Skip to main content
SpringerLink
Log in

Retinal-Based Anion Pump from the Cyanobacterium Tolypothrix campylonemoides

  • Published:
Biochemistry (Moscow) Aims and scope Submit manuscript

Abstract

In this work, TcaR rhodopsin from the cyanobacterium Tolypothrix campylonemoides was characterized. Analysis of the amino acid sequence of TcaR revealed that this protein possesses a TSD motif that differs by only one amino acid from the TSA motif of the known halorhodopsin chloride pump. The TcaR protein was expressed in E. coli, purified, and incorporated into proteoliposomes and nanodiscs. Functional activity was measured by electric current generation through the planar bilayer lipid membranes (BLMs) with proteoliposomes adsorbed on one side of the membrane surface, as well as by fluorescence using the voltage-dependent dye oxonol VI. We have shown that TcaR rhodopsin functions as a powerful anion pump. Our results show that the novel microbial anion transporter, TcaR, deserves deeper investigation and may be of interest both for fundamental studies of membrane proteins and as a tool for optogenetics.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4.
Fig. 5.
Fig. 6.

Similar content being viewed by others

Abbreviations

BLM:

planar bilayer lipid membrane

CCCP:

carbonyl cyanide m-chlorophenyl hydrazone

PLs:

proteoliposomes

References

  1. Gushchin, I., and Gordeliy, V. (2018) Microbial rhodopsins, Subcell Biochem., 87, 19-56, https://doi.org/10.1007/978-981-10-7757-9_2.

    Article  CAS  PubMed  Google Scholar 

  2. Gomez-Consarnau, L., Raven, J. A., Levine, N. M., Cutter, L. S., Wang, D., Seegers, B., Aristegui, J., Fuhrman, J. A., Gasol, J. M., and Sanudo-Wilhelmy, S. A. (2019) Microbial rhodopsins are major contributors to the solar energy captured in the sea, Sci. Adv., 8, eaaw8855, https://doi.org/10.1126/sciadv.aaw8855.

    Article  CAS  Google Scholar 

  3. Spudich, J. L., Sineshchekov, O. A., and Govorunova, E. G. (2014) Mechanism divergence in microbial rhodopsins, Biochim. Biophys. Acta, 1837, 546-552, https://doi.org/10.1016/j.bbabio.2013.06.006.

    Article  CAS  PubMed  Google Scholar 

  4. Mukherjee, S., Hegemann, P., and Broser, M. (2019) Enzymerhodopsins: novel photoregulated catalysts for optogenetics, Curr. Opin. Struct. Biol., 57, 118-126, https://doi.org/10.1016/j.sbi.2019.02.003.

    Article  CAS  PubMed  Google Scholar 

  5. Kirpichnikov, M. P. and Ostrovskiy, M. A. (2019) Optogenetics and vision, Herald Russ. Acad. Sci., 89, 34-38, https://doi.org/10.1134/S1019331619010039.

    Article  Google Scholar 

  6. Luecke, H., Schobert, B., Richter, H. T., Cartailler, J. P., and Lanyi, J. K. (1999) Structure of bacteriorhodopsin at 1.55 A resolution, J. Mol. Biol., 291, 899-911, https://doi.org/10.1006/jmbi.1999.3027.

    Article  CAS  PubMed  Google Scholar 

  7. Das, S., Singh, D., Madduluri, M., Chandrababunaidu, M. M., Gupta, A., Adhikary, S. P., and Tripathy, S. (2015) Draft genome sequence of bioactive-compound-producing cyanobacterium Tolypothrix campylonemoides strain VB511288, Genome Announc., 3, e00226-15, https://doi.org/10.1128/genomeA.00226-15.

    Article  PubMed  PubMed Central  Google Scholar 

  8. Hasemi, T., Kikukawa, T., Watanabe, Y., Aizawa, T., Miyauchi, S., Kamo, N., and Demura, M. (2019) Photochemical study of a cyanobacterial chloride-ion pumping rhodopsin, Biochim. Biophys. Acta, 1860, 136-146, https://doi.org/10.1016/j.bbabio.2018.12.001.

    Article  CAS  Google Scholar 

  9. Yun, J. H., Park, J. H., Jin, Z., Ohki, M., Wang, Y., Lupala, C. S., Liu, H., Park, S. Y., and Lee, W. (2020) Structure-based functional modification study of a cyanobacterial chloride pump for transporting multiple anions, J. Mol. Biol., 432, 5273-5286, https://doi.org/10.3390/ma13143061.

