Biophysics and Physicobiology
Online ISSN : 2189-4779
ISSN-L : 2189-4779
Regular Article (Invited)
Algal rhodopsins encoding diverse signal sequence holds potential for expansion of organelle optogenetics
Kumari SushmitaSunita SharmaManish Singh KaushikSuneel Kateriya
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2023 Volume 20 Issue Supplemental Article ID: e201008

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Abstract

Rhodopsins have been extensively employed for optogenetic regulation of bioelectrical activity of excitable cells and other cellular processes across biological systems. Various strategies have been adopted to attune the cellular processes at the desired subcellular compartment (plasma membrane, endoplasmic reticulum, Golgi, mitochondria, lysosome) within the cell. These strategies include-adding signal sequences, tethering peptides, specific interaction sites, or mRNA elements at different sites in the optogenetic proteins for plasma membrane integration and subcellular targeting. However, a single approach for organelle optogenetics was not suitable for the relevant optogenetic proteins and often led to the poor expression, mislocalization, or altered physical and functional properties. Therefore, the current study is focused on the native subcellular targeting machinery of algal rhodopsins. The N- and C-terminus signal prediction led to the identification of rhodopsins with diverse organelle targeting signal sequences for the nucleus, mitochondria, lysosome, endosome, vacuole, and cilia. Several identified channelrhodopsins and ion-pumping rhodopsins possess effector domains associated with DNA metabolism (repair, replication, and recombination) and gene regulation. The identified algal rhodopsins with diverse effector domains and encoded native subcellular targeting sequences hold immense potential to establish expanded organelle optogenetic regulation and associated cellular signaling.

Significance

In our current study, rhodopsin(s) possessing effector domains associated with diverse biological processes were identified which would enable to expand optogenetic modulation of cellular processes within the cell. Encoded native signal sequences for subcellular targeting such as nucleus, mitochondria, lysosome, vacuole, and cilia were deciphered in several algal rhodopsins. The current research would enable to achieve the functional expression of rhodopsin to the desired subcellular compartment and their applications in organelle optogenetics. Our analysis would expand the applicability of microbial-type rhodopsins of algae as a preferred optogenetic tool for cilia biology, ciliopathies, and channelopathies.

Introduction

Rhodopsins widespread in many taxa of life are light-sensing proteins using retinal as a chromophore. Retinal configuration divides the rhodopsins into two broad categories-Type 1 (microbial rhodopsins) and Type 2 (Animal rhodopsins). Type 1 rhodopsins with diverse functions have been pioneered in diverse organisms like-archaea, bacteria, algae, fungi, and viruses. Type 1 rhodopsins function as light-gated ion pumps, light-driven ion channels, sensors [1], and enzymerhodopsin [2,3]. Among light gated ion-pumps, proton [4,5], sodium [6], and chloride pumps [7] have been discovered so far. Light-gated ion channels include cation channelrhodopsins [8,9] and anion channelrhodopsins [10]. Recently, bacteriorhodopsins like-cation channelrhodopsins from cryptophytes have been discovered [11]. Light-gated ion channels and pumps have been utilized for modulating cellular processes across different biological systems in a spatiotemporal manner [12]. Despite the discovery of a myriad of rhodopsins yet there remain many unexplored groups of rhodopsins with unique features and properties. Characterization of identified rhodopsins are required to expand the optogenetic application of Type 1 rhodopsins. Though the optogenetic field has gained advancement, the major challenge is to achieve the expression and trafficking of optogenetic protein to the desired subcellular compartment and integration into the membrane. Attempts have been made in this direction to target the rhodopsin to desired subcellular compartments like plasma membrane, ER, Golgi, and lysosome of the cell [13]. Type 1 rhodopsins have also been targeted to the confined compartments in neuronal cells like- the axonal compartment, somato-dendritic compartment, postsynaptic dendrites, spines, synaptic vesicles, and presynaptic terminals [13]. The functional expression of Type 1 rhodopsins at the plasma membrane has been enriched by the addition of signal peptide at the N-terminus, ER export, and/or Golgi targeting signal at the C-terminus [14,15]. Subcellular targeting is further achieved by coupling rhodopsins with peptides or the introduction of a specific interaction site destined for a specific organelle or compartment [16,17]. Previously, bacteriorhodopsin was targeted to the mitochondria by the addition of a signal sequence from COX IV [18]. The targeting of rhodopsins to the desired subcellular compartment could be simplified if the rhodopsins itself possesses the targeting sequences. We have already reported the modular algal rhodopsins and optogenetic potential of encoded effector domains [19]. In the present study, rhodopsin sequences were identified from diverse algal groups and subcellular targeting signals were analyzed in detail. The relatedness and structure-function of rhodopsins were analyzed via sequence alignment and mapping was performed for the crucial residues responsible for its function. Based on the above analysis, identified rhodopsins were categorized into light-gated ion channels or pumps, histidine-kinase rhodopsins (HKRs), and a group possessing unique retinal binding pocket diverged from ChRs.

Subcellular targeting sequences were predicted using available web-based tools. Targeting sequences for the nucleus, mitochondria, lysosome, vacuoles, endosomes, and cilia were identified in multiple rhodopsins. Rhodopsins with effector domains were segregated and proteins interacting with effector domains have been speculated for dissection of its optogenetic potential. Interestingly, nuclear and mitochondrial targeting sequences were identified in rhodopsins with effector domains associated with gene regulation and oxidative transformation.

In Type 2 rhodopsins, the ciliary targeting sequences (CTSs)-VxPx and FR motif at the C-terminus have been reported for their trafficking to the outer segment in photoreceptor cells [20]. CTSs play a very important role in different types of ciliopathies and channelopathies. Mutation of CTSs in different proteins causes inherited diseases such as retinitis pigmentosa and polycystic kidney disease [2123]. Recently, a protein sorting motif have been reported in ChR1 and are essential for their interaction with GTPases involved in protein trafficking [24]. CTSs in terms of VxPx and AXXXQ motifs were identified in various rhodopsins indicating their targeting to the cilia [25,26]. Our current analysis would expand the existing optogenetic application of Type 1 rhodopsins for targeting to destined subcellular compartments. Rhodopsin with CTSs could be utilized for the optogenetic modulation of the protein trafficking in the cell and treatment of different ciliary defects.

