Division of Soft Matter, Graduate School of Life Science, Hokkaido University, Sapporo, Japan.
Division of Macromolecular Functions, Department of Biological Science, School of Science, Hokkaido University, Sapporo, Japan.
J Biol Chem. 2023 Nov;299(11):105305. doi: 10.1016/j.jbc.2023.105305. Epub 2023 Sep 29.
Previous research of anion channelrhodopsins (ACRs) has been performed using cytoplasmic domain (CPD)-deleted constructs and therefore have overlooked the native functions of full-length ACRs and the potential functional role(s) of the CPD. In this study, we used the recombinant expression of full-length Guillardia theta ACR1 (GtACR1_full) for pH measurements in Pichia pastoris cell suspensions as an indirect method to assess its anion transport activity and for absorption spectroscopy and flash photolysis characterization of the purified protein. The results show that the CPD, which was predicted to be intrinsically disordered and possibly phosphorylated, enhanced NO transport compared to Cl transport, which resulted in the preferential transport of NO. This correlated with the extended lifetime and large accumulation of the photocycle intermediate that is involved in the gate-open state. Considering that the depletion of a nitrogen source enhances the expression of GtACR1 in native algal cells, we suggest that NO transport could be the natural function of GtACR1_full in algal cells.
先前对阴离子通道视紫红质（ACRs）的研究使用的是细胞质结构域（CPD）缺失的构建体，因此忽略了全长 ACRs 的天然功能以及 CPD 的潜在功能作用。在这项研究中，我们使用重组表达全长的Guillardia theta ACR1（GtACR1_full），通过毕赤酵母细胞悬浮液中的 pH 值测量，作为一种间接方法来评估其阴离子转运活性，并对纯化蛋白进行吸收光谱和闪光光解特性研究。结果表明，CPD 可能是内在无序的，并且可能被磷酸化，与 Cl 转运相比，CPD 增强了 NO 转运，导致 NO 的优先转运。这与参与门控开放状态的光循环中间体的延长寿命和大量积累相关。考虑到氮源的消耗会增强天然藻类细胞中 GtACR1 的表达，我们认为 NO 转运可能是 GtACR1_full 在藻类细胞中的天然功能。
