title: The C-terminal cytoplasmic portion of the NhaP2 cation-proton antiporter from Vibrio cholerae affects its activity and substrate affinity.
abstract: In this work, we report the phenotypic and biochemical effects of deleting the C-terminal cytoplasmic portion of the NhaP2 cation/proton antiporter from Vibrio cholerae. While the deletion changed neither the expression nor targeting of the Vc-NhaP2 in an antiporter-less Escherichia coli strain, it resulted in a changed sensitivity of the host to sodium ions at neutral pH, indicating an altered Na(+) transport through the truncated variant. When assayed in inside-out sub-bacterial vesicles, the truncation was found to result in greatly reduced K(+)/H(+) and Na(+)/H(+) antiport activity at all pH values tested and a greater than fivefold decrease in the affinity for K(+) (measured as the apparent K m) at pH 7.5. Being expressed in trans in a strain of V. cholerae bearing a chromosomal nhaP2 deletion, the truncated nhaP2 gene was able to complement its inability to grow in potassium-rich medium at pH 6.0. Thus the residual K(+)/H(+) antiport activity associated with the truncated Vc-NhaP2 was still sufficient to protect cells from an over-accumulation of K(+) ions in the cytoplasm. The presented data suggest that while the cytoplasmic portion of Vc-NhaP2 is not involved in ion translocation directly, it is necessary for optimal activity and substrate binding of the Vc-NhaP2 antiporter.
authors.affiliation: Department of Chemistry, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada.
authors.forename: Evan J
authors.initials: EJ
authors.lastname: Wiens
authors.forename: Judith L
authors.initials: JL
authors.lastname: Winogrodzki
authors.forename: Craig T
authors.initials: CT
authors.lastname: Resch
authors.forename: George L
authors.initials: GL
authors.lastname: Orriss
authors.forename: Jörg
authors.initials: J
authors.lastname: Stetefeld
authors.forename: Pavel
authors.initials: P
authors.lastname: Dibrov
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002412:Cations / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods; D003593:Cytoplasm / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D011188:Potassium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*; D014734:Vibrio cholerae / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D002412:Cations; D017923:Sodium-Hydrogen Exchangers; D012964:Sodium; D011188:Potassium