Materials and Methods

Sequence Retrieval, Identification, and Domain Analysis of Algal Rhodopsins

Rhodopsin sequences were retrieved from the JGI Phycocosm database (https://phycocosm.jgi.doe.gov/phycocosm/home) [27]. Bacteriorhodopsin from Halobacterium salinarum [PDB ID: 1KGB], channelrhodopsin 1 [PDB ID: 3UG9] and 2 [PDB ID: 6EID], and histidine-kinase rhodopsin (accession no: AAQ16277.3) from Chlamydomonas reinhardtii were used as the template for sequence retrieval by BLAST tool. Sequence identity has been summarized in “Supplementary Table S1”. Domains encoded within the retrieved sequences were predicted by the InterPro (https://www.ebi.ac.uk/interpro), Expasy Prosite server (https://prosite.expasy.org/), conserved domain architecture retrieval tool (CDART) [28] and the conserved domain database (CDD) [29]. Sequence encoding at least five-transmembrane helices with conserved lysine in the seventh helix were considered further for homology, relatedness analysis, and presence of cellular targeting signals. Domains showing significant presence in rhodopsin sequences after the analysis with at least two conserved domain analysis servers and with low e-value were considered for further analysis.

Sequence Relatedness and Evolutionary Pattern of Rhodopsin Domain

Rhodopsin domain of well-characterized microbial-type rhodopsins and identified algal rhodopsins were aligned by the ClustalW program using default parameters [30] and phylogenetic analysis was performed by maximum-likelihood method in MEGA7 software [31]. Based on phylogeny, rhodopsin sequences were categorized into distinct groups-algal proton pumps, channelrhodopsins, schizorhodopsins, and HKRs. Sequence alignment was performed with regard to channelrhodopsin, bacteriorhodopsin, and other well-characterized rhodopsins. Identity shading and color editing were performed via the BioEdit tool (http://en.bio-soft.net/format/BioEdit.html). Critical residues forming the retinal binding pocket(s) were mapped in all rhodopsins. Channelrhodopsin-like, algal proton pumps-like, and schizorhodopsins-like sequences were analyzed in detail in terms of residues important for ion channel kinetics and/or proton pumping function.

Protein Network Analysis of Unique Domains Coupled with Algal Rhodopsins

To study the interacting partners of unique effector domains coupled to algal modular rhodopsins, protein-protein interaction analysis for each of the unique domains was performed. Protein network for BAH (bromo adjacent homology), EI24 (etoposide-induced protein 2.4 homolog), 2OG-Fe(II)-oxygenase (2OG-Fe(II)-Oxy), PPR (pentatricopeptide repeat) and Spo11 (DNA topoisomerase subunit VI A) from Arabidopsis thaliana (for BAH and EI24), Homo sapiens (for Spo11 domain), Escherichia coli, Saccharomyces cerevisiae, and Volvox carteri, respectively, was predicted using the String version 11 [32]. STRING predicted the protein network based on Gene Ontology, KEGG, high-throughput text-mining as well as on hierarchical clustering of the association network. The evidences for the connections between two proteins were used from independent data sources. The analysis generates interaction scores as well as the FDR (false discovery rate) value, representing confidence (on a scale of zero to one) and significance of the association, respectively. The STRING output was further subjected to betweenness analysis using Cytoscape 3.7.2 [33].

Identification of Encoded Cellular Trafficking Signals within New Algal Rhodopsins

Rhodopsin sequences with at least five transmembrane helices and showing a low e-value score were selected among all the rhodopsin sequences. Mitochondrial and chloroplastic targeting sequences were predicted using MitoFates [34] and ChloroP 1.1 server [35], respectively. Signal peptide sequences were predicted using various servers such as SignalP-5.0 [36], DeepSig [37], and BUSCA [38]. Nuclear targeting signal sequences were predicted using Euk-mPLoc 2.0 [39], INSP [40], and LocNES [41] servers. Different CTSs were analyzed in rhodopsin sequences based on available literature.

Results

Phylogeny Indicates the Functional Diversification of Algal Rhodopsins

The transmembrane helices of the rhodopsin domain from newly identified sequences were aligned and phylogeny was studied in comparison to rhodopsins with pump, channel, sensory and enzymatic functions. Different colour code was assigned to clusters formed with chlorophyte channelrhodopsins (ChR), algal proton pumping rhodopsins, schizorhodopsins (SzRs), and histidine-kinase rhodopsins (HKRs) as depicted in “Figure 1”. Few rhodopsin sequences could not show explicit clustering, referred to as unknown in text and tables since a function could not be hypothesized. A group of rhodopsins branched with ChR (blue in “Figure 1”) was hypothesized to function as a light-gated ion channel. None of the rhodopsin grouped along with cation channelrhodopsin (CCR) or anion channel rhodopsin (ACR) and therefore, no further categorization was done. Another distinct clade (purple in “Figure 1”) was observed for light-gated algal proton pumps (Coccomyxa subellipsoidea rhodopsin-CsR and Acetabularia acetabulum rhodopsin-ARII), cyanophosins (CpR1 and CpR2) and cryptophyte sensor rhodopsin (CR1) suggesting a group of light-gated outward ion pumps. The clustering of rhodopsin sequences with SzRs (pink in Figure 1) indicates their functioning as light-gated inward ion-pump. The largest cluster was found to be associated with HKRs (green in “Figure 1”) suggesting their role in enzymatic function. Sequences were further analyzed for structure-function relationship.

