Topological study of Vibrio alginolyticus NhaB Na+/H+ antiporter using gene fusions in Escherichia coli cells.
NhaB, an Na+/H+ antiporter, of Vibrio alginolyticus is a 528-amino-acid protein. Hydropathy profile-based computer analysis predicted that the NhaB might contain up to 13 membrane-spanning domains. To examine this hypothesis, we applied the phoA fusion method to the cloned nhaB gene. Eighteen plasmid-borne nhaB-phoA fusion genes were constructed in Escherichia coli cells and the alkaline phosphatase activity and expression level of the fusion proteins analyzed. These results and the results obtained with additional constructs indicated that V. alginolyticus NhaB has a unique topology consisting of nine transmembrane segments with the N-terminus in the cytoplasm and the C-terminus in the periplasm.
Enomoto
H
H
Laboratory of Membrane Biochemistry, Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263, Japan.
Unemoto
T
T
Nishibuchi
M
M
Padan
E
E
Nakamura
T
T
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D019677:Artificial Gene Fusion; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D002462:Cell Membrane / Q000737:chemistry; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000138:chemical synthesis; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D014733:Vibrio / Q000737:chemistry / Q000235:genetics*
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; C491047:NhaB protein, E coli; D011993:Recombinant Fusion Proteins; D017923:Sodium-Hydrogen Exchangers; D000469:Alkaline Phosphatase