PMID:8943289
| Citation |
Rothman, A, Padan, E and Schuldiner, S (1996) Topological analysis of NhaA, a Na+/H+ antiporter from Escherichia coli. J. Biol. Chem. 271:32288-92 |
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| Abstract |
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. |
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| Keywords |
Alkaline Phosphatase/metabolism; Amino Acid Sequence; Carboxypeptidase B; Carboxypeptidases/metabolism; Escherichia coli/chemistry; Escherichia coli Proteins; Molecular Sequence Data; Protein Conformation; Protein Structure, Secondary; Sodium-Hydrogen Antiporter/chemistry |
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