Figure 1 

Sequence relatedness analysis of rhodopsin domain. Aligned 1–7 transmembrane helices of rhodopsin were employed for constructing phylogeny. Well characterized rhodopsins taken as reference are marked in red colour. Identified novel rhodopsin sequences clustered along with different reference sequences are marked in different colours. Light-gated ion channels (channelrhodopsin): blue; light-gated inward and outward ion-pumps: pink and purple respectively; and Histidine-kinase rhodopsins: green. Sequence identity - BR [PDB ID: 1KGB]; ChR1 [PDB ID: 3UG9]; ChR2 [PDB ID: 6EID]; ARII [PDB ID: 3AM6]; CsR [PDB ID: 6GYH]; HsHR [PDB ID: 1E12]; ClR [PDB ID: 5B2N]; SRII [PDB ID: 1H2S]; KR2 [PDB ID: 6RF6_A]; GtACR1 [PDB ID: 6CSM]; GtACR2: AKN63095.1; CrHKR1: AAQ16277.3; CrHKR2: Cre11.g467678.t1.1; CpR1 ACV05065.1; CpR2: ACZ04975.1; CR1: ABA08439.1; PoXeR: WP_051881467.1; RmXeR: WP_094549673.1; ASR: 1XIO; NpXeR: 6EYU_1; SRI: WP_010903211.1; AR1: P69051.1 & AR2: P29563.1; NpHR: AAA72222.1; SzR1: TFG18381.1; SzR2: QBQ84358.1; GtCCR2: ANC73518.1 & 4: ARQ20888.1; GLR: EHQ02967.1; BPR: Q9AFF7.2; GPR: Q9F7P4.1; XR: WP_011404249.1; OlPVRII: 6SQG; BeGC1: AIC07007.1; CaRhGC: AVZ03094.1; RhPDE: XP_004998010.1.

Effector Domains are Encoded at C- and N-terminus of Algal Rhodopsins

Our extensive database search for rhodopsins fetched us rhodopsin sequences encoding unique effector domains at C- and N-terminus. Four rhodopsins were found to possess effector domains at C-terminus (“Supplementary Figure S4A”). Two of the rhodopsins clustering with HKRs were found to be unique-(1) MesvirRh3 directly coupled to sterile alpha motif (SAM) domain at the C-terminus of rhodopsin and (2) SceobRh2 possessing HK, RR, cyclase (Cyc), and squamosa promoter binding protein (SBP) in tandem at the C-terminus of rhodopsin. Two of the rhodopsin (CyapaRh2 and 4) with no explicit classification possess bromo adjacent homology (BAH) domain.

Four rhodopsins with effector domain at N-terminus were identified in the study (“Supplementary Figure S4B”). SymmicRh1 and 2, encode pentatricopeptide repeats (PPRs) and 2-oxoglutarate-Fe(II) oxygenase (2-OG-Fe(II)-oxy) domain, respectively. ChrveliRh16 possesses etoposide-induced protein 2.4 (EI24) domain. ChleuRh1 grouped with ChR accommodates DNA topoisomerase VI subunit A (Spo11) at N-terminus. The putative function of the encoded effector domain is described in “Table 1”. In addition, several rhodopsins possess different domains represented in “Supplementary Figure S1”.

Table 1  Putative function of the effector domain(s) coupled with rhodopsins.
Algal rhodopsin Effector domains Putative function
ChrveliRh16
(unknown)
EI24, Rh P53 regulated apoptosis
SymmicRh1
(unknown)
PPR, Rh RNA binding module
ChleuRh1
(ChR)
Spo11, Rh Replication, repair, and recombination
SymmicRh2
(unknown)
2OG-Fe (II)-Oxy, Rh Oxidative transformations
CyapaRh2/CyapaRh4
(unknown)
Rh, BAH Transcriptional regulation
SceobRh2
(Modular)
Rh, HK, RR, Cyc, SMC, SBP Transcription factor
MesvirRh3
(Modular)
Rh, SAM Protein-protein interaction

Rhodopsin from Chlamydomonas eustigma (ChleuRh1) is a Channelrhodopsin with Spo11 Domain

Homology analysis was performed for rhodopsins categorized with ChR and key residue(s) for their functions were mapped to ChR2. Among these rhodopsins, DesarRh1, UlvmuRh, ChlscRh1, and TetstrRh1 lack the first transmembrane helix. In addition, UlvmuRh, ChlscRh1, and TetstrRh1 also lack initial amino acids of the second transmembrane helix including the important glutamate residues. The conserved helices 2–7 are depicted in “Figure 2”, and key residues responsible for ion selectivity and kinetics (E90, K93, and E97) corresponding to ChR2 were marked by the black arrow. Key residues for deprotonation and reprotonation of retinal Schiff base (RSB) have been marked by the red arrow. These key residues have been mapped and compared in “Supplementary Table S2”. Mutation of residues (E90, K93, and E97) reveals their role in ion selectivity [42]. Mutation E97A showed reduced photocurrent despite its high expression and was proposed to be important for ion conductance [43].

Figure 2 

Homology and comparative analysis of identified algal ChRs with ChR1 and ChR2. Key residues mapped with ChR2 for ion selectivity, channel kinetics, and photocycle are marked with black arrows. Key residues responsible for deprotonation and reprotonation of retinal Schiff base (RSB) have been marked by the red arrow.

D156 and D253 are the proton donor and proton acceptors to and from the RSB in ChR2 [44]. C128 is hydrogen-bonded with D156 (shown as D158 in "Figure 2") and forms a DC gate that acts as a switch for the movement of ions [45]. Mutation of C128T delays the closure of the channel and therefore remains in conducting state for a longer period [46]. This mutation has enhanced the property of ChR2 to be used as an optogenetic tool. Mutation L132C increases the permeability of Ca2+ ions and leads to a large stationary current [47].

Mapping of key residues suggests that C of the DC pair is conserved in all rhodopsin but D is not conserved in four of identified rhodopsin in our analysis. E90 crucial for the lifetime of the channel was found to be mostly conserved. ChlscRh1, closest to ChR2 (phylogenetic analysis) belongs to Chlamydomonas schloesseri, closely resembles Chlamydomonas reinhardtii. ChlscRh1 lacks the first transmembrane helix and also the DC pair suggesting the alternate gating mechanism might exist. ChleuRh1 encoding Spo11 at N-terminus retains the amino acids for ion-channel activity. In addition, encoded Spo11 domain shows homology with Spo11 of yeast and archaea. Tyrosine (Y), glutamate-rich stretch and DXD motif essential for topoisomerase activity were well conserved in Spo11 domain of ChleuRh1 (“Figure 3”). Conservation of motifs led us to speculate it to be functional and therefore holds great optogenetic potential for DNA modification.

Figure 3 

Alignment of topoisomerase 6A subunit coupled to ChleuRh1 and topoisomerase 6A subunit from yeast and archaeal system. Conserved tyrosine (Y), glutamate-rich region, and DxD motif required for its enzymatic action are highlighted in the red box by the arrow. Number marked above arrow corresponds to Methanocaldococcus jannaschii.

