Topological analysis of NhaA, a Na+/H+ antiporter from Escherichia coli

Andrea Rothman, Etana Padan, Shimon Schuldiner*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

60 Scopus citations

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-β-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.

Original languageEnglish
Pages (from-to)32288-32292
Number of pages5
JournalJournal of Biological Chemistry
Volume271
Issue number50
DOIs
StatePublished - 1996

Fingerprint

Dive into the research topics of 'Topological analysis of NhaA, a Na+/H+ antiporter from Escherichia coli'. Together they form a unique fingerprint.

Cite this