Genetic transfer of the pigment bacteriorhodopsin into the eukaryote Schizosaccharomyces pombe

Abstract

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.

Functional expression of bacteriorhodopsin in oocytes allows direct measurement of voltage dependence of light induced H+ pumping

“…Although functional expression in S. pombe was already dcmonstrated a few years ago [15,22] it was still surprising that tl~ archaebacterial protein bacteriorhodopsin could be expl essed in an amphibian cell. It was additionally satisfying from tl-e electrophysiological investigations that the direction of light in :luced current flow was always positive, i.e.…”

“…Plasmid pGEMHE contains the 3' and 5' untranslated regions of the b globin gene from Xenopus [14]. A promoter-free bop gene, encoding bacterioopsin including its signal sequence, was isolated from pBSbop [15] by cleavage with BamHI and HindIII and ligated into pGEMHE, behind the T7 RNA-polymerase promoter. E. coli was transformed by the RbCI method [16].…”

Approaches for biological and biomimetic energy conversion

“…Sitespecific mutants of bacteriorhodopsin were able to reproduce this behavior (16), showing that the ion specificity can be designed. Functional bacteriorhodopsin has also been transfected into naturally nonphotoactive organisms (17,18), introducing the possibility of producing specialized organisms that are optimized for solar energy conversion.…”

Photoactive mitochondria: in vivo transfer of a light-driven proton pump into the inner mitochondrial membrane of Schizosaccharomyces pombe.

“…In previous papers, it was shown that the fission yeast Schizosaccharomyces pombe is the only heterologous organism suitable for functional expression of bacteriorhodopsin (bR) in high yield (8,9), without isolation and reconstitution of the protein in artificial membranes as is necessary for the Escherichia coli expression system (3). The retinal protein bR expressed in S. pombe with its original leader sequence is located in the cell membrane as in the natural host Halobacterium salinarium.…”

“…The retinal protein bR expressed in S. pombe with its original leader sequence is located in the cell membrane as in the natural host Halobacterium salinarium. To obtain functional bR in the fission yeast, bacterioopsin (bO) is regenerated in vivo by the addition of retinal to the culture medium (8,9).…”