Botanik I, Julius-Maximilians-Universität Würzburg, Biozentrum, Julius-von-Sachs-Platz 2, 97082, Würzburg, Germany.
Department of Cellular and Developmental Biology of Plants, University of Bielefeld, Universitätsstr. 25, 33615, Bielefeld, Germany.
BMC Biol. 2018 Dec 6;16(1):144. doi: 10.1186/s12915-018-0613-5.
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.
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.
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.
绿藻莱茵衣藻和团藻是研究光感知和反应的重要模式生物，它们表达了许多不同的光受体。在莱茵衣藻中报道了超过 10 种视蛋白，然而仅有两种——通道视紫红质被功能表征。新视蛋白的表征将有助于理解绿藻的光生物学，并为光遗传学开发新的工具。
在这里，我们从这些绿藻中报告了一个新型视蛋白家族的特征：通过类似于双组分的磷酸转移调节的光抑制鸟苷酸环化酶，称为“双组分环化酶视蛋白”（2c-Cyclops）。我们证明了这种视蛋白在莱茵衣藻和团藻中的存在，并表明它们具有细胞质 N 端和 C 端，暗示了一个八次跨膜螺旋结构。我们还证明 cGMP 的产生既受光抑制又依赖于 ATP。Cr2c-Cyclop1 的环化酶活性在黑暗中通过组氨酸激酶向应答调节因子的持续磷酸化和磷酸转移保持功能，这通过突变证明。光的吸收抑制环化酶活性，很可能通过抑制磷酸转移。在团藻中过量表达 Vc2c-Cyclop1 蛋白会导致 cGMP 水平显著升高，证明了 Vc2c-Cyclop1 在体内的鸟苷酸环化酶活性。在团藻中对 YFP 标记的 Vc2c-Cyclop1 的活细胞成像显示，在核的直接外围存在发育依赖性的层状结构和细胞外围的密集斑点。
Cr2c-Cyclop1 和 Vc2c-Cyclop1 是受光抑制和 ATP 依赖的鸟苷酸环化酶，具有 I 型视蛋白结构域的异常八次跨膜螺旋结构，我们建议将其分类为 Ib 型，与 7TM 型 Ia 视蛋白相反。在团藻中过量表达 Vc2c-Cyclop1 蛋白会导致 cGMP 显著增加，证明了酶在其起源生物体中的功能。荧光活细胞成像显示 Vc2c-Cyclop1 位于细胞核的外围和细胞外围的限定区域。
