Proton-transporting heliorhodopsins from marine giant viruses
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Evaluation Summary:
Hososhima et al. characterize a marine virus Heliorhodopsin as the first of its class to show ion transport activity. These bacteriorhodopsin homologs have been recently described and the present careful characterization of V2HeR3 represents an important step in the understanding of these interesting membrane proteins. Though the experiments are carried out carefully and the results, in general, support the conclusions, some experiments are needed and the interpretation of results needs to be clarified.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Rhodopsins convert light into signals and energy in animals and microbes. Heliorhodopsins (HeRs), a recently discovered new rhodopsin family, are widely present in archaea, bacteria, unicellular eukaryotes, and giant viruses, but their function remains unknown. Here, we report that a viral HeR from Emiliania huxleyi virus 202 (V2HeR3) is a light-activated proton transporter. V2HeR3 absorbs blue-green light, and the active intermediate contains the deprotonated retinal Schiff base. Site-directed mutagenesis study revealed that E191 in TM6 constitutes the gate together with the retinal Schiff base. E205 and E215 form a PAG of the Schiff base, and mutations at these positions converted the protein into an outward proton pump. Three environmental viral HeRs from the same group as well as a more distantly related HeR exhibited similar proton-transport activity, indicating that HeR functions might be diverse similarly to type-1 microbial rhodopsins. Some strains of E. huxleyi contain one HeR that is related to the viral HeRs, while its viruses Eh V-201 and Eh V-202 contain two and three HeRs, respectively. Except for V2HeR3 from Eh V-202, none of these proteins exhibit ion transport activity. Thus, when expressed in the E. huxleyi cell membranes, only V2HeR3 has the potential to depolarize the host cells by light, possibly to overcome the host defense mechanisms or to prevent superinfection. The neuronal activity generated by V2HeR3 suggests that it can potentially be used as an optogenetic tool, similarly to type-1 microbial rhodopsins.
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Author Response
Reviewer #1 (Public Review):
This manuscript provides the first experimental evidence that some members of the newly discovered heliorhodopsins can function as proton channels. The authors provide evidence of this transport function as well as a characterization of the photocycle. The authors also demonstrate that these heliorhodopsin proton channels can be utilized as optogenetic tools. These findings should be of interest to a wide audience interested in membrane biophysics as well as in the development of tools for neuroscience.
The authors present a very thorough characterization of several biophysical aspects of the transport properties and the photocycle of V2HeR3, as well as a phylogenetic analysis. Furthermore, the authors demonstrate that the V2HeR3 protein can be used as an optogenetic tool, albeit with …
Author Response
Reviewer #1 (Public Review):
This manuscript provides the first experimental evidence that some members of the newly discovered heliorhodopsins can function as proton channels. The authors provide evidence of this transport function as well as a characterization of the photocycle. The authors also demonstrate that these heliorhodopsin proton channels can be utilized as optogenetic tools. These findings should be of interest to a wide audience interested in membrane biophysics as well as in the development of tools for neuroscience.
The authors present a very thorough characterization of several biophysical aspects of the transport properties and the photocycle of V2HeR3, as well as a phylogenetic analysis. Furthermore, the authors demonstrate that the V2HeR3 protein can be used as an optogenetic tool, albeit with limited capabilities.
Though the experiments are carried out carefully and the results, in general, support the conclusions, some procedures and interpretation of results need to be expanded and/or clarified for a more general readership as well as for specialized readers.
The manuscript will likely impact our understanding of the biophysics of bacteriorhodopsins in general and these new heliorhodopsins in particular, as well as serve as a platform to engineer these proton transporters for future use as tools in biotechnology and neuroscience.
