Genetic transfer of the pigment bacteriorhodopsin into the eukaryote Schizosaccharomyces pombe.
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. coli, 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 protoplasts 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.
Hildebrandt
V
V
Freie Universität Berlin, Institut für Physik, Abt. Biophysik, Arnimalle, Germany.
Ramezani-Rad
M
M
Swida
U
U
Wrede
P
P
Grzesiek
S
S
Primke
M
M
Büldt
G
G
D001436:Bacteriorhodopsins / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology; D015153:Blotting, Western; D005798:Genes, Bacterial; D005822:Genetic Vectors; D006217:Halobacterium / Q000235:genetics*; D008565:Membrane Proteins / Q000235:genetics; D010777:Photochemistry; D010957:Plasmids; D011487:Protein Conformation; D011498:Protein Precursors / Q000378:metabolism; D004718:Saccharomycetales / Q000235:genetics*; D012568:Schizosaccharomyces / Q000235:genetics*; D013057:Spectrum Analysis / Q000379:methods; D014162:Transfection
D008565:Membrane Proteins; D011498:Protein Precursors; D001436:Bacteriorhodopsins