    Article  CAS  PubMed  Google Scholar 

  10. Astashkin, R., Kovalev, K., Bukhdruker, S., Vaganova, S., Kuzmin, A., Alekseev, A., Balandin, T., Zabelskii, D., Gushchin, I., Royant, A., Volkov, D., Bourenkov, G., Koonin, E., Engelhard, M., Bamberg, E., and Gordeliy, V. (2022) Structural insights into light-driven anion pumping in cyanobacteria, Nat. Commun., 13, 6460, https://doi.org/10.1038/s41467-022-34019-9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Oesterhelt, D., Tittor, J., and Bamberg, E. (1992) A unifying concept for ion translocation by retinal proteins, J. Bioenerg. Biomembr., 24, 181-191, https://doi.org/10.1007/BF00762676.

    Article  CAS  PubMed  Google Scholar 

  12. Drachev, L. A., Jasaitis, A. A., Kaulen, A. D., Kondrashin, A. A., Liberman, E. A., Nemecek, I. B., Ostroumov, S. A., Semenov, A. Yu., and Skulachev, V. P. (1974) Direct measurement of electric current generation by cytochrome oxidase, H+-ATPase and bacteriorhodopsin, Nature, 249, 321-324, https://doi.org/10.1038/249321a0.

    Article  CAS  PubMed  Google Scholar 

  13. Drachev, L. A., Frolov, V. N., Kaulen, A. D., Liberman, E. A., Ostroumov, S. A., Plakunova, V. G., Semenov, A. Y., and Skulachev, V. P. (1976) Reconstitution of biological molecular generators of electric current. Bacteriorhodopsin, J. Biol. Chem., 251, 7059-7065, https://doi.org/10.1016/S0021-9258(17)32940-X.

    Article  CAS  PubMed  Google Scholar 

  14. Bamberg, E., Apell, H. J., Dencher, N. A., Sperling, W., Stieve, H., and Lauger, P. (1979) Photocurrents generated by bacteriorhodopsin on planar bilayer membranes, Biophys. Struct. Mech., 5, 277-292, https://doi.org/10.1007/BF02426663.

    Article  CAS  Google Scholar 

  15. Bamberg, E., Butt, H. J., Eisenrauch, A., and Fendler, K. (1993) Charge transport of ion pumps on lipid bilayer membranes, Q. Rev. Biophys., 26, 1-25, https://doi.org/10.1017/s0033583500003942.

    Article  CAS  PubMed  Google Scholar 

  16. Friedrich, T., Geibel, S., Kalmbach, R., Chizhov, I., Ataka, K., Heberle, J., Engelhard, M., and Bamberg, E. (2002) Proteorhodopsin is a light-driven proton pump with variable vectoriality, J. Mol. Biol., 321, 821-838, https://doi.org/10.1016/s0022-2836(02)00696-4.

    Article  CAS  PubMed  Google Scholar 

  17. Shevchenko, V., Mager, T., Kovalev, K., Polovinkin, V., Alekseev, A., Juettner, J., Chizhov, I., Bamann, C., Vavourakis, C., Ghai, R., Gushchin, I., Borshchevskiy, V., Rogachev, A., Melnikov, I., Popov, A., Balandin, T., Rodriguez-Valera, F., Manstein, D. J., Bueldt, G., Bamberg, E., and Gordeliy, V. (2017) Inward H+ pump xenorhodopsin: mechanism and alternative optogenetic approach, Sci. Adv., 3, e1603187, https://doi.org/10.1126/sciadv.1603187.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Schuler, M. A., Denisov, I. G., and Sligar, S. G. (2013) Nanodiscs as a new tool to examine lipid-protein interactions, Methods Mol. Biol., 974, 415-433, https://doi.org/10.1007/978-1-62703-275-9_18.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Rokitskaya, T. I., Maliar, N. L., Siletsky, S. A., Gordeliy, V., and Antonenko, Y. N. (2022) Electrophysiological characterization of microbial rhodopsin transport properties: electrometric and ΔpH measurements using planar lipid bilayer, collodion film, and fluorescent probe approaches, Methods Mol. Biol., 2501, 259-275, https://doi.org/10.1007/978-1-0716-2329-9_12.

    Article  CAS  PubMed  Google Scholar 

  20. Rokitskaya, T. I., Maliar, N., Kovalev, K. V., Volkov, O., Gordeliy, V. I., and Antonenko, Y. N. (2021) Rhodopsin channel activity can be evaluated by measuring the photocurrent voltage dependence in planar bilayer lipid membranes, Biochemistry (Moscow), 86, 409-419, https://doi.org/10.1134/S0006297921040039.

    Article  CAS  PubMed  Google Scholar 

  21. Selwyn, M. J., Dawson, A. P., Stockdale, M., and Gains, N. (1970) Chloride-hydroxide exchange across mitochondrial, erythrocyte and artificial lipid membranes mediated by trialkyl-and triphenyltin compounds, Eur. J. Biochem., 14, 120-126, https://doi.org/10.1111/j.1432-1033.1970.tb00268.x.

    Article  CAS  PubMed  Google Scholar 

  22. Antonenko, Y. N. (1990) Electrically silent anion transport through bilayer lipid membrane induced by tributyltin and triethyllead, J. Membr. Biol., 113, 109-113, https://doi.org/10.1007/BF01872884.

    Article  CAS  Google Scholar 

  23. Sato, T., Konno, H., Tanaka, Y., Kataoka, T., Nagai, K., Wasserman, H. H., and Ohkuma, S. (1998) Prodigiosins as a new group of H+/Cl– symporters that uncouple proton translocators, J. Biol. Chem., 273, 21455-21462, https://doi.org/10.1074/jbc.273.34.21455.

    Article  CAS  PubMed  Google Scholar 

  24. Bamberg, E., Hegemann, P., and Oesterhelt, D. (1984) Reconstitution of the light-driven electrogenic ion-pump halorhodopsin in black lipid membranes, Biochim. Biophys. Acta, 773, 53-60, https://doi.org/10.1016/0005-2736(84)90549-2.

    Article  CAS  Google Scholar 

  25. Bamberg, E., Tittor, J., and Oesterhelt, D. (1993) Light-driven proton or chloride pumping by halorhodopsin, Proc. Natl. Acad. Sci. USA, 90, 639-643, https://doi.org/10.1073/pnas.90.2.639.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Antonenko, Y. N., Denisov, S. S., Silachev, D. N., Khailova, L. S., Jankauskas, S. S., Rokitskaya, T. I., Danilina, T. I., Kotova, E. A., Korshunova, G. A., Plotnikov, E. Y., and Zorov, D. B. (2016) A long-linker conjugate of fluorescein and triphenylphosphonium as mitochondria-targeted uncoupler and fluorescent neuro- and nephroprotector, Biochim. Biophys. Acta, 1860, 2463-2473, https://doi.org/10.1016/j.bbagen.2016.07.014.

    Article  CAS  PubMed  Google Scholar 

  27. Steiner, M., Oesterhelt, D., Ariki, M., and Lanyi, J. K. (1984) Halide binding by the purified halorhodopsin chromoprotein. I. Effects on the chromophore, J. Biol. Chem., 259, 2179-2184, https://doi.org/10.1016/S0021-9258(17)43334-5.

    Article  CAS  PubMed  Google Scholar 

Download references

Funding

This work was financially supported by the Russian Science Foundation (grants 23-24-00038, 21-64-00018) and by the Ministry of Science and Higher Education of the Russian Federation (project AAAA-A19-119031390114-5).

Author information

Authors and Affiliations

  1. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991, Moscow, Russia

    Tatyana I. Rokitskaya & Yuri N. Antonenko

  2. Moscow Institute of Physics and Technology, 141701, Dolgoprudny, Moscow Region, Russia

    Aleksey A. Alekseev, Fedor M. Tsybrov & Sergej M. Bukhalovich

  3. Institut de Biologie Structurale (IBS), Université Grenoble Alpes, CEA, CNRS, Grenoble, France

    Valentin I. Gordeliy

Authors
  1. Tatyana I. Rokitskaya
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Aleksey A. Alekseev
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Fedor M. Tsybrov
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sergej M. Bukhalovich
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yuri N. Antonenko
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Valentin I. Gordeliy
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

V.I.G. concept of the study; A.A.A., T.I.R., Y.N.A., S.M.B., F.M.Ts. conducting experiments; Y.N.A., V.I.G., T.I.R. writing text of the paper. All authors participated in discussion of the obtained results and editing of the final text of the paper.

Corresponding authors

Correspondence to Tatyana I. Rokitskaya, Yuri N. Antonenko or Valentin I. Gordeliy.

Ethics declarations

The authors declare no conflict of interests in financial or any other sphere. This article does not contain any studies with human participants or animals performed by any of the authors.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Rokitskaya, T.I., Alekseev, A.A., Tsybrov, F.M. et al. Retinal-Based Anion Pump from the Cyanobacterium Tolypothrix campylonemoides. Biochemistry Moscow 88, 1571–1579 (2023). https://doi.org/10.1134/S0006297923100127

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1134/S0006297923100127

Keywords

Search

Navigation