We thank all the reviewers for careful reading of our manuscript and for providing valuable opinions. As we described, this is the first demonstration that the some of heliorhodopsins (HeRs) exhibit light-activated ion transport. By combining electrophysiology and spectroscopic experiments of wt and mutants V2HeR3, we describe here molecular mechanism of ion transport. However, as all three reviewers pointed out, we did not pay much attention to the substantial peak photocurrent (I0) in our electrophysiological recordings. Thus, we performed additional experiment to characterize the I0 in more detail, which are shown in Figure S6 and S7. Besides this, we have corrected and modified the entire manuscript. I hope the revised version has improved the readability and would suit for publication in eLife.
Reviewer #3 (Public Review):
The manuscript from Hososhima et al. entitled "Proton-transporting heliorhodopsins from marine giant viruses" reports for the first time proton-translocation activity for heliorhodopsins. Heliorhodopsin (HeR) is a newly discovered family of opsin proteins that are distinct from either type-1 or type-2 rhodopsins and are found in Archaea, Bacteria and Eukarya as well as giant viruses (Pushkareve et al. 2018). A unique feature of HeR is their inverted topology compared to the microbial and type-2 opsins. Despite the availability of detailed structural information on members of the HeR family (Kovalev et al. 2020, Lue et al. 2020 and Shihoya et al. 2019), their function and mode of action remain unknown. In this manuscript, the authors use the heterologous expression of synthesized HeR genes from giant viruses (V1HeR1-2,V2HeR1-3) to investigate the ion transporting properties of these viral HeRs (VHeR). Authors demonstrate that one of the viral HeR genes (V2HeR3) exhibits a unique photon-induced current that translocates protons across the membrane. Interestingly all other tested viral HeR do not show any proton-translocating activity (similarly to previously tested HeR such as Ehux-HeR, TaHeR or HeR 48C12) potentially pointing to enzymatic/signalling function of these members. Furthermore, the authors characterized the basic electrophysiological parameters of these photocurrent components in terms of their light sensitivity, kinetics, ion selectivity and more. A mutational study identifies key residues that are likely controlling the direction of ion transport. Protein purification and UV-VIS spectroscopy further reveal a prototypical slow photocycle that is similar to other HeR with maximum absorption of around 500 nm. The authors identify the M-state as a putative conducting state.
Overall, the work demonstrates nicely the mode of operation for a member of the HeR family that will pave the way to understanding the biological role and evolution of these rhodopsins. Also, the absence of any ion-translocating activity for the other HeR genes potentially underlines the diverse functions that lie within this new opsins family. The authors hand-wavingly discuss the functional role of a proton-transport activity for V2HeR3 as either depolarizing the host cell and thereby facilitating entry into the cell, or preventing superinfection.
The authors carefully chose their wording in the title as " ... proton-transporting", but then focused very much on channel activity on V2HeR3. Yet, the contribution of the passive conductance (I1 and I2) is rather small compared to the pump current I0. Could the author add some information on the initial pumping current in terms of kinetic (on- and off-kinetic for I0 are also important parameters to evaluate the potential application for HeR), ion selectivity, or spectral properties? Authors should also show wavelength dependence for all components (I0 - I2). Does it follow the spectroscopic absorption? I was a bit puzzled by the light intensity curve for the I0 component - why is the pump current not saturating at such high light powers (off-kinetic/photocycle does not look so fast to account for that!).
We thank for valuable comments on Io component which actually exhibited the largest amplitude in the photocurrent recording. We reanalyzed data and performed additional experiments to characterize the I0 more carefully concerning ion selectivity and kinetics (Fig. S6 and Fig. S7). As shown in Fig. S5, I-V plots of I0 under various ionic conditions suggest that I0 amplitude is dependent on intracellular pH. Thus, we proposed that H+ is permeant ion of I0. We also demonstrated that no other cation/anion is transported. As for kinetics, we showed in Fig. S7 that the peak time of I0 is about 0.9 ms regardless of membrane voltage, and the off-kinetics analysis revealed involvement of two time constants. We added explanation of above in the text (lines 145-158).
