CaltechAUTHORS
  A Caltech Library Service

In Vitro Functional Characterization of BtuCD-F, the Escherichia coli ABC Transporter for Vitamin B_(12) Uptake

Borths, Elizabeth L. and Poolman, Bert and Hvorup, Rikki N. and Locher, Kaspar P. and Rees, Douglas C. (2005) In Vitro Functional Characterization of BtuCD-F, the Escherichia coli ABC Transporter for Vitamin B_(12) Uptake. Biochemistry, 44 (49). pp. 16301-16309. ISSN 0006-2960. doi:10.1021/bi0513103. https://resolver.caltech.edu/CaltechAUTHORS:20141212-144459778

Full text is not posted in this repository. Consult Related URLs below.

Use this Persistent URL to link to this item: https://resolver.caltech.edu/CaltechAUTHORS:20141212-144459778

Abstract

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.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1021/bi0513103DOIArticle
http://pubs.acs.org/doi/abs/10.1021/bi0513103PublisherArticle
ORCID:
AuthorORCID
Poolman, Bert0000-0002-1455-531X
Rees, Douglas C.0000-0003-4073-1185
Contact Email Address:phoebe@caltech.edu
Alternate Title:In Vitro Functional Characterization of BtuCD-F, the Escherichia coli ABC Transporter for Vitamin B12 Uptake
Additional Information:© 2005 American Chemical Society Received July 8, 2005; Revised Manuscript Received October 4, 2005 We wish to thank Profs. A. Varshavsky and P. Patterson and their groups for the use of their ultracentrifuge and gamma counter, respectively, in these studies. This research was supported in part by the Swiss National Science Foundation, Grant No. 3100A0-103765 (K.P.L.), a Fulbright grant from NACEE to support the stay of B.P. in the laboratory of D.C.R., and the Howard Hughes Medical Institute (D.C.R.).
Funders:
Funding AgencyGrant Number
Swiss National Science Foundation (SNSF)3100A0-103765
NACEEUNSPECIFIED
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Issue or Number:49
DOI:10.1021/bi0513103
Record Number:CaltechAUTHORS:20141212-144459778
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20141212-144459778
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:52781
Collection:CaltechAUTHORS
Deposited By: SWORD User
Deposited On:22 Dec 2014 21:28
Last Modified:10 Nov 2021 19:43

Repository Staff Only: item control page