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.
Highlights
The green algae Chlamydomonas reinhardtii and Volvox carteri are important models for studying light perception and response, expressing many different photoreceptors
We demonstrated that RhoPDE is light-regulated via a light-sensitive Michaelis-Menten constant [18]
Live cell imaging of Vc2c-Cyclop1 in V. carteri For in vivo localization of the guanylyl cyclase in the source organism, we focused on Vc2c-Cyclop1 because we expected better localization results in Volvox for reasons of cell size: The largest cells in Volvox, the reproductive cells, are approximately 50 μm in diameter, whereas Chlamydomonas cells usually are only about 10 μm in diameter, which corresponds to a 125-fold difference in volume
Summary
The green algae Chlamydomonas reinhardtii and Volvox carteri are important models for studying light perception and response, expressing many different photoreceptors. The family of microbial rhodopsins expanded remarkably after the first demonstration of bacteriorhodopsin as a light-activated proton pump [1, 2] in the archaeon Halobacterium halobium [3] (later correctly identified as H. salinarum). Tian et al BMC Biology (2018) 16:144 and C-termini and most likely eight transmembrane helices (TMs), as first proposed and demonstrated by us [12] and later confirmed by Trieu et al with a different method [15]. Another new enzymatic microbial rhodopsin is the rhodopsin phosphodiesterase, abbreviated as RhPDE [16] or RhoPDE [17, 18]. We propose to classify the microbial or type I rhodopsins in two subtypes: type Ia with 7 TM helices and an extracellular N-terminus and type Ib with 8 TM helices and a cytosolic N-terminus
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