As the reviewer suggested, it is important to assess the wavelength dependency of photocurrent components. However, our equipment (a light source) for the action spectrum measure does not have sufficient light power to obtain sufficient photocurrent of V2HeR3 (0.5~1 mW/mm2 is needed as judged from Fig. S2).
As the reviewer pointed out, the I0 component does not saturate even at 25 mW/mm2, while I1 and I2 are saturated at 0.5~1.0 mW/mm2 already (Fig. S2). This result indicates that the light sensitivity of the each current component differs. The molecular basis of these observation is still unknown. But we previously reported a similar property in a Na+ pump rhodopsin, KR2 (Please refer Fig. S1 in Hosohima et al. PlosOne 2021 Sep 10;16(9):e0256728,).
Authors should reconsider their terminology of the photocurrent components. I find peak photocurrent for I1 misleading, especially since I0 is called transient photocurrent. Maybe authors should stick to I0, I1, and I2? Also, is I1 an independent component? Ion selectivity looks very similar to I2, and maybe the observed overshoot at positive potentials (figure 1 C red arrow I1) is an effect of a slightly higher [H+] in the vicinity of HeR after the pumping current?
Thank you for your important comment. We analyzed the I0 component in the revised manuscript. Although I0 presumably involves ion-transport, it is difficult to distinguish ionic current from an intramolecular charge displacement, as mentioned in the revised manuscript. We thus do no change the terminology (I0, I1, I2). Even though the ion selectivity of I1 and I2 are similar, we tentatively consider I1 is independent from I2.
In figure 1D authors should check if changes in Erev or photocurrent for NaCl_e and KCl_e are significantly different when compared to NMG at pH_e 7.4.
As the authors claim that there is an extracellular binding site for Cl- based on their results in figure S4. The larger photocurrent for Na2SO4 is a bit puzzling. So there is also a binding site for SO42- or did the authors not correct for double the amount of sodium? Table S7 is potentially useful in this respect but would need to be filled out completely.
We have rechecked the Erev and the current amplitude for NMG+, Na+ and K+ in Fig. 1D. Although no significant difference in Erev in Na+ and K+, there is a statistically difference in current amplitude in K+. This indicates that H+ transport is somehow enhanced in the presence of K+ in the extracellular side. We explained this in the text (lines 115-117).
We anticipated the Cl- binding site, because the current is reduced in the presence of the Cl- (also Br- and NO3-), i.e. Cl- binding (also Br- and NO3- binding) somehow modulates the H+ transport. However, the current amplitude in the presence of SO42- is in a similar level compared to the Asp-, indicating no effect of SO42-. Thus we conclude the Cl- binding site, but not SO42-.
We thank the reviewer to point out the Table S7, which was totally empty in the submitted version. We filled is in the revised version.
I believe that the use of HeR as an optogenetic tool is limited; the authors should not try to build such an artificial link to such an application. I believe their finding is of high value independent of optogenetic use. Yet, if the authors believe that it is hard to foresee how the community will embrace HeR, I suggest a more vigorous analysis. First, the expression in ND7/23 looks very cytoplasmatic (Fig 1B). Could the authors provide images from cortical neurons they use for the measurements in figure 1E (the image quality of all figures is very poor in the manuscript - it needs to be improved)?
We thank for the opinion on the scientific significance of our study and on the optogenetics application. We agree that the use of V2HeR for optical neuronal stimulation is limited because of the small ion conductance and the long photocycle. Temporal resolution is limited only up to 1 Hz, whereas ChR2 and its variants enable much higher frequency (40 Hz and even higher).
But there could be a room for V2HeR3 application for a specific use. Oppermann et al. reported anion channelrhodopsin (MerMAIDs), which exhibit a rapid and strong desensitization in its anion conductance (Ref.1 below). Such feature could be a disadvantage when continuous optical silencing is needed. But they demonstrated that MerMAIDS allows a transient suppression of individual action potentials without affecting subsequent spiking. Thus, V2HeR3 could be applicable for some specific purposes.
