The use of gene fusions to examine the membrane topology of the L-subunit of the photosynthetic reaction center and of the cytochrome b subunit of the bc1 complex from Rhodobacter sphaeroides.
The topology of the cytochrome b subunit of the bc1 complex from Rhodobacter sphaeroides has been examined by generating gene fusions with alkaline phosphatase. Gene fusions were generated at random locations within the fbcB gene encoding the cytochrome b subunit. These fusion products were expressed in Escherichia coli and were screened for alkaline phosphatase activity on chromogenic plates. 33 in-frame fusions which showed activity were further characterized. The fusion junctions of all those fusions which had a high specific activity were clustered in three regions of the cytochrome b polypeptide, and thus these regions were tentatively assigned as being near the periplasmic surface. The data are consistent with a model containing eight transmembrane helices. In order to explore the validity of the gene fusion approach for a protein not normally expressed in E. coli, the topology of the L-subunit of the photosynthetic reaction center from R. sphaeroides was also explored using phoA gene fusions. A similar protocol was used as with the cytochrome b subunit. The gene fusions with high specific activity were shown to be in regions of the L-subunit polypeptide known to be at or near the periplasmic surface, as defined by the high resolution structure determined by X-ray crystallography. These data demonstrate the utility of this approach for determining membrane protein topology and extend potential applications to include at least some proteins not normally expressed in E. coli.
Yun
C H
CH
Department of Physiology and Biophysics, University of Illinois, Urbana 61801.
Van Doren
S R
SR
Crofts
A R
AR
Gennis
R B
RB
D000469:Alkaline Phosphatase / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D003573:Cytochrome b Group / Q000737:chemistry / Q000235:genetics*; D014450:Electron Transport Complex III / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry / Q000235:genetics*; D010957:Plasmids; D011487:Protein Conformation; D015183:Restriction Mapping; D012242:Rhodobacter sphaeroides / Q000235:genetics / Q000378:metabolism*; D014961:X-Ray Diffraction
D003573:Cytochrome b Group; D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D045322:Photosynthetic Reaction Center Complex Proteins; D000469:Alkaline Phosphatase; D014450:Electron Transport Complex III