Two-component cyclase opsins of green algae are ATP-dependent and light-inhibited guanylyl cyclases

Abstract

Background: The green algae Chlamydomonas reinhardtii and Volvox carteri are important models for studying light perception and response, expressing many different photoreceptors. More than 10 opsins were reported in C. reinhardtii, yet only two-the channelrhodopsins-were functionally characterized. Characterization of new opsins would help to understand the green algae photobiology and to develop new tools for optogenetics.

Results: Here we report the characterization of a novel opsin family from these green algae: light-inhibited guanylyl cyclases regulated through a two-component-like phosphoryl transfer, called "two-component cyclase opsins" (2c-Cyclops). We prove the existence of such opsins in C. reinhardtii and V. carteri and show that they have cytosolic N- and C-termini, implying an eight-transmembrane helix structure. We also demonstrate that cGMP production is both light-inhibited and ATP-dependent. The cyclase activity of Cr2c-Cyclop1 is kept functional by the ongoing phosphorylation and phosphoryl transfer from the histidine kinase to the response regulator in the dark, proven by mutagenesis. Absorption of a photon inhibits the cyclase activity, most likely by inhibiting the phosphoryl transfer. Overexpression of Vc2c-Cyclop1 protein in V. carteri leads to significantly increased cGMP levels, demonstrating guanylyl cyclase activity of Vc2c-Cyclop1 in vivo. Live cell imaging of YFP-tagged Vc2c-Cyclop1 in V. carteri revealed a development-dependent, layer-like structure at the immediate periphery of the nucleus and intense spots in the cell periphery.

Conclusions: Cr2c-Cyclop1 and Vc2c-Cyclop1 are light-inhibited and ATP-dependent guanylyl cyclases with an unusual eight-transmembrane helix structure of the type I opsin domain which we propose to classify as type Ib, in contrast to the 7 TM type Ia opsins. Overexpression of Vc2c-Cyclop1 protein in V. carteri led to a significant increase of cGMP, demonstrating enzyme functionality in the organism of origin. Fluorescent live cell imaging revealed that Vc2c-Cyclop1 is located in the periphery of the nucleus and in confined areas at the cell periphery.

Keywords: Chlamydomonas reinhardtii; Chlamyopsin; Optogenetics; Two-component system; Volvox carteri.

Fig. 1

Gene structures, mRNA structures, and protein domain compositions of Cr2c-Cyclop1 and Vc2c-Cyclop1. A1, B1. The genetic maps of Cr2c-Cyclop1 and Vc2c-Cyclop1 genes show all exon and intron segments and a number of restriction enzyme cleavage sites. ATG, the translation start site; TAG or TGA, the translation stop site; the filled boxes represent exons; thick horizontal bars indicate untranslated regions; and the thinner horizontal bars represent upstream and downstream sequences. A2, B2. Arrowheads indicate the position of the numerous introns within the genetic maps of the Cr2c-Cyclop1 and Vc2c-Cyclop1 mRNAs. The specified restriction enzyme cleavage sites facilitate orientation. AUG, the translation start site; UAG or UGA, the translation stop site; filled black box, the open reading frame; thick horizontal bar, the 5′- and 3′-UTRs; AAA, the poly A tail. A3, B3. Cr2c-Cyclop1 and Vc2c-Cyclop1 protein domain compositions. Opsin, the rhodopsin domain; H kinase, the histidine kinase domain; RR, the response regulator domain; GC, the guanylyl cyclase domain. Gray text in A3 shows where Cr2c-Cyclop has been shortened to produce Cr2c-Cyclop1

Fig. 2

2c-Cyclops possess cytosolic N-termini and a likely 8 transmembrane helices topology. a Schematic model of BiFC (bimolecular fluorescence complementation) experiments. The opsin domain was N- and C-terminally fused to the two parts of split YFP (YFP_C = aa155–238 of YFP, YFP_N = aa1–154 of YFP). b Fluorescence pictures show the following: control oocyte (control), oocytes expressing YFP_C::Cr2c-Cyclop1/opsin::YFP_N (Cr2c-Cyclop1), YFP_C::Vc2c-Cyclop1/opsin::YFP_N (Vc2c-Cyclop1), and YFP_C::Cop5/opsin::YFP_N (Cop5(HKR)) constructs. The fusion sequence of YFP_C and YFP_N was designed according to the YFP structure to facilitate the fluorescence complementation. The fluorescence images were taken by a confocal microscope 3 dpi (days post injection) with 30 ng cRNA injection into Xenopus oocytes. Scale bars 250 μm

Fig. 3

Cr2c-Cyclop1 and Vc2c-Cyclop1 are light-inhibited guanylyl cyclase opsins. a Light (532 nm, ~ 20 μW/mm²) and dark activities of Cr2c-Cyclop1 + 1 μM ATR, Cr2c-Cyclop1-YFP + 1 μM ATR, and Cr2c-Cyclop1-YFP without ATR. The cGMP production ability was normalized to one oocyte membrane. n = 3, error bars = SD. b Action spectrum of Cr2c-Cyclop1. n = 3, error bars = SD. c Light sensitivity of Cr2c-Cyclop1 to three different wavelengths of light. Different intensities of blue light (473 nm), green light (532 nm), and orange light (596 nm) were applied. n = 3, error bars = SD. d Light (532 nm, ~ 20 μW/mm²) and dark activities of Vc2c-Cyclop1. One micromolar of ATR was added. The cGMP production ability was normalized to one oocyte membrane. n = 4, error bars = SD. e Action spectrum of Vc2c-Cyclop1. n = 4, error bars = SD. f Light sensitivity of Vc2c-Cyclop1 to 556 nm light. n = 4, error bars = SD. For b, c, e, and f, inhibition percentage was calculated by (dark activity − light activity)/dark activity

Fig. 4

Characterization of Cr2c-Cyclop1 activity under different conditions. a Dynamic activity of Cr2c-Cyclop1. All three samples were under initial dark stage for 30 s and then put to constant dark (blue square), constant green light illumination (532 nm, ~ 20 μW/mm², green rhombus), and 3-min green light followed by constant dark (gray dot). Samples were collected and measured at different time points indicated in the figure. n = 3, error bars = SD. b Cr2c-Cyclop1 activity influenced by ATP, Mg²⁺, and Ca²⁺. Mg²⁺ were depleted (no MgCl₂), replaced by Ca²⁺ (5 mM CaCl₂) or ATP depleted (no ATP) from the standard reaction buffer. n = 4, error bars = SD. c Cr2c-Cyclop1 activity at different temperatures (10, 20, 30, and 40 °C). n = 3, error bars = SD. d Cr2c-Cyclop1 activity at different pHs (6.3, 7.3, 8.3). n = 3, error bars = SD. Illumination conditions were same for a–d

Fig. 5

Mutation analysis of Cr2c-Cyclop1. a All mutations were made based on Cr2c-Cyclop1-YFP construct. No ATP, ATP depleted from the standard reaction buffer. With AMP-PNP, 0.25 mM AMP-PNP was added to replace ATP. Illumination condition, 532 nm, ~ 20 μW/mm². Activities of different constructs were adjusted to the same protein amount based on the fluorescence emission value, n = 4, error bars = SD. wt, Cr2c-Cyclop1-YFP wild-type. b Co-expression of Cr2c-Cyclop1-YFP and H352F mutant. Total protein amounts were controlled to be the same based on the fluorescence emission value. Illumination condition, 532 nm, ~ 20 μW/mm². n = 3, error bars = SD. c The relative fluorescence emission values of different constructs were determined to ensure similar total protein amount. n = 3, error bars = SD

Fig. 6

Schematic of the 2c-Cyclop working model. a Scheme of the Cr2c-Cyclop1 structure with important amino acids. Rhodopsin domain is embedded in the membrane with both termini in the cytosolic side. The key K298 residue, located in the last transmembrane helix, binds retinal covalently. Histidine kinase domain is depicted with DHp (dimerization and histidine phosphotransferase domain) and CA (catalytic and ATPase domain) in the red modules, including key residues H352, T356, and G533. Response regulator is drawn in a green module with key D1092 residue to accept phosphoryl group. Guanylyl cyclase (GC) domain is illustrated in the purple module, producing cGMP from substrate GTP. b A model for the cascade reaction within 2c-Cyclops. Green light is detected by the 8 TM rhodopsin, which in turn inhibits the histidine kinase. Without inhibition, the histidine kinase performs autophosphorylation using a phosphoryl group from ATP and then it transfers the phosphoryl group to the response regulator. The phosphorylated response regulator in turn activates the guanylyl cyclase to produce cGMP from GTP. The cGMP then acts as an effector molecule to trigger cellular processes

