Membrane topology of the beta-subunit of the oxaloacetate decarboxylase Na+ pump from Klebsiella pneumoniae.
The topology of the beta-subunit of the oxaloacetate Na+ pump (OadB) was probed with the alkaline phosphatase (PhoA) and beta-galactosidase (lacZ) fusion technique. Additional evidence for the topology was derived from amino acid alignments and comparative hydropathy profiles of OadB with related proteins. Consistent results were obtained for the three N-terminal and the six C-terminal membrane-spanning alpha-helices. However, the two additional helices that were predicted by hydropathy analyses between the N-terminal and C-terminal blocks did not conform with the fusion results. The analyses were therefore extended by probing the sideness of various engineered cysteine residues with the membrane-impermeant reagent 4-acetamido-4'-maleimidylstilbene-2, 2'-disulfonate. The results were in accord with those of the fusion analyses, suggesting that the protein folds within the membrane by a block of three N-terminal transmembrane segments and another one with six C-terminal transmembrane segments. The mainly hydrophobic connecting segment is predicted not to traverse the membrane fully, but to insert in an undefined manner from the periplasmic face. According to our model, the N-terminus is at the cytoplasmic face and the C-terminus is at the periplasmic face of the membrane.
Jockel
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P
Mikrobiologisches Institut der Eidgenössischen Technischen Hochschule, ETH-Zentrum, Zürich, Switzerland.
Di Berardino
M
M
Dimroth
P
P
D000469:Alkaline Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001693:Biological Transport, Active; D002262:Carboxy-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D018844:Cyclin-Dependent Kinases / Q000235:genetics; D003545:Cysteine / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D005798:Genes, Bacterial; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000235:genetics; D007763:Lac Operon; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012964:Sodium / Q000378:metabolism*; D013267:Stilbenes / Q000737:chemistry; D013451:Sulfonic Acids / Q000737:chemistry; D001616:beta-Galactosidase / Q000378:metabolism
C000618862:4-acetamido-4'-maleimidylstilbene-2,2'-disulfonate; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D013267:Stilbenes; D013451:Sulfonic Acids; D012964:Sodium; D018844:Cyclin-Dependent Kinases; D000469:Alkaline Phosphatase; D001616:beta-Galactosidase; D002262:Carboxy-Lyases; C021916:oxaloacetate decarboxylase; D003545:Cysteine