A revised model for the structure and function of the lactose permease. Evidence that a face on transmembrane segment 2 is important for conformational changes

Aileen L Green; Ethan J Anderson; Robert J Brooker

doi:10.1074/jbc.M909202199

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A revised model for the structure and function of the lactose permease. Evidence that a face on transmembrane segment 2 is important for conformational changes

Journal article

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A revised model for the structure and function of the lactose permease. Evidence that a face on transmembrane segment 2 is important for conformational changes

Aileen L Green, Ethan J Anderson and Robert J Brooker

The Journal of biological chemistry, Vol.275(30), pp.23240-23246

07/28/2000

DOI: 10.1074/jbc.M909202199

PMID: 10807929

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Abstract

The lactose permease is an integral membrane protein that cotransports H(+) and lactose into the bacterial cytoplasm. Previous work has shown that bulky substitutions at glycine 64, which is found on the cytoplasmic edge of transmembrane segment 2 (TMS-2), cause a substantial decrease in the maximal velocity of lactose uptake without significantly affecting the K(m) values (Jessen-Marshall, A. E., Parker, N. J., and Brooker, R. J. (1997) J. Bacteriol. 179, 2616-2622). In the current study, mutagenesis was conducted along the face of TMS-2 that contains glycine-64. Single amino acid substitutions that substantially changed side-chain volume at codons 52, 57, 59, 63, and 66 had little or no effect on transport activity, whereas substitutions at codons 49, 53, 56, and 60 were markedly defective and/or had lower levels of expression. According to helical wheel plots, Phe-49, Ser-53, Ser-56, Gln-60, and Gly-64 form a continuous stripe along one face of TMS-2. Several of the TMS-2 mutants (S56Y, S56L, S56Q, Q60A, and Q60V) were used as parental strains to isolate mutants that restore transport activity. These mutations were either first-site mutations or second-site suppressors in TMS-1, TMS-2, TMS-7 or TMS-11. A kinetic analysis showed that the suppressors had a higher rate of lactose transport compared with the corresponding parental strains. Overall, the results of this study are consistent with the notion that a face on TMS-2, containing Phe-49, Ser-53, Ser-56, Gln-60, and Gly-64, plays a critical role in conformational changes associated with lactose transport. We hypothesize that TMS-2 slides across TMS-7 and TMS-11 when the lactose permease interconverts between the C1 and C2 conformations. This idea is discussed within the context of a revised model for the structure of the lactose permease.

Mutagenesis, Site-Directed

Membrane Proteins - genetics

Carbohydrate Metabolism

Structure-Activity Relationship

Membrane Transport Proteins - chemistry

Escherichia coli Proteins

Biological Transport

Membrane Proteins - chemistry

Membrane Transport Proteins - genetics

Monosaccharide Transport Proteins

Symporters

Membrane Transport Proteins - metabolism

Protein Conformation

Membrane Proteins - metabolism

Details

Title: Subtitle: A revised model for the structure and function of the lactose permease. Evidence that a face on transmembrane segment 2 is important for conformational changes
Creators: Aileen L Green - Department of Genetics, Cell Biology, and Development and the BioProcess Technology Institute, University of Minnesota, St. Paul, Minnesota 55108, USA
Ethan J Anderson
Robert J Brooker
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.275(30), pp.23240-23246
DOI: 10.1074/jbc.M909202199
PMID: 10807929
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Publisher: United States
Grant note: GM53259 / NIGMS NIH HHS
Language: English
Date published: 07/28/2000
Academic Unit: Pharmaceutical Sciences and Experimental Therapeutics; Fraternal Order of Eagles Diabetes Research Center; Health, Sport, and Human Physiology
Record Identifier: 9984065691702771

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