Govorunova Elena G, Jung Kwang-Hwan, Sineshchekov Oleg A, Spudich John L
Center for Membrane Biology, Department of Biochemistry and Molecular Biology and Department of Microbiology and Molecular Genetics, University of Texas Medical School, Houston, Texas 77030, USA.
Biophys J. 2004 Apr;86(4):2342-9. doi: 10.1016/S0006-3495(04)74291-5.
Two retinylidene proteins, CSRA and CSRB, have recently been shown by photoelectrophysiological analysis of RNAi-transformants to mediate phototaxis signaling in Chlamydomonas reinhardtii. Here we report immunoblot detection of CSRA and CSRB apoproteins in C. reinhardtii cells enabling assessment of the cellular content of the receptors. We obtain 9 x 10(4) CSRA and 1.5 x 10(4) CSRB apoprotein molecules per cell in vegetative cells of the wild-type strain 495, a higher value than that for functional receptor cellular content estimated previously from photosensitivity measurements and retinal extraction yields. Exploiting our ability to control the CSRA/CSRB ratio by transformation with receptor gene-directed RNAi, we report analysis of the CSRA and CSRB roles in the photophobic response of the organism by action spectroscopy with automated cell tracking/motion analysis. The results show that CSRA and CSRB each mediate the photophobic swimming response, a second known retinal-dependent photomotility behavior in C. reinhardtii. Due to the different light saturation and spectral properties of the two receptors, CSRA is dominantly responsible for photophobic responses, which appear at high light intensity.
最近通过对RNAi转化体的光电生理分析表明，两种视黄醛蛋白CSRA和CSRB介导莱茵衣藻的趋光信号传导。在此，我们报告了在莱茵衣藻细胞中对CSRA和CSRB脱辅基蛋白的免疫印迹检测，从而能够评估受体的细胞含量。在野生型菌株495的营养细胞中，我们每个细胞获得9×10⁴个CSRA和1.5×10⁴个CSRB脱辅基蛋白分子，该值高于先前通过光敏性测量和视网膜提取产量估计的功能性受体细胞含量。利用我们通过受体基因定向RNAi转化来控制CSRA/CSRB比率的能力，我们通过自动细胞跟踪/运动分析的作用光谱法报告了CSRA和CSRB在生物体避光反应中的作用分析。结果表明，CSRA和CSRB各自介导避光游泳反应，这是莱茵衣藻中第二种已知的视网膜依赖性光运动行为。由于这两种受体的光饱和和光谱特性不同，CSRA主要负责在高光强度下出现的避光反应。
