Topological analysis of NhaA, a Na+/H+ antiporter from Escherichia coli.
Analysis of the hydropathic profile of the amino acid sequence of NhaA, a Na+/H+ antiporter from Escherichia coli has previously suggested the existence of 11 putative transmembrane segments (Taglicht, D., Padan, E., and Schuldiner, S. (1991) J. Biol. Chem. 266, 11289-11294). In the present work to test the location of the C terminus, right-side-out and inside-out membrane vesicles were digested with carboxypeptidase B and probed with an antibody raised against a synthetic peptide whose sequence was based on the C terminus sequence. The results demonstrate that the C terminus is facing the cell interior because it is available for digestion only from the inside. Previous evidence from an NhaA-beta-galactosidase fusion to loop 5 of NhaA indicated that this loop is also facing the cytoplasm (Karpel, R., Alon, T., Glaser, G., Schuldiner, S., and Padan, E. (1991) J. Biol. Chem. 266, 21753-21759) and therefore was not consistent with the position of the C terminus in an 11-transmembrane segment model. Therefore, the model was re-evaluated. For this purpose, 10 nhaA'-'phoA gene fusions were constructed and assayed for alkaline phosphatase activity. The results support a 12-transmembrane segment model with the N and C termini located in the cytoplasm. The evidence indicates that two very short segments, 14 and 16 amino acids long, must cross the membrane in an unknown conformation.
Rothman
A
A
Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel. shimons@leonardo.ls.huji.ac.il
Padan
E
E
Schuldiner
S
S
D000469:Alkaline Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D043424:Carboxypeptidase B; D002268:Carboxypeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry*
D029968:Escherichia coli Proteins; C102839:NhaA protein, E coli; D017923:Sodium-Hydrogen Exchangers; D000469:Alkaline Phosphatase; D002268:Carboxypeptidases; D043424:Carboxypeptidase B