A bipartite S unit of an ECF-type cobalt transporter.
ECF-class transporters comprise abundant importers for micronutrients such as vitamins and transition-metal ions, and for intermediates of salvage pathways in bacteria and archaea. They are composed of ABC ATPases (A units), a conserved transmembrane protein (T unit) and a substrate-specific transmembrane protein (S unit or core transporter). Here we analyzed the function of an ECF-type Co(2+) transporter (CbiMNQO) and, in particular, the derived bipartite S unit CbiMN. CbiMN was characterized as the minimal unit that functions as a Co(2+) transporter. Neither the solitary CbiM nor a tripartite CbiMQO complex was active, indicating an essential role for CbiN. CbiN was loosely bound in CbiMNQO and CbiMN complexes, and did not copurify with its partners. Generating a contiguous reading frame resulted in a Cbi(MN) fusion protein that displayed Co(2+)-transport activity and interacted with CbiQO in vivo. Sixteen variants of Cbi(MN) with modifications in the strongly conserved N-terminal stretch of ten amino-acid residues were constructed and analyzed for transport activity. The results indicate that the length and sequence of this region are critical for functioning of the core transporter. Specifically, they point to essential roles of His₂ and the distance of His₂ to the amino group of the peptide chain in metal recognition.
Siche
Stefanie
S
Institut für Biologie/Mikrobiologie, Humboldt-Universität zu Berlin, Chausseestraße 117, 10115 Berlin, Germany. st.siche@web.de
Neubauer
Olivia
O
Hebbeln
Peter
P
Eitinger
Thomas
T
D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000378:metabolism*; D003035:Cobalt / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016354:Rhodobacter capsulatus / Q000201:enzymology* / Q000235:genetics
D018528:ATP-Binding Cassette Transporters; D050505:Mutant Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D003035:Cobalt