Hoffmann A, Hildebrandt V, Heberle J, Büldt G
Forschungszentrum Jülich, Institut für Biologische Informationsverarbeitung, Germany.
Proc Natl Acad Sci U S A. 1994 Sep 27;91(20):9367-71. doi: 10.1073/pnas.91.20.9367.
The light-driven proton pump bacteriorhodopsin (bR) from Halobacterium salinarium has been genetically transferred into the inner mitochondrial membrane (IM) of the eukaryotic cell Schizosaccharomyces pombe, where the archaebacterial proton pump replaces or increases the proton gradient usually formed by the respiratory chain. For targeting and integration, as well as for the correct orientation of bR in the IM, the bacterioopsin gene (bop) was fused to signal sequences of IM proteins. Northern and Western blot analysis proved that all hybrid gene constructs containing the bop gene and a mitochondrial signal sequence were expressed and processed to mature bR. Fast transient absorption spectroscopy showed photocycle activity of bR integrated in the IM by formation of the M intermediate. Experiments with the pH-sensitive fluorescence dye 2',7'-bis(2-carboxyethyl)-5 (and -6)-carboxyfluorescein revealed bR-mediated proton pumping from the mitochondrial matrix into the intermembrane space. Glucose uptake measurements under anaerobic conditions showed that yeast cells containing photoactive mitochondria need less sugar under illumination. In summary, our experiments demonstrate the functional genetic transfer of a light energy converter to a naturally nonphotoactive eukaryotic organism.
盐生盐杆菌的光驱动质子泵细菌视紫红质（bR）已被基因转移到真核细胞粟酒裂殖酵母的线粒体内膜（IM）中，在那里古细菌质子泵替代或增加了通常由呼吸链形成的质子梯度。为了实现靶向和整合，以及使bR在IM中正确定向，细菌视蛋白基因（bop）与IM蛋白的信号序列融合。Northern和Western印迹分析证明，所有包含bop基因和线粒体信号序列的杂交基因构建体均被表达并加工成成熟的bR。快速瞬态吸收光谱显示，通过形成M中间体，整合在IM中的bR具有光循环活性。使用pH敏感荧光染料2'，7'-双（2-羧乙基）-5（和-6）-羧基荧光素进行的实验表明，bR介导质子从线粒体基质泵入膜间隙。厌氧条件下的葡萄糖摄取测量表明，含有光活性线粒体的酵母细胞在光照下需要的糖分更少。总之，我们的实验证明了光能转换器向天然无光活性真核生物的功能性基因转移。
