Photochemical and biophysical properties

The photochemical properties of microbial sensory rhodopsins SRI and NpSRII have been studied in the presence and absence of their transducers (9). Also, an interesting photochromic phenomena of ASR has been reported (31,32). The Chlamydomonas sensory rhodopsins have been expressed in Xenopus oocytes, mammalian neurons, human HEK293 cells, autaptic hippocampal neurons, PC12 cells, and mouse retinal neurons (20,21,38–41). Unfortunately, we have so far been unable to pursue photochemical studies of these rhodopsins.

SRI in H. salinarum is an attractant photoreceptor for orange light (587 nm) and a repellent photoreceptor for UV (373 nm) together with orange light. This is because the photochemical reactions are produced by either one-photon (orange light) or two-photon (orange + UV light) excitations of SRI (Fig. 4a). As characterized by kinetic flash spectroscopy, SRI exists in three spectrally distinct forms: SR587max = 587 nm) species is a dark-adapted state and orange light absorption by SR587 initiates a series of reactions which produce the long-lived S373max = 373 nm) species; S373 contains a deprotonated Schiff base linkage like M state in BR. S373 thermally returns to SR587 with a halftime of ∼1000 ms, completing the SRI photocycle (Fig. 4a). The decay of S373 is pH-dependent in the absence of its transducer HtrI (43). S373 is also photoreactive and converts to a second long-lived species Sb510 (the superscript denotes the photoconversion “back” to SR587, λmax = 510 nm), which is the repellent signaling state (44,45). The S373 species is the attractant signaling state (18) that, during its transient existence, transmits through HtrI, signals to inhibit swimming reversals and the Sb510 state is the repellent signaling state for inducing the flagellar reversals by CheA and CheY phosphorylation (46). CheA and CheY are a histidine kinase and the response regulator, respectively, in the most common two-component signal transduction system of prokaryotes and eukaryotes outside of the animal kingdom. The photocycle of NpSRII is slow compared to the photocycle of BR (Fig. 4b). The ground state of NpSRII produces M and O photo-intermediates after green light illumination. NpHtrII binding accelerates the rate of formation of the M and the decay of the O intermediates (47–50). Recent studies show the formation of a late M intermediate is correlated with the formation of the signaling state for the photophobic response to green light (51,52).

Figure 4.

 The photocycle of SRI (a), NpSRII (b) and ASR (c). Superscripts M, O, and C indicate correspondence to similar thermal intermediate states of the BR photocycle. Subscripts are the wavelength maxima observed for the pigments or as calculated for their photo-intermediates from flash photolysis analysis. The decay rate of each photo-intermediate is indicated by milliseconds (ms).

The photochemical characteristic of ASR is atypical because the light/dark equilibrium of all trans/13-cis retinal interconversion shows an opposite ratio from that found in BR (31,32). The K, L, M, and O intermediates are produced from ASR which contains the all-trans retinal form and the rate of long-lived O decay is about 260 ms. The ASR of 13-cis retinal, however, produces a red-shifted photo-intermediate (C in BR) with a decay of about 750 ms (Fig. 4c) (47). Unlike BR and NpSRII, the proton acceptor of ASR is not Asp75 (which corresponds to the position of Asp 85 in BR) (29,53). Residue Asp217 in the cytoplasm serves as a proton acceptor as identified by mutant analysis using FTIR spectroscopy (30). Another unusual characteristic of ASR is the slow rate of M formation which could be explained by this long distance between the protonated Schiff base and Asp217. The direction of proton movement, from protonated Schiff base to Asp217, is supported by a study of the negative photo-electric signal from pigments expressed in intact E. coli cells (54). The rate of the photocycle is enhanced by nearly 20% in the presence of its soluble 14 kDa protein.

The CSRA and CSRB pigments (also termed Channel rhodopsins 1 and 2) cannot be expressed using a typical heterologous expression system (20,21). As only 104–105 molecules of rhodopsin are in a Chlamydomonas cell, attempts to purify the Chlamydomonas sensory rhodopsins were not successful, but they can be detected using antibodies against CSRA (24). These techniques showed the localization of the pigment at a small part of cytoplasmic membrane confined in the eyespot, which is a carotenoid-rich disk-shaped organelle (42). CSRA (ChR1) and CSRB (ChR2) were successfully expressed in the Xenopus oocytes and light-induced passive proton and cation (H+, Na+, K+, Ca2+) movement across the membrane were observed (25,55).

