RESUMO
Bacterial ATP-binding cassette (ABC) importers are primary active transporters that are critical for nutrient uptake. Based on structural and functional studies, ABC importers can be divided into two distinct classes, type I and type II. Type I importers follow a strict alternating access mechanism that is driven by the presence of the substrate. Type II importers accept substrates in a nucleotide-free state, with hydrolysis driving an inward facing conformation. The ribose transporter in Escherichia coli is a tripartite complex consisting of a cytoplasmic ATP-binding cassette protein, RbsA, with fused nucleotide binding domains; a transmembrane domain homodimer, RbsC2; and a periplasmic substrate binding protein, RbsB. To investigate the transport mechanism of the complex RbsABC2, we probed intersubunit interactions by varying the presence of the substrate ribose and the hydrolysis cofactors, ATP/ADP and Mg(2+). We were able to purify a full complex, RbsABC2, in the presence of stable, transition state mimics (ATP, Mg(2+), and VO4); a RbsAC complex in the presence of ADP and Mg(2+); and a heretofore unobserved RbsBC complex in the absence of cofactors. The presence of excess ribose also destabilized complex formation between RbsB and RbsC. These observations suggest that RbsABC2 shares functional traits with both type I and type II importers, as well as possessing unique features, and employs a distinct mechanism relative to other ABC transporters.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Periplásmicas de Ligação/metabolismo , Ribose/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Transporte Biológico/genética , Western Blotting , Dicroísmo Circular , Espectroscopia de Ressonância de Spin Eletrônica , Proteínas de Escherichia coli/genética , Magnésio/metabolismo , Proteínas de Membrana Transportadoras/genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Proteínas Periplásmicas de Ligação/genética , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismoRESUMO
The topology of RbsC, the membrane component of the ribose transporter in Escherichia coli, has been determined by using 34 single-cysteine mutants and a modified fluorescence labeling technique designated multiplex labeling. This technique gives topology, expression, and localization information for a membrane protein from a single batch of bacterial cells. The results indicate that RbsC contains 10 transmembrane-spanning helices, with the N and C termini being in the cytosol. This topology matches predictions from the latest prediction programs and the topology of the similar, recently crystallized membrane protein BtuC.