Taken the reviewer’s words, we further analyzed the results. First, expression and membrane localization in ND7/23 cells was visualized by anti cMyc-AB staining in Fig. 1B. The eGFP image in Fig. 1B is observed in cytoplasmic side, because eGFP domain is truncated from the V2HeR3 domain with P2A signal peptide located between two domains (We have added text to explain this, lines 90-97).
We are not able to provide image of cortical neurons unfortunately.
Ref. 1. MerMAIDs: a family of metagenomically discovered marine anion-conducting and intensely desensitizing channelrhodopsins. Oppermann J, Fischer P, Silapetere A, Liepe B, Rodriguez-Rozada S, Flores-Uribe J, Peter E, Keidel A, Vierock J, Kaufmann J, Broser M, Luck M, Bartl F, Hildebrandt P, Wiegert JS, Béjà O, Hegemann P, Wietek J. Nat Commun. 2019 Jul 25;10(1):3315.
Secondly, I do not agree that the experimental design the authors chose to test neuronal fitness after overexpression of HeR is appropriate. The electrical induction of APs (300pA, pulse width?) is not a good read-out for neuronal excitability levels (or alteration of those). Therefore the authors should measure rheobase (current steps or ramps). Additionally, parameters such as Ri, Cm, Vm, or Rm should be used to evaluate the fitness of cells.
We thank for the suggestions for neuronal experiments. We injected 300 pA current for 10 ms. We followed two articles listed below for experimental condition of current injection. The authors in Ref.1 electrically stimulated neurons by current injection from 300 to 500 pA (Fig. 5b, c, d). In Ref.2, the authors also injected from 300 to 500 pA (Fig. 5c. e. f). Thus, we believe the 300 pA would be relevant. However we further analyzed our neuronal data and characterized properties of action potentials. We revised the Figures and text (Fig. 1E-H and Fig. S8, lines 165-76).
Ref. 1. MerMAIDs: a family of metagenomically discovered marine anion-conducting and intensely desensitizing channelrhodopsins. Oppermann J, Fischer P, Silapetere A, Liepe B, Rodriguez-Rozada S, Flores-Uribe J, Peter E, Keidel A, Vierock J, Kaufmann J, Broser M, Luck M, Bartl F, Hildebrandt P, Wiegert JS, Béjà O, Hegemann P, Wietek J. Nat Commun. 2019 Jul 25;10(1):3315.
Ref. 2. Grimm C, Silapetere A, Vogt A, Bernal Sierra YA, Hegemann P. “Electrical properties, substrate specificity and optogenetic potential of the engineered light-driven sodium pump eKR2.” Sci Rep. 2018, 8(1):9316
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Evaluation Summary:
Hososhima et al. characterize a marine virus Heliorhodopsin as the first of its class to show ion transport activity. These bacteriorhodopsin homologs have been recently described and the present careful characterization of V2HeR3 represents an important step in the understanding of these interesting membrane proteins. Though the experiments are carried out carefully and the results, in general, support the conclusions, some experiments are needed and the interpretation of results needs to be clarified.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
This manuscript provides the first experimental evidence that some members of the newly discovered heliorhodopsins can function as proton channels. The authors provide evidence of this transport function as well as a characterization of the photocycle. The authors also demonstrate that these heliorhodopsin proton channels can be utilized as optogenetic tools. These findings should be of interest to a wide audience interested in membrane biophysics as well as in the development of tools for neuroscience.
The authors present a very thorough characterization of several biophysical aspects of the transport properties and the photocycle of V2HeR3, as well as a phylogenetic analysis. Furthermore, the authors demonstrate that the V2HeR3 protein can be used as an optogenetic tool, albeit with limited capabilities.
