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.

The gene encoding for bacterio-opsin (bop gene) from Halobacterium halobium has been introduced in a yeast expression vector. After transformation in Schizosaccharomyces pombe, bacterio-opsin (BO) is expressed and was detected by antisera. The precursor protein of BO (pre-BO) is processed by cleavage of amino acids at the N-terminal end as in H. halobium. Addition of the chromophore, retinal, to the culture medium results in a slight purple colour of the yeast cells indicating the in vivo regeneration of BO to bacteriorhodopsin (BR) and its incorporation into membranes. Therefore, in contrast to the expression in E. coil, isolation of the membrane protein and reconstitution in lipid vesicles is not necessary for functional analysis. The kinetics of the ground state signal of the photocycle BR in protoplasm is demonstrated by flash spectroscopy and is comparable to that of the natural system. The present investigation shows for the first time the transfer of an energy converting protein from archaebacteria to eukaryotes by genetic techniques. This is a basis for further studies on membrane biogenesis, genetics, and bioenergetics by analysis of in vivo active mutants.

A likely conformation of Rho EF involves some extension of helices E and F, with the tip of the loop lying over helix C and projecting towards the C terminus. This is consistent with mutagenesis data showing the TTQ transducin-binding motif close to loop CD, and cysteine cross-linking data indicating the C-terminal part of Rho EF to be close to the CD loop.

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.

The purple pigment bacteriorhodopsin (BR) acts as a light-driven proton pump in the cytoplasmic membrane of the archaebacterium Halobacterium halobium. The original gene encoding for the precursor of bacterio-opsin (bop-gene) and a shortened gene, where the first nucleotides encoding for the presequence are deleted, were introduced in yeast expression vectors. Northern blot analysis of transformed Schizosaccharomyces pombe cells revealed the strong transcription of the archaebacterial DNA directed by the constitutive alcohol dehydrogenase promoter of the fission yeast. The translated precursor and the second construction without the presequence were accumulated in the plasma membrane fraction of the eukaryote. Yeast cells containing the mature BR showed a weaker color than those harboring the precursor protein, if the cells were grown in minimal medium with 2% glucose. At higher glucose concentrations (7%) the expression of BR increased with and without presequence. The overexpressed precursor leads to coryneform fission yeast, whereas cells transformed with the vector containing the bop-gene without the presequence, producing the mature protein, retained the rod-shaped form.