Abstract
Rhodopsins with enzymatic activity were found in microbes, in 2004 hypothetically from sequence data and since 2014 by experimental proof. So far three different types are known: light-activated guanylyl cyclase opsins (Cyclop) in fungi, light-inhibited two-component guanylyl cyclase opsins (2c-Cyclop) in green algae, and rhodopsin phosphodiesterases (RhoPDE) in choanoflagellates. They are integral membrane proteins with eight transmembrane helices (TM), different to the other microbial (type I) rhodopsins with 7 TM. Therefore, we propose a classification as type Ib rhodopsins for opsins with 8 TM and type Ia for the ones with 7 TM. To characterize those rhodopsins or their mutants, the expression in Xenopus laevis oocytes proved to be an efficient strategy. Functional analysis was initially performed “in oocyte” (in vivo), but more detailed characterization can be obtained with an in vitro assay. In this chapter, we describe procedures how to extract membranes from oocytes after cRNA microinjection and heterologous protein expression. Enzymatic activity of these membranes is then analyzed under different illumination conditions. In addition, fluorescent labeling of the rhodopsins is employed to quantify the expression level and the absolute activity of designed mutants. We discuss strengths and pitfalls, associated with this expression system, and strategies for selecting potentially useful optogenetic tools.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Gurdon JB, Lane CD, Woodland HR et al (1971) Use of frog eggs and oocytes for the study of messenger RNA and its translation in living cells. Nature 233:177–182
Wagner CA, Friedrich B, Setiawan I et al (2000) The use of Xenopus laevis oocytes for the functional characterization of heterologously expressed membrane proteins. Cell Physiol Biochem 10:1–12
Knox BE, Khorana H, Nasi E (1993) Light-induced currents in Xenopus oocytes expressing bovine rhodopsin. J Physiol 466:157–172
Nagel G, Möckel B, Büldt G et al (1995) Functional expression of bacteriorhodopsin in oocytes allows direct measurement of voltage dependence of light induced H+ pumping. FEBS Lett 377:263–266
Nagel G, Ollig D, Fuhrmann M et al (2002) Channelrhodopsin-1: a light-gated proton channel in green algae. Science 296:2395–2398
Nagel G, Szellas T, Huhn W et al (2003) Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc Natl Acad Sci 100:13940–13945
Schröder-Lang S, Schwärzel M, Seifert R et al (2007) Fast manipulation of cellular cAMP level by light in vivo. Nat Methods 4:39–42
Avelar GM, Schumacher RI, Zaini PA et al (2014) A rhodopsin-guanylyl cyclase gene fusion functions in visual perception in a fungus. Curr Biol 24:1234–1240
Gao S, Nagpal J, Schneider MW et al (2015) Optogenetic manipulation of cGMP in cells and animals by the tightly light-regulated guanylyl-cyclase opsin CyclOp. Nat Commun 6:1–12
Scheib U, Stehfest K, Gee CE et al (2015) The rhodopsin–guanylyl cyclase of the aquatic fungus Blastocladiella emersonii enables fast optical control of cGMP signaling. Sci Signal 8:rs8
Kateriya S, Nagel G, Bamberg E et al (2004) “Vision” in single-celled algae. Physiology 19:133–137
Tian Y, Gao S, von der Heyde EL et al (2018) Two-component cyclase opsins of green algae are ATP-dependent and light-inhibited guanylyl cyclases. BMC Biol 16:1–18
Tian Y, Nagel G, Gao S (2021) An engineered membrane-bound guanylyl cyclase with light-switchable activity. BMC Biol 19:1–9
Tian Y, Gao S, Yang S et al (2018) A novel rhodopsin phosphodiesterase from Salpingoeca rosetta shows light-enhanced substrate affinity. Biochem J 475:1121–1128
Yoshida K, Tsunoda SP, Brown LS et al (2017) A unique choanoflagellate enzyme rhodopsin exhibits light-dependent cyclic nucleotide phosphodiesterase activity. J Biol Chem 292:7531–7541
Lamarche LB, Kumar RP, Trieu MM et al (2017) Purification and characterization of RhoPDE, a retinylidene/phosphodiesterase fusion protein and potential optogenetic tool from the choanoflagellate Salpingoeca rosetta. Biochemistry 56:5812–5822
Ikuta T, Shihoya W, Sugiura M et al (2020) Structural insights into the mechanism of rhodopsin phosphodiesterase. Nat Commun 11:1–12
Richter DJ, Fozouni P, Eisen MB et al (2018) Gene family innovation, conservation and loss on the animal stem lineage. elife 7:e34226
Brunet T, Larson BT, Linden TA et al (2019) Light-regulated collective contractility in a multicellular choanoflagellate. Science 366:326–334
Sugiura M, Tsunoda SP, Hibi M et al (2020) Molecular properties of new enzyme rhodopsins with phosphodiesterase activity. ACS Omega 5:10602–10609
Tian Y, Yang S, Gao S (2020) Advances, perspectives and potential engineering strategies of light-gated phosphodiesterases for optogenetic applications. Int J Mol Sci 21:7544
Möller S, Croning MD, Apweiler R (2001) Evaluation of methods for the prediction of membrane spanning regions. Bioinformatics 17:646–653
Krogh A, Larsson B, Von Heijne G et al (2001) Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305:567–580
Drozdetskiy A, Cole C, Procter J et al (2015) JPred4: a protein secondary structure prediction server. Nucleic Acids Res 43:W389–W394
Kerppola TK (2006) Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells. Nat Protoc 1:1278–1286
Kerppola TK (2008) Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells. Annu Rev Biophys 37:465–487
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Tian, Y., Gao, S., Nagel, G. (2022). In Vivo and In Vitro Characterization of Cyclase and Phosphodiesterase Rhodopsins. In: Gordeliy, V. (eds) Rhodopsin. Methods in Molecular Biology, vol 2501. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2329-9_16
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2329-9_16
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2328-2
Online ISBN: 978-1-0716-2329-9
eBook Packages: Springer Protocols