Topological analysis of the Rhodobacter capsulatus PucC protein and effects of C-terminal deletions on light-harvesting complex II.
A theoretical model for the cytoplasmic membrane topology of the Rhodobacter capsulatus PucC protein was derived and tested experimentally with pucC'::pho'A gene fusions. The alkaline phosphatase (AP) activities of selected fusions were assayed, and the resultant pattern of high and low activity was compared with that of the theoretical model. High AP activity correlated well with fusion joints located in regions predicted to be periplasmic, and most fusions in predicted cytoplasmic loops yield approximately 1/20th as much activity. Replacement of pho'A with lac'Z in nine of the fusions confirmed the topology, as beta-galactosidase activities were generally reciprocal to the corresponding AP activity. On the basis of the theoretical analysis and the information provided by the activities of fusions, a model for PucC topology in which there are 12 membrane-spanning segments and both the N and C termini are located in the cytoplasm is proposed. Translationally out-of-frame pucC::phoA fusions were expressed in an R. capsulatus delta pucC strain. None of the fusions missing only one or two of the proposed C-terminal transmembrane segments restored the wild-type phenotype, suggesting that the C terminus of PucC is important for function.
LeBlanc
H N
HN
Department of Microbiology & Immunology, University of British Columbia, Vancouver, Canada.
Beatty
J T
JT
D000469:Alkaline Phosphatase / Q000096:biosynthesis / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002462:Cell Membrane / Q000378:metabolism; D005798:Genes, Bacterial; D045342:Light-Harvesting Protein Complexes; D008961:Models, Structural; D008969:Molecular Sequence Data; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000378:metabolism*; D045332:Photosystem II Protein Complex; D010957:Plasmids; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016354:Rhodobacter capsulatus / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D013053:Spectrophotometry; D001616:beta-Galactosidase / Q000378:metabolism
D001426:Bacterial Proteins; D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; C100892:PucC protein, Rhodobacter; D011993:Recombinant Fusion Proteins; C081485:light-harvesting protein complex II, Rhodobacter capsulatus; D000469:Alkaline Phosphatase; D001616:beta-Galactosidase