Seven Identified Rhodopsins Correspond to the Proton Pumping Coccomyxa subellipsoidea Rhodopsin (CsR)

A group of newly identified rhodopsin(s) was found in clade possessing algal proton-pumping rhodopsin, CsR ([PDB ID: 6GYH], Coccomyxa subellipsoidea rhodopsin) and ARII ([PDB ID: 3AM6], Acetabularia acetabulum rhodopsin) in phylogenetic analysis and were predicted to function as a light-driven ion pump. The key residues responsible for photocycle and ion pumping activity were compared with algal proton-pumping rhodopsin (CsR). The proton pumping mechanism of algal proton-pumping rhodopsin is similar to the BR and hence the conserved residues. Residues responsible for deprotonation and reprotonation of RSB, and release of protons were mapped and marked by the arrow in “Figure 4” and summarized in “Supplementary Table S3”. D86 and D97 are the proton acceptor and donor from and to the RSB, respectively. R83, Y84, T90, T91, and D211 form part of proton transfer and stabilize the complex during transfer. E193 and E203 are key residues responsible for the release of protons toward the extracellular environment of the cell. Crucial residues like D86 and D97 were conserved in all rhodopsin except EnacosRh1 for which D97 is replaced by E. E193 and E203 are well conserved in all rhodopsins. Besides these common conserved residues, CsR possesses conserved Y14 which is hydrogen bonded with highly conserved R83 [48]. Y14 was also conserved in five out of seven algal ion-pumping rhodopsins. Another unique feature of CsR includes the presence of R77 in the interconnection loop between the TM2 and TM3 forming the bond with E203 and I198 [48]. Conservation of key residues responsible for ion-pumping suggests unique light-driven ion-pumping rhodopsin from the algal group.

Figure 4 

Sequence comparison of identified rhodopsins clustered with algal proton pumping rhodopsins (CsR and ARII). Residues corresponding to photocycle and ion-pumping of CsR are marked with the arrow. Helices 1–7 are shown in the red box in the alignment as in CsR: Coccomyxa subellipsoidea rhodopsin.

Some of the algal rhodopsin sequences clustered with cyanophopsin1 and 2 (CpR1: ACV05065.1 and CpR2: ACZ04975.1) and cryptophyte sensor rhodopsin (CR1: ABA08439.1). Since, the key residues for CpRs and CR1 are not yet defined, the residues were also mapped with algal proton-pumping rhodopsins (CsR and ARII) represented in “Supplementary Figure S2” and “Supplementary Table S3”. Crucial residue D86 (proton acceptor) in algal proton pumping rhodopsin is mostly conserved in algal sequences clustering with CpR1, CpR2 and CR1. CpR1 and CR1 possess N at position 97 instead of D (proton acceptor in proton pumping rhodopsins), While CpR2 possesses S at this position. Rhodopsin of this cluster majorly possesses D or E as proton donor and acceptor resembling CsR. CR1 possesses H instead of highly conserved R residue at position 83 (corresponding to CsR). E193 and E203 in CsR form proton release complex. E193 is replaced by T in CpR1 and by Q in CR1. Proton release complex (E193 and E203) was mostly conserved in cluster. Y14 forming a hydrogen bond with R83 in CsR is not conserved in these sequences. Whereas R77 uniquely forms a bond with E203 and I198 in CsR is conserved in these sequences. Conservation of proton pumping residues suggests that many of the rhodopsins may act as light-gated proton pumps while others showing variability might have different functions or other mechanism of proton transfer.

Rhodopsins Corresponding to Schizorhodopsins in Phylogeny Lack Functional Key Residues

Schizorhodopsins (SzRs) are light-gated inward proton pumps first found in Asgard archaea, closest to the eukaryotes. Light-gated inward ion pumps have also been identified in bacteria and other microorganisms named as xenorhodopsins (XeRs). The mechanism of proton transfer in SzRs differs from XeRs. In our analysis, many algal rhodopsins clustered close to the SzRs and none with the XeRs. Therefore, algal rhodopsins clustered close to SzRs were aligned and key residues were mapped with SzR1 (TFG18381.1), SzR2 (QBQ84358.1), SzR3 (TFF95899.1), and SzR4 (TFG21677.1, [PDB ID: 7E4G]) (“Supplementary Figure S3” and “Supplementary Table S4”). One of the unique features of SzRs is F corresponds to D85 (proton acceptor) in BR [49], In algal SzR-like sequence, this position is taken by Q. SzRs possess C at position 90 with respect to BR, which also corresponds to the C of DC pair in ChRs. This position is conserved in SzR-like algal sequences. The crystal structure of SzR4 suggests that E81 is critical for inward proton release. Mutation of E81 to Q in SzR4 resulted in the loss of H+ transport activity. In algal SzR-like sequences, E81 is replaced by the Q. N100 and V103 in SzR4 are the positions responsible for the spectral tuning of SzRs. In algal SzRs-like sequences, N100 is mostly replaced by T and V103 by L, M, T, and C. Other most conserved residues in SzRs are compared in algal sequences and marked by the arrow in (“Supplementary Figure S3”). Some of the positions were observed to be conserved while some with variability. Algal SzR-like sequences might have light-mediated inward ion pumping activity with mechanism different than SzR since these lack E81 but are found closest to SzR in phylogenetic analysis.

Since HKR(s) is the least studied group and key residues for photocycle are yet to be defined. The key residues for HKRs have been mapped with respect to BR and have been discussed previously [19].

Encoded Native Organelle Targeting Signal Sequences in Algal Rhodopsins and Their importance in Organelle optogenetics

Signal sequences and motifs are employed for protein enrichment and trafficking to desired subcellular compartments for advancements in subcellular optogenetic applications (shown in “Figure 5”) [13]. For protein targeting to various subcellular locations, a large number of targeting signal sequences have been known in different organisms (mentioned in “Table 2”). Different signal sequences for various subcellular compartments were predicted in identified algal rhodopsins using different software. Rhodopsin sequences showing high accuracy score for valid prediction, are shown in “Table 2”.

Figure 5 

Schematic summary of cellular route of protein trafficking to different cell organelles guided by encoded signal sequences for specific organelles. Rhodopsin could be targeted to the plasma membrane and different subcellular compartments directed via encoded signal sequences such as ETMs (endosome targeting motifs), NTSs (nuclear targeting signal sequences), SPs (signal peptides), MTSs (mitochondrial targeting sequence motifs), VTMs (Vacuolar targeting motifs) and CTSs (ciliary targeting sequences). ECM: extracellular matrix.

