Topological analysis of the membrane-localized redox-responsive sensor kinase PrrB from Rhodobacter sphaeroides 2.4.1.
Photosynthesis gene expression in Rhodobacter sphaeroides is controlled in part by the two-component (Prr) regulatory system composed of a membrane-bound sensor kinase (PrrB) and a response regulator (PrrA). Hydropathy profile-based computer analysis predicted that the PrrB polypeptide could contain six membrane-spanning domains at its amino terminus and a hydrophilic, cytoplasmic carboxyl terminus. Both the localization and the topology of the PrrB sensor kinase have been studied by generating a series of gene fusions with the Escherichia coli periplasmically localized alkaline phosphatase and the cytoplasmic beta-galactosidase. Eighteen prrB-phoA and five prrB-lacZ fusions were constructed and expressed in both E. coli and R. sphaeroides. Enzymatic activity assays and immunoblot analyses were performed to identify and to localize the different segments of PrrB in the membrane. The data obtained in E. coli generally correlated with the data obtained in R. sphaeroides and support the computer predictions. On the basis of the theoretical model and the results provided by these studies, a topological model for the membrane localization of the PrrB polypeptide is proposed.
Ouchane
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Department of Microbiology and Molecular Genetics, University of Texas Medical School, Houston, Texas 77030, USA.
Kaplan
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D000469:Alkaline Phosphatase / Q000235:genetics; D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D000071677:Histidine Kinase; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins; D012242:Rhodobacter sphaeroides / Q000201:enzymology* / Q000235:genetics; D015398:Signal Transduction; D001616:beta-Galactosidase / Q000235:genetics
D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D011494:Protein Kinases; D000071677:Histidine Kinase; D000469:Alkaline Phosphatase; D001616:beta-Galactosidase