In vitro functional characterization of BtuCD-F, the Escherichia coli ABC transporter for vitamin B12 uptake.
BtuCD is an ATP binding cassette (ABC) transporter that facilitates uptake of vitamin B(12) into the cytoplasm of Escherichia coli. The crystal structures of BtuCD and its cognate periplasmic binding protein BtuF have been recently determined. We have now explored BtuCD-F function in vitro, both in proteoliposomes and in various detergents. BtuCD reconstituted into proteoliposomes has a significant basal ATP hydrolysis rate that is stimulated by addition of BtuF and inhibited by sodium ortho-vanadate. When using different detergents to solubilize BtuCD, the basal ATP hydrolysis rate, the ability of BtuF to stimulate hydrolysis, and the extent to which sodium ortho-vanadate inhibits ATP hydrolysis all vary significantly. Reconstituted BtuCD can mediate transport of vitamin B(12) against a concentration gradient when coupled to ATP hydrolysis by BtuD in the liposome lumen and BtuF outside the liposomes. These in vitro studies establish the functional competence of the BtuCD and BtuF preparations used in the crystallographic analyses for both ATPase and transport activities. Furthermore, the tight binding of BtuF to BtuCD under the conditions studied suggests that the binding protein may not dissociate from the transporter during the catalytic cycle, which may be relevant to the mechanisms of other ABC transporter systems.
Borths
Elizabeth L
EL
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.
Poolman
Bert
B
Hvorup
Rikki N
RN
Locher
Kaspar P
KP
Rees
Douglas C
DC
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D001692:Biological Transport; D003902:Detergents / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008081:Liposomes; D008958:Models, Molecular; D033902:Periplasmic Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D014805:Vitamin B 12 / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; C050021:BtuC peptide, E coli; C050022:BtuD peptide, E coli; D003902:Detergents; D029968:Escherichia coli Proteins; D008081:Liposomes; D033902:Periplasmic Binding Proteins; C446048:btuF protein, E coli; D000251:Adenosine Triphosphatases; D014805:Vitamin B 12