Table 2  Summary of signal sequences in identified algal rhodopsins, targeting different cell organelles such as mitochondria, chloroplast, nucleus, and secretory pathways.
Organism Sequence name Nuclear targeting signals (NTS)
Chlamydomonas eustigma NIES-2499 ChleuRh1
ChR
LRKLK [82]
Edaphochlamys debaryana CCAP 11/70 EdadeRh1
ChR
RKKTK [83,84]
Cyanophora paradoxa CyapaRh2
unknown
SFSEDGRKLRVRRFLRARRMARVQ [85]
Cyanophora paradoxa CyapaRh4
unknown
NKKIK [84]
Chlamydomonas schloesseri ChlscRh3
HKR
KGALKRKSSFREKGTKVRVL, EATARWIKAKRGMH [51,85]
Mesostigma viride NIES-296 MesvirRh1
HKR
PSLGPRRRKPHSFDDRAF [51,86]
Chlorokybus atmophyticus CCAC 0220 ChlatRh
HKR
GKRKL [51,85]
Bathycoccus prasinos RCC1105 BatpraRh
HKR
EQKRKVPP [87,88]
Tetraselmis striata TetstrRh3
HKR
VRRLRI [86]
Micractinium conductrix SAG 241.80 MicondRh
HKR
FERRPVKRQL [89]
Picochlorum soloecismus DOE101 PicspRh
HKR
ARRKI [51]
Symbiochloris reticulata SymretAfRh3
HKR
DKKRL [82]
Trebouxia sp. A1-2 TrebspRh2
HKR
LAKSKKHLPKRAA [90]
Chlamydomonas eustigma NIES-2499 ChleuRh2
HKR
RKLREL [91]
Chlamydomonas incerta ChlinRh2
HKR
KGALKRKSSFRKG, RWSKAKRGMH
Edaphochlamys debaryana CCAP 11/70 EdadeRh3
HKR
VKGALKRKSSFRE
Scenedesmus sp. ScespRh
HKR
VGARRPRYR, LGQLFRRRLSKKGD [91,92]
Scenedesmus obliquus SceobRh5
HKR
VQRKLRAG [51]
Organism Sequence name Signal peptide (SP)
Chlamydomonas incerta SAG 7.73 ChlinRh1
ChR
MAYVMNWIAFAISVVVLAWYAYEA
Cleavage site: 24–25
Vitrella brassicaformis VitbrasRh1
BR
MRSSVVVCVLLALVAVTRA
Cleavage site: 19–20
Chromera velia CCMP2878 ChrveliRh1
BR
MKSAQSFVIALCVLAALVSA
Cleavage site: 20–21
Symbiodinium microadriaticum SymmicRh2
unknown
MGVLRAILLLASLSKLAYA
Cleavage site: 19–20
Organism Sequence name Mitochondrial targeting sequence (MTS)
Enallax costatus EnacosRh2
ChR
HHVMRSLLNE (HHBPHH); MPP: 15 [93,94]
Phaeocystis globosa PhagloRh
BR
VSHFVPGDGQLV; MPP: 31 [93,95]
Symbiodinium. microadriaticum SymmicRh2
unknown
LLLASLSKLA, WLRLC; MPP: 33 [93,95]
Organism Sequence name Vacuolar/endosome/lysosome targeting signals
Chromera velia CCMP2878 ChrveliRh1
BR
EGASLI, EQGGLL, LGFILL, DLGLL, DTELL, YSQY, YKQF, YKQF, YYDW
Cyanophora paradoxa CCMP329 CyapaRh1
BR
YADY, YVMF, YSPF, YIDW, DLALL
Phaeocystis globosa PhagloRh
BR
EVPCLL, ENLDLL, YIDW, YQTF
Chromera velia CCMP2878 ChrveliRh16
unknown
DQSLI, LATFLL, YAVF, YVYF, YVYF
Phaeocystis antarctica CCMP1374 v2.2 PhaantRh
BR
YIDW
Trebouxia sp. A1-2 TrebspRh1
BR
YVDW, DLXLL
Scenedesmus obliquus SceobR5
HKR
DRGLLL, DNVRLI, EVARLL, LVQELL, LQVLLL, LSTALL, LQQQLL, LQQVLL, LLAGLL, LAASLL, YVQW
Symbiodinium microadriaticum CCMP2467 SymmicRh2
unknown
LRAILL, DLNGLL, EFCELL, LASVLL, LREELL, DVGELL, LQHPLL, YDGF, YIDW, YLKW
Chlamydomonas schloesseri ChlscRh3
unknown
NPLY, DQENLL, YCFY, YVQW,
Chlamydomonas incerta SAG 7.73 ChlinRh1
ChR
NPLY, YPLL, YEAL, YGDI, YPDL, YCAF, YGWI, YHMV, YSAM, LFSKLL
Edaphochlamys debaryana CCAP 11/70 EdadeRh1
ChR
NPLY, LIFFLL, YETW, YVAL, YAEW, YHTV, YTFF, YVRL, YELV, YGDI

Several rhodopsin sequences were predicted for having the single consensus sequence of basic amino acids (K/R) 4–6, monopartite or as two small clusters separated by other amino acid residues (K/R)2 X10-12 (K/R)3, bipartite NLS (nuclear localization signal sequences) [50,51]. Therefore, rhodopsin sequences showing the significant presence of nuclear targeting signal sequences (NTSs) can be employed for nuclear trafficking and enrichment of proteins.

Among ChR-like rhodopsins ChleuRh1 and EdadeRh1 from Chlamydomonas eustigma and Edaphochlamys debaryana, respectively possess NTS. Two of the rhodopsin (CyapaRh2 and 4) also possess NTS. Other rhodopsin possessing NTS belongs to rhodopsin possessing histidine kinase (HK) and response regulator (RR) in common. HK and RR form a part of two-component signaling and are reported to be involved in gene regulation.