Reviewer #1 (Public Review):
This manuscript provides the first experimental evidence that some members of the newly discovered heliorhodopsins can function as proton channels. The authors provide evidence of this transport function as well as a characterization of the photocycle. The authors also demonstrate that these heliorhodopsin proton channels can be utilized as optogenetic tools. These findings should be of interest to a wide audience interested in membrane biophysics as well as in the development of tools for neuroscience.
The authors present a very thorough characterization of several biophysical aspects of the transport properties and the photocycle of V2HeR3, as well as a phylogenetic analysis. Furthermore, the authors demonstrate that the V2HeR3 protein can be used as an optogenetic tool, albeit with limited capabilities.
Though the experiments are carried out carefully and the results, in general, support the conclusions, some procedures and interpretation of results need to be expanded and/or clarified for a more general readership as well as for specialized readers.The manuscript will likely impact our understanding of the biophysics of bacteriorhodopsins in general and these new heliorhodopsins in particular, as well as serve as a platform to engineer these proton transporters for future use as tools in biotechnology and neuroscience.
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Reviewer #2 (Public Review):
Heliorhodopsins form a vast superfamily of retinylidene proteins recently discovered by the authors. Only very few heliorhodopsins have so far been characterized in any detail, and no biological function could have been assigned to any of them. The Authors show that some heliorhodopsins possess a proton-transporting ability, and present a detailed analysis of one such protein, V2HeR3, by several complementary methods, including patch clamp electrophysiology, pH measurements, UV/Vis spectroscopy, flash photolysis, FTIR spectroscopy, and chromophore extraction. In addition, they have created and tested several mutants of the key residues and tested several wild-type homologs of V2HeR3. The authors have succeeded in the detailed characterization of a completely new type of protein, which provided a major …
Reviewer #2 (Public Review):
Heliorhodopsins form a vast superfamily of retinylidene proteins recently discovered by the authors. Only very few heliorhodopsins have so far been characterized in any detail, and no biological function could have been assigned to any of them. The Authors show that some heliorhodopsins possess a proton-transporting ability, and present a detailed analysis of one such protein, V2HeR3, by several complementary methods, including patch clamp electrophysiology, pH measurements, UV/Vis spectroscopy, flash photolysis, FTIR spectroscopy, and chromophore extraction. In addition, they have created and tested several mutants of the key residues and tested several wild-type homologs of V2HeR3. The authors have succeeded in the detailed characterization of a completely new type of protein, which provided a major conceptual advance in the field. However, the authors' interpretation of some of their results does not seem to be justified. Despite this relatively minor problem, this study is a major breakthrough in our understanding of heliorhodopsins and is also important in the wider contexts of photobiology and membrane protein research.
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Reviewer #3 (Public Review):
The manuscript from Hososhima et al. entitled "Proton-transporting heliorhodopsins from marine giant viruses" reports for the first time proton-translocation activity for heliorhodopsins. Heliorhodopsin (HeR) is a newly discovered family of opsin proteins that are distinct from either type-1 or type-2 rhodopsins and are found in Archaea, Bacteria and Eukarya as well as giant viruses (Pushkareve et al. 2018). A unique feature of HeR is their inverted topology compared to the microbial and type-2 opsins. Despite the availability of detailed structural information on members of the HeR family (Kovalev et al. 2020, Lue et al. 2020 and Shihoya et al. 2019), their function and mode of action remain unknown. In this manuscript, the authors use the heterologous expression of synthesized HeR genes from giant viruses …
Reviewer #3 (Public Review):
The manuscript from Hososhima et al. entitled "Proton-transporting heliorhodopsins from marine giant viruses" reports for the first time proton-translocation activity for heliorhodopsins. Heliorhodopsin (HeR) is a newly discovered family of opsin proteins that are distinct from either type-1 or type-2 rhodopsins and are found in Archaea, Bacteria and Eukarya as well as giant viruses (Pushkareve et al. 2018). A unique feature of HeR is their inverted topology compared to the microbial and type-2 opsins. Despite the availability of detailed structural information on members of the HeR family (Kovalev et al. 2020, Lue et al. 2020 and Shihoya et al. 2019), their function and mode of action remain unknown. In this manuscript, the authors use the heterologous expression of synthesized HeR genes from giant viruses (V1HeR1-2,V2HeR1-3) to investigate the ion transporting properties of these viral HeRs (VHeR). Authors demonstrate that one of the viral HeR genes (V2HeR3) exhibits a unique photon-induced current that translocates protons across the membrane. Interestingly all other tested viral HeR do not show any proton-translocating activity (similarly to previously tested HeR such as Ehux-HeR, TaHeR or HeR 48C12) potentially pointing to enzymatic/signalling function of these members. Furthermore, the authors characterized the basic electrophysiological parameters of these photocurrent components in terms of their light sensitivity, kinetics, ion selectivity and more. A mutational study identifies key residues that are likely controlling the direction of ion transport. Protein purification and UV-VIS spectroscopy further reveal a prototypical slow photocycle that is similar to other HeR with maximum absorption of around 500 nm. The authors identify the M-state as a putative conducting state.
