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.
Highlights
The predicted amino acid sequence of NhaA suggested that the antiporter consists of 362 amino acids [6]
RSO and ISO membrane vesicles from E. coli in which NhaA was labeled with [35S]methionine were prepared
The vesicle preparations were digested for various time periods with 40 g/ml carboxypeptidase B
Summary
Bacterial Strains and Plasmids—Bacterial strains used in this study are E. coli K-12 derivatives. Plasmids pGM36 carrying the wild-type nhaA [6] and pT7–5/lacY-phoA carrying the phoA [12] were used for construction of the nhaAЈ-ЈphoA fusions. Plasmids pDT82 carrying the wild-type nhaA [4] and pGP1–2 [13] were used for exclusive labeling of NhaA, whereas plasmids pT7–5/nhaA-phoA carrying the nhaAЈ-ЈphoA fusions and pGP1–2 were used for labeling the alkaline phosphatase fusions. The final construct pT7–5/nhaA-phoA encodes full-length NhaA with alkaline phosphatase attached in frame to its C terminus. All other nhaAЈ-ЈphoA fusions were made by polymerase chain reaction as described above, with a reverse primer that generates XbaI sites in desired regions of nhaA (Table I). Alkaline Phosphatase Activity Analysis—E. coli (TA15) cells carrying plasmid pGP1–2 were transformed with the nhaAЈ-ЈphoA fusion constructs for determination of alkaline phosphatase activity and labeling with [35S]methionine. The additional sequences have been introduced to create the XbaI restriction site
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