Four of the algal rhodopsin sequences (ChlinRh1, VitbrasRh1, ChrveliRh1, SymmicRh2) were also predicted for the significant presence of signal peptides (SPs). Three algal rhodopsin sequences (EnacosRh2, PhagloRh, and SymmicRh2) were predicted to possess mitochondrial targeting sequences (MTSs). SymmicRh2 is coupled with the 2OG-Fe (II)-Oxy domain responsible for carrying out oxidative transformations and might be localized on the mitochondrial membrane. Chloroplast targeting signal motifs (CTMs) were not present in any rhodopsin sequences.

Vacuolar/endosome/lysosome targeting motifs (VTMs/ETMs/LTMs) such as [DE]XXXL[LI] or DXXLL, LXXXLL, YXXΦ and NPXY/Φ (X: any amino acid residue; Φ: bulky hydrophobic residue Y, F, W, V, L, I, M) [52] were found in several rhodopsin sequences. Sequence motifs YXXΦ and NPXY were predicted in several rhodopsin sequences which direct clathrin-coated endocytosis from the plasma membrane and targeting to different endosomal compartments, vacuole, and lysosome [53]. Sequence motifs [DE]XXXL[LI] or DXXLL were found to bind with adaptor proteins and involved in the endocytosis of various receptor proteins from the plasma membrane [54] predicted in ChrveliRh1, CyapaRh1, TrebspRh1, SceobRh5, PhagloRh, SymmicRh2, ChlscRh3. Therefore, the rhodopsin sequences with remarkable signal sequence motifs for different organelles such as the nucleus, mitochondria, ER, Golgi, lysosome, vacuole, and plasma membrane could be further employed as an important optogenetic tool for enrichment and trafficking of different proteins in specific cell organelles.

Encoded Ciliary Targeting Sequences and Their Potential in Optogenetic Modulation of Cilia Biology

Proteins found in specified organelles contain various cis-acting targeting sequences for directing their localization in destined cell organelles. For ciliary targeting, ciliary proteins contain diverse ciliary targeting sequences (CTSs) in different species which speculate their role in protein trafficking through interaction with various proteins in cilia [26]. CTSs could be utilized for the optogenetic applications to study cell signaling, ciliogenesis [55,56], and treating various diseases called channelopathies [57] and ciliopathies [55,56,58]. Mutation in the conserved CTSs leads to various diseases like autosomal dominant polycystic kidney disease (ADPKD), retinitis pigmentosa, and other retinal degenerative diseases [21,23,26]. Hence, in this research we focus on the rhodopsins that have CTSs (summarized in “Table 3”). Various ciliopathies and channelopathies can be studied through these different rhodopsins.

Table 3  Summary of different ciliary targeting sequences (CTSs) in different algal rhodopsins.
Organism Rhodopsin Ciliary targeting Sequences
Chromeravelia CCMP2878 ChrveliRh6
Unknown
VQPD, FP
Symbiodinium microadriaticum CCMP2467 SymmicRh1
unknown
VQPT, YP
Chlamydomonas eustigma NIES-2499 ChleuRh1
ChR
VNPL, YP
Chlamydomonas incerta SAG 7.73 ChlinRh1
ChR
VAPS, YP
Desmodesmus armatus UTEX B 2533 DesarRh1
ChR
VQPG, FP
Edaphochlamys debaryana CCAP 11/70 EdadeRh1
ChR
VSPG, FP
Pelagophyceae sp. CCMP2097 PelagoRh
BR
VRPE, YP
Vitrella brassicaformis VitbrasRh1
BR
VIPV, YP
Cyanophora paradoxa CCMP329 CyapaRh2
unknown
VRPP, YP
Edaphochlamys debaryana EdadeRh3
HKR
VTPA, VGPG, FP
Scenedesmus sp. NREL 46B-D3 ScespRh
HKR
VIPV
Tetradesmus obliquus UTEX B 72 TetobRh4
HKR
VTPM, FG
Chromochloris zofingiensis SAG 211-14 ChrzofiRh
HKR
VVPV, FP
Scenedesmus obliquus EN0004 v1.0 SceobRh5
HKR
VGPS
Chlamydomonas schloesseri ChlscRh4
HKR
VSPG, FP
Chlorokybus atmophyticus CCAC 0220 ChlatRh
HKR
VSPR, FP
Bathycoccus prasinos RCC1105 BatpraRh
HKR
VGPE, FP
Micractinium conductrix SAG 241.80 MicondRh
HKR
VAPT, FP
Chlamydomonas incerta SAG 7.73 ChlinRh2
HKR
VSPG, FP
Chromera velia CCMP2878 ChrveliRh16
(unknown)
ARALQ
Chromera velia CCMP2878 ChrveliRh9
(unknown)
ARAFQ
Chromera velia CCMP2878 ChrveliRh2
BR
YP
Tetraselmis striata TetstrRh3
HKR
FP
Picochlorum soloecismus DOE101 PicspRh
HKR
FP
Symbiochloris reticulata SymretAfRh3
HKR
YP
Trebouxia sp. A1-2 TrebspRh1
BR
YP
Cyanophora paradoxa CCMP329 CyapaRh3
BR
YP
Cryptophyceae sp. CCMP2293 CryptoRh1
BR
YP
Pavlovales sp. CCMP2436 PavlovRh2
BR
YP
Phaeocystis antarctica CCMP1374 PhaantRh11
BR
YP
Phaeocystis globosa PhagloRh
BR
YP
Chromera velia CCMP2878 ChrveliRh1
BR
YP
Chromera velia CCMP2878 ChrveliRh2
BR
YP
Symbiodinium microadriaticum CCMP2467 SymmicRh2 unknown YP
Chlamydomonas eustigma NIES-2499 ChleuRh2
HKR
FP
Enallax costatus EnacosRh2
ChR
FP
Mesostigma viride NIES-296 MesvirRh4
HKR
YP
Mesostigma viride NIES-296 MesvirRh1
HKR
FG, AVSSQ
Tetradesmus deserticola SNI-2 TetdesRh
HKR
FP

