Three two-component transporters with channel-like properties have monovalent cation/proton antiport activity

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 104; no. 33; pp. 13289 - 13294
• Main Authors: Fujisawa, Makoto, Ito, Masahiro, Krulwich, Terry A
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 14.08.2007; National Acad Sciences
• Subjects: Adducts; Ammonia; Antiporters; Bacillus; Bacillus subtilis; Bacteriology; Biochemistry; Biological Sciences; Cations, Monovalent - metabolism; Cloning, Molecular; DNA; E coli; Escherichia coli; Fluorescence; Glutathione - physiology; Membrane proteins; Membrane Transport Proteins - metabolism; NAD - metabolism; P branes; Plasmids; Proteins; Protons; Respiration; String theory
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0703709104

Properties of four two-component bacterial transport systems of the cation/proton antiporter-2 (CPA2) family led to suggestions that this CPA2 subset may use a channel rather than an antiport mechanism [see Booth IR, Edwards MD, Gunasekera B, Li C, Miller S (2005) in Bacterial Ion Channels, eds Kubalski A, Martinac B (Am Soc Microbiol, Washington, DC), pp 21-40]. The transporter subset includes the intensively studied glutathione-gated K⁺ efflux systems from Escherichia coli, KefGB, and KefFC. KefG and KefF are ancillary proteins. They are peripheral membrane proteins that are encoded in operons with the respective transporter proteins, KefB and KefC, and are required for optimal efflux activity. The other two-component CPA2 transporters of the subset are AmhMT, an NH₄⁺ (K⁺) efflux system from alkaliphilic Bacillus pseudofirmus OF4; and YhaTU, a K⁺ efflux system from Bacillus subtilis. Here a K⁺/H⁺ antiport capacity was demonstrated for YhaTU, AmhMT, and KefFC in membrane vesicles from antiporter-deficient E. coli KNabc. The apparent Km for K⁺ was in the low mM range. The peripheral protein was required for YhaU- and KefC-dependent antiport, whereas both AmhT and AmhMT exhibited antiport. KefFC had the broadest range of substrates, using Rb⁺[almost equal to]K⁺>Li⁺>Na⁺. Glutathione significantly inhibited KefFC-mediated K⁺/H⁺ antiport in vesicles. The inhibition was enhanced by NADH, which presumably binds to the KTN/RCK domain of KefC. The antiport mechanism accounts for the H⁺ uptake involved in KefFC-mediated electrophile resistance in vivo. Because the physiological substrate of AmhMT in the alkaliphile is NH₄⁺, the results also imply that AmhMT catalyzes NH₄⁺/H⁺ antiport, which would prevent net cytoplasmic H⁺ loss during NH₄⁺ efflux.