The archaebacterial membrane protein bacterio-opsin is expressed and N-terminally processed in the yeast Saccharomyces cerevisiae.
The bop gene codes for the membrane protein bacterio-opsin (BO), which on binding all-trans-retinal, constitutes the light-driven proton pump bacteriorhodopsin (BR) in the archaebacterium Halobacterium salinarium. This gene was cloned in a yeast multi-copy vector and expressed in Saccharomyces cerevisiae under the control of the constitutive ADH1 promoter. Both the authentic gene and a modified form lacking the precursor sequence were expressed in yeast. Both proteins are incorporated into the membrane in S. cerevisiae. The presequence is thus not required for membrane targeting and insertion of the archaebacterial protein in budding yeast, or in the fission yeast Schizosaccharomyces pombe, as has been shown previously. However, in contrast to S. pombe transformants, which take on a reddish colour when all-trans-retinal is added to the culture medium as a result of the in vivo regeneration of the pigment, S. cerevisiae cells expressing BO do not take on a red colour. The precursor of BO is processed to a protein identical in size to the mature BO found in the purple membrane of Halobacterium. The efficiency of processing in S. cerevisiae is dependent on growth phase, as well as on the composition of the medium and on the strain used. The efficiency of processing of BR is reduced in S. pombe and in a retinal-deficient strain of H. salinarium, when retinal is present in the medium.
Lang-Hinrichs
C
C
Technische Universität Berlin, FG Mikrobiologie und Genetik, Germany.
Queck
I
I
Büldt
G
G
Stahl
U
U
Hildebrandt
V
V
D000426:Alcohol Dehydrogenase / Q000235:genetics; D001436:Bacteriorhodopsins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D015153:Blotting, Western; D003001:Cloning, Molecular / Q000379:methods; D015870:Gene Expression; D005798:Genes, Bacterial; D006217:Halobacterium / Q000235:genetics* / Q000378:metabolism*; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012172:Retinaldehyde / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
D008565:Membrane Proteins; D011994:Recombinant Proteins; D001436:Bacteriorhodopsins; C017827:bacterio-opsin; D000426:Alcohol Dehydrogenase; D012172:Retinaldehyde