Overall, the work demonstrates nicely the mode of operation for a member of the HeR family that will pave the way to understanding the biological role and evolution of these rhodopsins. Also, the absence of any ion-translocating activity for the other HeR genes potentially underlines the diverse functions that lie within this new opsins family. The authors hand-wavingly discuss the functional role of a proton-transport activity for V2HeR3 as either depolarizing the host cell and thereby facilitating entry into the cell, or preventing superinfection.The authors carefully chose their wording in the title as " ... proton-transporting", but then focused very much on channel activity on V2HeR3. Yet, the contribution of the passive conductance (I1 and I2) is rather small compared to the pump current I0. Could the author add some information on the initial pumping current in terms of kinetic (on- and off-kinetic for I0 are also important parameters to evaluate the potential application for HeR), ion selectivity, or spectral properties? Authors should also show wavelength dependence for all components (I0 - I2). Does it follow the spectroscopic absorption? I was a bit puzzled by the light intensity curve for the I0 component - why is the pump current not saturating at such high light powers (off-kinetic/photocycle does not look so fast to account for that!).
Authors should reconsider their terminology of the photocurrent components. I find peak photocurrent for I1 misleading, especially since I0 is called transient photocurrent. Maybe authors should stick to I0, I1, and I2? Also, is I1 an independent component? Ion selectivity looks very similar to I2, and maybe the observed overshoot at positive potentials (figure 1 C red arrow I1) is an effect of a slightly higher [H+] in the vicinity of HeR after the pumping current?
In figure 1D authors should check if changes in Erev or photocurrent for NaCl_e and KCl_e are significantly different when compared to NMG at pH_e 7.4.
As the authors claim that there is an extracellular binding site for Cl- based on their results in figure S4. The larger photocurrent for Na2SO4 is a bit puzzling. So there is also a binding site for SO42- or did the authors not correct for double the amount of sodium? Table S7 is potentially useful in this respect but would need to be filled out completely.I believe that the use of HeR as an optogenetic tool is limited; the authors should not try to build such an artificial link to such an application. I believe their finding is of high value independent of optogenetic use. Yet, if the authors believe that it is hard to foresee how the community will embrace HeR, I suggest a more vigorous analysis. First, the expression in ND7/23 looks very cytoplasmatic (Fig 1B). Could the authors provide images from cortical neurons they use for the measurements in figure 1E (the image quality of all figures is very poor in the manuscript - it needs to be improved)? Secondly, I do not agree that the experimental design the authors chose to test neuronal fitness after overexpression of HeR is appropriate. The electrical induction of APs (300pA, pulse width?) is not a good read-out for neuronal excitability levels (or alteration of those). Therefore the authors should measure rheobase (current steps or ramps). Additionally, parameters such as Ri, Cm, Vm, or Rm should be used to evaluate the fitness of cells.
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