The most frequently established CTS in rhodopsin is the VxPx motif which is found at the C-terminus of the rhodopsin sequence [20]. The VxPx motif and its modifications are the primary CTS in rhodopsins and are very essential for ciliary targeting of rhodopsins [21,26] and other proteins such as RPGR (retinitis pigmentosa GTPase regulator) [59], polycystin-1 (PC1) [23], polycystin-2 (PC2) [22], etc. This motif was found to be present in most of the newly discovered rhodopsin sequences which include several ChR-like sequences such as ChleuRh1, ChlinRh1, DesarRh1, and EdadeRh1; BR-like sequences such as PelagoRh and VitbrasRh1. Other rhodopsin sequences such as EdadeRh3, ScespRh, TetobRh, ChrzofiRh, SceobRh5, ChlscRh4, ChlatRh, BatpraRh, MicondRh, ChlinRh2 with VxPx motif belong to HK and RR in common. Another ciliary targeting motif AXXXQ in the 3rd intracellular loop of some vertebrate rhodopsins specialized for vertebrates GPCRs ciliary targeting [26], was found only in some of the rhodopsin sequences like ChrveliRh16, ChrveliRh9, and MesvirRh1. Next unexplored putative CTS, first identified in cytoplasmic helix H8 of GPCRs is the FR motif and its modified form [60], found in most of the identified rhodopsin sequences including ChrveliRh6, SymmicRh1, ChleuRh1, ChlinRh1, DesarRh1, EdadeRh1, PelagoRh, VitbrasRh1, CyapaRh2, EdadeRh3, TetobRh4, ChrzofiRh, ChlscRh4, ChlatRh, BatpraRh, MicondRh, ChlinRh2, ChrveliRh2, TetstrRh3, PicspRh, SymretAfRh3, TrebspRh1, CyapaRh3, CryptoRh1, PavlovRh2, PhaantRh, PhagloRh, ChrveliRh1, ChrveliRh2, SymmicRh2, ChleuRh2, EnacosRh2, MesvirRh1, MesvirRh4, TetdesRh. CTSs have already been utilized as an optogenetic tool for targeting the sensor proteins like SACY (soluble adenylate cyclase) without restraining protein function [56,61].

A protein sorting putative motif NPXXY interact with ADP-ribosylation factor (ARF), a GTPase involved in membrane trafficking. It is present in cytoplasmic domain of the seventh transmembrane helix of GPCRs [62,63], was found in two of the new modular rhodopsin sequences like TetdsRh, MesvirRh1, and one another rhodopsin sequence i.e., ChlscRh3.

Therefore, CTSs could be utilized as an important optogenetic tool for the modulation of protein trafficking in cilia for the treatment of different ciliopathies and channelopathies.

Protein-Protein Network Regulation Mediated with Modular Domain(s) of Algal Rhodopsin

Out of a variety of modular rhodopsin-coupled effector domains of functional importance, we have selected seven domains i.e., BAH, EI24, 2OG-Fe (II) oxygenase, PPR, Spo11, and SAM, which have not yet been characterized in the algal system. We have performed the protein-protein interaction analysis and identified the potential interacting partners for the selected domains (“Figure 6A-F”). The BAH domain for A. thaliana majorly interacts with different cysteine-rich receptor-like kinases (CRKs), 2-oxoglutarate, and Fe (II) dependent oxygenase superfamily protein and an uncharacterized protein At5g63220 (“Figure 6A”). Expression of CRKs also involves in the regulation of defense-related genes in response to the attack of plant pathogens [64,65]. The induced expression of CRK19 and CRK20 also trigger hypersensitive response-like cell death in transgenic plants [66,67]. The protein-protein interaction analysis of the EI24 domain from A. thaliana interacts with autophagy and cell cycle regulation related proteins along with some of the proteins that belong to a family of unknown function (DUF572) and uncharacterized protein At1g52720 (“Figure 6B”). The 2OG-Fe (II) oxygenase domain from A. thaliana interacts with the protein partners associated with the process of DNA modification, energy metabolism, and protein transport (“Figure 6C”). Furthermore, the interactome analysis of a PPR domain-containing protein, CCM1, from S. cerevisiae interacts with proteins generally involves in the regulation of mitochondrial–nuclear incompatibility responsible for reproductive isolation between species or hybrid incompatibility (“Figure 6D”). The interacting partners of Spo11 from H. Sapiens were involved in the regulation of the meiotic recombination process (“Figure 6E”). We have used the SAM (sterile alpha motif) domain SAMD1 from H. sapiens and predicted its interacting proteins which were found to be the pleckstrin homology domain-containing family member 1 (PLEKN1), gamma-aminobutyric acid receptor subunit delta (GABRD), nucleolar complex protein 2 homolog (NOC2L), RNA-binding protein 33 (RBM33) and RNA polymerase II subunit A C-terminal domain phosphatase SSU72 SSU72 (“Figure 6F”).

Figure 6 

Interactome showing interacting partners for BAH (bromo adjacent homology) domain (A), EI24 (etoposide-induced protein 2.4 homolog domain) (B), 2OG-Fe(II) oxygenase domain (C), PPR (pentatricopeptide repeat) (D), Spo11 (E), SAM (sterile alpha motif) (F) containing proteins from Arabidopsis thaliana (for BAH, EI24, and 2OG-Fe(II) oxygenase domain), Homo sapiens (for Spo11 and SAM domain) and Saccharomyces cerevisiae (for PPR domain), respectively. The interactions were predicted using String version 11 (https://string-db.org/) and output data were further analyzed using CytoScape 3.7.2. The size of the nodes represents number of interactions for each node and colour scale ranging from red-orange-yellow-green to blue represents confidence level from low (<0.4) to high (>0.7) values.

Discussion

Algal rhodopsins with unique modular domain organization hold the potential for expanding the optogenetic toolkit for modulating diverse cellular processes. Based on the relatedness analysis and mapping of key residues responsible for photocycle, newly identified algal rhodopsin could be categorized as ChR, HKRs, algal outward and inward proton pumping rhodopsin. Detailed analysis of rhodopsin domain organization led to the identification of rhodopsin sequences coupled with the unique effector domain. Among all domains, Spo11 coupled with a ChR-like sequence (ChleuRh1) from Chlamydomonas eustigma with highest E-value is of utmost importance. Spo11 is a DNA topoisomerase with conserved residues required for DNA metabolism. Interestingly, ChleuRh1 possesses targeting sequence(s) for the nucleus. Our analysis fetched sequence coupled to diverse effector domains like-bromo adjacent homology domain (BAH) and squamosa promoter binding protein domain (SBP) associated with gene regulation. However, no nuclear targeting sequence was present in these rhodopsins. Therefore, three mechanisms have been postulated for gene regulation by these modular rhodopsins, (i) light-mediated modulation of membrane potential leads to structural change in the effector domain allowing it to interact with an intermediate transcription factor that might migrate to the nucleus. Alternatively, the effector domain undergoes post-translational modification initiates the signaling cascade for migration of other transcription factors to the nucleus; (ii) the associated effector domain might get cleaved and move to the nucleus to regulate gene expression; (iii) since, some of the rhodopsins possess the targeting sequences for the nucleus and other organelle membranes, it might be localized in the nuclear membrane. The identified algal rhodopsins in addition to the nuclear targeting sequences, also possess signal peptides targeted for other subcellular compartments like vacuole, lysosome, mitochondria, Golgi, and cilia. Therefore, these identified algal rhodopsins hold the potential to modulate cellular response at a desired location in the cell, overcoming the major hurdle of the cellular optogenetic field.

