Photoactive mitochondria: in vivo transfer of a light-driven proton pump into the inner mitochondrial membrane of Schizosaccharomyces pombe.
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.
Hoffmann
A
A
Forschungszentrum Jülich, Institut für Biologische Informationsverarbeitung, Germany.
Hildebrandt
V
V
Heberle
J
J
Büldt
G
G
D001436:Bacteriorhodopsins / Q000096:biosynthesis / Q000378:metabolism*; D002458:Cell Fractionation; D005452:Fluoresceins; D005456:Fluorescent Dyes; D018014:Gene Transfer Techniques; D006217:Halobacterium / Q000235:genetics / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism* / Q000648:ultrastructure; D007700:Kinetics; D008027:Light; D008928:Mitochondria / Q000378:metabolism* / Q000648:ultrastructure; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012568:Schizosaccharomyces / Q000378:metabolism* / Q000502:physiology / Q000648:ultrastructure
D005452:Fluoresceins; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D001436:Bacteriorhodopsins; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein