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

Abstract

BackgroundThe 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.ResultsHere 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.ConclusionsCr2c-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.Electronic supplementary materialThe online version of this article (10.1186/s12915-018-0613-5) contains supplementary material, which is available to authorized users.

Babo1, formerly Vop1 and Cop1/2, is no eyespot photoreceptor but a basal body protein illuminating cell division in Volvox carteri

“…The LHCBM1 gene is a chlorophyll a / b binding protein in the light‐harvesting complex II. In previous RNA‐seq studies, the LHCBM1 promoter demonstrated strong expression and the expression levels were similar in both cell types (Klein et al , ; Tian et al , ). A 0.9 kb DNA fragment containing the LHCBM1 promoter region was introduced in front of the fused genes using Kpn I/ Apa I for babo1 / yfp and Xho I/ Xho I for cfp / tubB2 , resulting in the plasmids pBlue_LHCBM1_Babo1_YFP (Figure b) and pBlue_LHCBM1_CFP_TubB2 (Figure d).…”

“…However, no transmembrane spanning segments have been identified and all of these programs predicted that Babo1 is not a transmembrane protein. Moreover, when yellow fluorescent protein (YFP)‐tagged Babo1 of C. reinhardtii (formerly Cop1/2) was expressed in Xenopus oocytes, YFP fluorescence was exclusively found in the soluble fraction of oocyte extracts but not in the membrane fraction (Tian et al , ). As a consequence of these results, Babo1 cannot be an opsin, which would necessarily require a transmembrane structure with generally seven transmembrane helices.…”

“…Furthermore, when C.r. Babo1 was expressed in Xenopus oocytes, it was not found in the membrane fraction (Tian et al , ). Given that all rhodopsins known to date have at least seven transmembrane helices and also contain a conserved retinal binding site (Gao et al , ), it is obvious that Babo1 is not an opsin.…”

“…The nitA − descendant of Eve10 was generated by random mutagenesis and chlorate selection as previously described (Huskey et al , ; Harper et al , ; Adams et al , ). The obtained non‐revertible mutant strain, TNit‐1013 (Tian et al , ), contains a deletion of 1013 bp in the nit A gene and, therefore, is not able to grow in medium containing nitrate as the sole source of nitrogen. V. carteri strain TNit‐1013 was therefore grown in Volvox medium (Provasoli and Pintner, ; Starr, ) supplemented with 1 m m ammonium chloride (NH 4 Cl) as a nitrogen source.…”

“…The nit A − V. carteri strain TNit‐1013 (Tian et al , ) was grown on a larger scale in Volvox medium supplemented with 1 m m NH 4 Cl. In preparation of particle bombardment, 3 mg of gold microprojectiles (1.0 µm in diameter, Bio‐Rad, Hercules, CA, USA) were coated as previously described (Lerche and Hallmann, ; Lerche and Hallmann, ) using 5 µg of plasmid pVcNR15 (Gruber et al , ), which allows for expression of the nit A gene for selection, and 5 µg of each plasmid that expresses fused gene constructs for subcellular localization (Figure ).…”