Photosignaling mechanisms and biological functions

Phototaxis by a two-component regulatory system in Archaea

Archaeal sensory rhodopsins mediate color-sensitive phototaxis responses in halobacteria. Light signals from HsSRI and NpSRII are transferred to the cytoplasm through HsHtrI (56) and NpHtrII (57), the halobacterial transducers for sensory rhodopsins. After the genome sequencing of H. salinarum (58) and biochemical studies of the cheA, cheY, cheR, and cheB components, phosphorylation and methylation were shown to be involved in phototaxis responses as in the eubacterial chemotaxis system (46). The net migration of the cell is toward the higher intensity of attractant light and away from the repellent light to enable swimming into favorable regions, just like the eubacterial chemotaxis system (Fig. 2). Chimeric constructs have established the notion that the archaeal sensory rhodopsins SRI and SRII bind tightly to their membrane-embedded transducers via interaction with the transmembrane helices (4,59). An extensive membrane-embedded transducer-binding region has been observed in the X-ray structure of N. pharaonis SRII cocrystallized with its transducer fragments (60,61). Also, a short membrane proximal domain (also called the stimulus relay domain (18) or the HAMP domain — a conserved signal transduction domain in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases (62)) can interact with its transducer. Its influence on photochemical properties was shown by suppressor studies (7), Cys–Cys cross-linking, and in vitro binding experiments (7,63,64).

Phototaxis and photophobic response by calcium influx in green algae

Unicellular photosynthetic algae are phototactic (oriented movement) and exhibit photophobic (stop) responses that prevent them crossing a light/dark border (22). Two rhodopsins in Chlamydomonas (CSRA [ChR1] and CSRB [ChR2]) have been shown to be involved in the photomobility responses by a Ca2+ channel. The rapid electrical current changes through the outer membrane modulate an alteration of flagellar beating (23). There are two different ideas advanced to explain the photosignaling mechanisms via the sensory rhodopsins. One is a passive proton or cation influx by the activation of CSRA and CSRB which initiates a Ca2+ current by a voltage-gated Ca2+ channel (25); this may not be a result of the direct interactions of rhodopsin with the Ca2+ channel. A second hypothesis is that CSRA and CSRB use different mechanisms for signal triggering because of the different properties for electrical signals. The CSRA-induced current is so fast that it does not have a detectable delay. However, the CSRB-mediated current shows a light dependent delay and low saturation suggesting a soluble messenger (23,24).

Photochromicity by red/green light in cyanobacteria

Atomic-resolution structures for the Anabaena sensory rhodopsin and its putative transducer have been obtained (32,65). Moreover, the photochromic properties of ASR between all trans and 13-cis retinal interconversions have been studied (31). However, the physiological function of ASR is not known. The most probable function of ASR is chromatic adaptation (33,34). The interaction of the soluble 14 kDa protein, as a signal transducer, with the ASR has been suggested by affinity-enrichment measurements and by SPR (Surface Plasmon Resonance) interaction analyses (15). Therefore, the study of ASR and its transducer would extend the range of signal transduction mechanisms used by microbial sensory rhodopsins from membrane bound transducers to soluble transducers (13).

Conclusions

Most of the newly found rhodopsin genes have not been expressed and their functions remain unknown. Analysis of the new microbial rhodopsins and their expression and functional characterization is expected to reveal whether they perform as ion pumps or provide sensory functions. Also, they may be used in a variety of signaling mechanisms in nature by the type I sensory rhodopsins. A future goal of research on microbial sensory rhodopsins is to identify novel photosensory mechanisms and characterize the molecular basis of receptor/transducer interactions. It may also be possible to identify new molecular component(s) that regulate photosignaling pathways as in Chlamydomonas. The new microbial rhodopsins offer opportunities for studying the biophysical chemistry of signal generation and relay, in order to elucidate specifically the fundamental principles of sensory transduction, and more broadly the nature of dynamic interactions between receptors and their transducers.

Acknowledgements The review was supported by a Korea Research Foundation Grant (KRF 2004-042-C00113 to K.H.J.). I thank Ah Reum Choi and Soon-Goo Lee for help in drawing figures and alignment.

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