The algal ChR(s) and HKR(s) possess ciliary targeting sequence (CTS) at the C-terminus. Many ciliary disorders arise due to the mistrafficking of ciliary proteins. Rhodopsin with CTS would enable optogenetic regulation of cilia biology and ciliary ion channel, which would empower researchers to understand the ciliopathies and channelopathies. Taken together, our current analysis would enable us to control the vast range of cellular processes including gene regulation at the desired location. The interactome analysis has predicted potential interacting partners for the selected domains (such as BAH, EI24, 2OG-Fe (II) oxygenase, PPR, Spo11, SAM) involved in regulation of the relevant signalosome and their optogenetic use in this regard. Kuo and coworkers [68] reported the BAH domain (a methyl-lysine-binding module) which directly links the DNA replication machinery and histone methylation [68]. The interactome analysis for the BAH domain from A. thaliana revealed its association with different cysteine-rich receptor-like kinases (CRKs) and it transcriptionally activated during the pathogenic invasion and oxidative stress [69]. The interactome of the EI24 domain revealed its interaction with proteins related to autophagy and cell cycle regulation. EI24 acts as a target of TP53/p53 and plays a critical role in the negative regulation of cell growth [70]. Devkota et al [71] also demonstrated the crosstalk between autophagy and the Ubiquitin-Proteasome System (UPS). In Dictyostelium cells, overexpression of EI24 showed enhanced DNA-damage repair as well as G2/M arrest in the cell cycle which is important for growth, development, and cell differentiation [72]. The protein-protein interaction analysis of the 2OG-Fe (II) oxygenase domain displayed that this domain interacts with the protein partners associated with DNA modification and energy metabolism. The 2-oxoglutarate (2OG)-Fe(II)-dependent oxygenases are distributed among a wide diversity of organisms ranging from bacteria to eukaryotes [73,74]. The 2OG-Fe(II)-dependent oxygenase family is known to control a wide variety of biological processes including chromatin modification, transcription, mRNA demethylation, mRNA splicing, biosynthesis, and catabolism of cellular metabolites, which is also evident from its interactome [75]. In E. coli, Van Staalduinen et al [76] reported the crystal structure of YcfD protein, a member of the 2-oxoglutarate (2OG)-Fe(II)-dependent oxygenase family. It acts as a ribosomal oxygenase regulating bacterial ribosomal assembly. The DNA repair protein AlkB necessary for inhibiting alkylating agent mediated toxic DNA modification in E. coli and humans also belongs to the 2-oxoglutarate (2OG)-Fe(II)-dependent oxygenase family [74]. A pentatricopeptide repeat (PPR) domain was also found coupled with the algal modular rhodopsin. The members of the PPR family are modular RNA-binding proteins that control the post-transcriptional processes like processing, splicing, editing, stability, and translation of RNAs in organelles as well as in the nucleus [77]. We also came across a Spo11 domain coupled with the algal modular rhodopsin. The protein-protein interaction analysis revealed that the interacting partners of Spo11 were involved in the regulation of the meiotic recombination process. In S. cerevisiae, Spo11 covalently attached DNA double-strand breaks (DSBs) during the early meiotic phase initiates homologous recombination which leads to meiotic synapsis and the generation of genetic diversity [7880]. We have also predicted the interactome for the sterile alpha motif (SAM) domain containing protein from H. sapiens. The SAM domain is known as one of the most prominent interaction domains, in plants and animals, of proteins including scaffolding proteins, transcriptional regulators, translational regulators, tyrosine kinases and serine/threonine kinases, involved in diverse functions [81]. Thus, the analysis revealed algal rhodopsin sequences coupled to the effector domains, having important functions in different lineages. The successful expression, desired spatial targeting and functional nature of these reported algal rhodopsin in the given biological system would pave the road map to harness the untapped potential of organelles-specific optogenetic regulation.

Conclusion

In the current study, rhodopsin sequences were identified from the algal database and were subjected to sequence relatedness analysis with that of well characterized microbial-type rhodopsins. This analysis led to the categorization of identified rhodopsins into light-gated ion-channel/pump and histidine-kinase like rhodopsins. Further, domain analysis, and mapping of residues with well-defined rhodopsins provide insight into their properties and novelty. Subsequently, motif for subcellular targeting was searched within the identified rhodopsin sequences. Sequence targeting study enabled identification of subcellular targeting sequences for mitochondria, nucleus and cilia. To get an insight of the function of effector domains encoded along with rhodopsin sequences, interactome (protein-protein interaction network) of effector domain was constructed. Our analysis fetched us with many rhodopsin sequences possessing effector domain might establish new horizon of optogenetic applications. Subcellular targeting sequences within the diverse rhodopsin sequence could be exploited for improved organelle optogenetics across the different biological systems.

Conflict of Interest

The authors declare that they have no conflict of interests.

Author Contributions

KS had performed structure-function, and evolutionary analysis of identified algal rhodopsins. SS had done conserved domain identification and prediction of cellular targeting sequences in the identified rhodopsins. MSK has performed protein-protein interaction analysis. SK conceived MS and interpreted obtained data. KS, SS, MSK, and SK wrote and edited MS.

Acknowledgements

KS and SS are thankful to DBT and CSIR for the financial support, respectively. SK is thankful to DBT and SERB for providing extramural funds.

Data Availability

The amino-acid sequences data in the current study are available in J-STAGE Data (https://jstagedata.jst.go.jp/biophysico) with the DOI of https://doi.org/10.34600/data.biophysico.21973022. Analysed data and figure will be available upon request to corresponding author.


References
 
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