Hildebrandt V, Fendler K, Heberle J, Hoffmann A, Bamberg E, Büldt G
Department of Physics/Biophysics, Freie Universität Berlin, Germany.
Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3578-82. doi: 10.1073/pnas.90.8.3578.
Bacterioopsin (bO) from Halobacterium salinarium ("Halobacterium halobium") has been functionally expressed in a heterologous system, the fission yeast Schizosaccharomyces pombe. Regeneration of bO to bacteriorhodopsin (bR) in S. pombe has been achieved in vivo by addition of the chromophore retinal to the culture medium, as shown for a retinal-negative mutant of H. salinarium (JW5). Western blot analysis revealed that bR is more stable than bO against proteolysis in fission yeast and also in JW5. The light-driven proton pump is expressed in the eukaryotic organism and incorporated into the plasma membrane. Illumination of intact yeast cells leads to acidification of the external medium due to the translocation of H+ from inside to outside of the cell, indicating the same orientation of bR in the yeast plasma membrane as in H. salinarium. The kinetics of proton release into the water phase was observed with the optical pH indicator pyranine. Time-resolved absorbance changes of isolated plasma membrane measured by flash spectroscopy showed rise and decay of the M intermediate during the photocycle similar to those in the homologous system. Photocurrents and photovoltages were recorded with yeast plasma membrane attached to a planar lipid membrane and to a polytetrafluoroethylene (Teflon) film, respectively. Stationary currents measured in the presence of a protonophore showed continuous pumping activity of bR. The action spectrum of the photocurrent and the kinetics of the photovoltage were analyzed and compared with signals obtained from purple membranes. From all these different investigations we conclude that the integral membrane protein bR is correctly folded in vivo into the cytoplasmic membrane of the fission yeast S. pombe.
来自盐生盐杆菌（曾称“嗜盐盐杆菌”）的细菌视紫红质（bO）已在异源系统——裂殖酵母粟酒裂殖酵母中实现功能性表达。通过向培养基中添加发色团视黄醛，在粟酒裂殖酵母体内已实现bO再生为细菌视紫红质（bR），盐生盐杆菌的一个视黄醛阴性突变体（JW5）的情况即如此。蛋白质印迹分析表明，在裂殖酵母以及JW5中，bR比bO对蛋白水解更稳定。这种光驱动质子泵在真核生物中表达并整合到质膜中。完整酵母细胞受光照会导致外部培养基酸化，这是因为H⁺从细胞内部转运到外部，表明酵母质膜中bR的取向与盐生盐杆菌中的相同。使用光学pH指示剂吡喃黄素观察到质子释放到水相中的动力学。通过闪光光谱法测量分离的质膜的时间分辨吸光度变化，结果显示光循环过程中M中间体的上升和衰减，与同源系统中的情况相似。分别将酵母质膜附着在平面脂质膜和聚四氟乙烯（特氟龙）膜上记录光电流和光电压。在存在质子载体的情况下测量的稳态电流显示bR具有持续的泵浦活性。分析了光电流的作用光谱和光电压的动力学，并与从紫膜获得的信号进行比较。从所有这些不同的研究中我们得出结论，整合膜蛋白bR在体内正确折叠到裂殖酵母粟酒裂殖酵母的细胞质膜中。