Fig. 7

mRNA expression analysis and guanylyl cyclase activity of Vc2c-Cyclop1 in V. carteri. a Quantitative analysis of Vc2c-Cyclop1 mRNA expression in wild type (wt) and in transformants that express Vc2c-Cyclop1-YFP under the control of the LHCBM1 promoter. The mRNA quantification was done by quantitative real-time RT-PCR. The expression of Vc2c-Cyclop1 in wild type was used as a reference point (=1) for calculation of the relative expression level of each transformant. The error bars represent the standard deviation of three biological replicates each. b Quantitative analysis of cGMP production in wild type (wt) and in transformants that express Vc2c-Cyclop1-YFP. The cGMP concentration serves as a measure of guanylyl cyclase activity. Wild-type and transformant V. carteri algae were grown under standard conditions at 28 °C in a cycle of day and night and finally analyzed during the day phase. Cell lysates were prepared both from algae samples that were transferred to the dark for 10 min (dark) and from algae samples that remained in the light during these 10 min (light). The cGMP concentration was determined in the cell lysates as described in the “Methods” section. The error bars refer to the standard deviation of three biological replicates each

Fig. 8

In vivo localization of Vc2c-Cyclop1 in V. carteri. A1–E1 (column 1) YFP fluorescence of Vc2c-Cyclop1 fused to YFP (Vc2c-Cyclop1-YFP, green). A2–E2 (column 2) Overlay of the YFP fluorescence of Vc2c-Cyclop1-YFP (green) and the chlorophyll fluorescence (magenta). A3–E3 (column 3) Transmission-PMT image (transmitted light). A4–E4 (column 4) Overlay of transmission-PMT, YFP fluorescence of Vc2c-Cyclop1-YFP (green) and chlorophyll fluorescence (magenta). A1–A4 Overview of an entire V. carteri spheroid expressing Vc2c-Cyclop1-YFP under the control of the LHCBM1 promoter. Larger amounts of Vc2c-Cyclop1-YFP are located around the nuclei of reproductive cells. Note that tiny spots of Vc2c-Cyclop1-YFP location can be seen only with higher magnification (see below). V. carteri consists of approximately 2000 small, terminally differentiated, biflagellate somatic cells at the surface and approximately 16 large reproductive cells in the interior of a transparent sphere of glycoprotein-rich extracellular matrix. B1–B4 Close-up view of an optical cross section of somatic cells. Each somatic cell contains one to several tiny spots of Vc2c-Cyclop1-YFP location. The fluorescent spots are 0.5 to 1.0 μm in diameter (B1). C1–C4 Close-up view of the cell surface of a reproductive cell during the growth phase. There are numerous tiny spots of Vc2c-Cyclop1-YFP location close to the cell surface. The fluorescent spots are 0.5 to 1.0 μm in diameter (C1), just like the ones observed in somatic cells. The spots of Vc2c-Cyclop1-YFP location never overlap with the chlorophyll fluorescence (C2). MTOC, microtubule organizing center. D1–D4 Cross section of a reproductive cell during the growth phase. Most of Vc2c-Cyclop1-YFP is located close to the nucleus and appears as a diffuse cloud (D1, D2). Vc2c-Cyclop1-YFP also is located close to the cell surface (arrowheads in D1), which corresponds to the spots observed in C1–C4. E1–E4 Cross section of a reproductive cell shortly before onset of embryogenesis. Vc2c-Cyclop1-YFP forms as a distinct structure around the nucleus. It appears that the surface of the nucleus is studded with tiny beads of Vc2c-Cyclop1-YFP. In addition, Vc2c-Cyclop1-YFP is located close to the cell surface of the cell (arrowheads in E1). Note that this reproductive cell is more advanced in development and thus larger than the reproductive cell in D1–D4; however, the focal plane is not as deep inside the cell as in D1–D4, which makes it possible to detect several large, non-contractile vacuoles