The conserved motif in hydrophilic loop 2/3 and loop 8/9 of the lactose permease of Escherichia coli. Analysis of suppressor mutations

S. M. Cain, E. A. Matzke, Robert J Brooker

Research output: Contribution to journalArticle

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Abstract

The major facilitator superfamily (MFS) of transport proteins, which includes the lactose permease of Escherichia coli, contains a conserved motif G-X-X-X-D/E-R/K-X-G-R/K-R/K in the loops that connect transmembrane segments 2 and 3, and transmembrane segments 8 and 9. In three previous studies (Jessen-Marshall, A.E., and Brooker, R.J. 1996. J. Biol. Chem. 271:1400-1404; Jessen-Marshall, A.E., Parker, N., and Brooker, R.J. 1997. J. Bacteriol. 179:2616-2622; and Pazdernik, N., Cain, S.M., and Brooker, R.J. 1997. J. Biol. Chem. 272:26110-26116), suppressor mutations at twenty different sites were identified which restore function to mutant permeases that have deleterious mutations in the conserved loop 2/3 or loop 8/9 motif. In the current study, several of these second-site suppressor mutations have been separated from the original mutation in the conserved motif. The loop 2/3 suppressors were then coupled to a loop 8/9 mutation (P280L) and the loop 8/9 suppressors were coupled to a loop 2/3 mutation (i.e., G64S) to determine if the suppressors could restore function only to a loop 2/3 mutation, a loop 8/9 mutation, or both. The single parent mutations changing the first position in loop 2/3 (i.e., G64S) and loop 8/9 (i.e., P280L) had less than 4% lactose transport activity. Interestingly, most of the suppressors were very inhibitory when separated from the parent mutation. Two suppressors, A50T and G370V, restored substantial transport activity when individually coupled to the mutation in loop 2/3 and also when coupled to the corresponding mutation in loop 8/9. In other words, these suppressors could alleviate a defect imposed by mutations in either half of the permease. From a kinetic analysis, these suppressors were shown to exert their effects by increasing the V(max) values for lactose transport compared with the single G64S and P280L strains. These results are discussed within the context of our model in which the two halves of the lactose permease interact at a rotationally symmetrical interface, and that lactose transport is mediated by conformational changes at the interface.

Original languageEnglish (US)
Pages (from-to)159-168
Number of pages10
JournalJournal of Membrane Biology
Volume176
Issue number2
DOIs
StatePublished - Aug 19 2000

Fingerprint

Genetic Suppression
Escherichia coli
Mutation
Lactose
Membrane Transport Proteins
lactose permease
Carrier Proteins

Keywords

  • Conserved motif
  • Lactose permease
  • Sugar transporter
  • Symporter

Cite this

The conserved motif in hydrophilic loop 2/3 and loop 8/9 of the lactose permease of Escherichia coli. Analysis of suppressor mutations. / Cain, S. M.; Matzke, E. A.; Brooker, Robert J.

In: Journal of Membrane Biology, Vol. 176, No. 2, 19.08.2000, p. 159-168.

Research output: Contribution to journalArticle

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abstract = "The major facilitator superfamily (MFS) of transport proteins, which includes the lactose permease of Escherichia coli, contains a conserved motif G-X-X-X-D/E-R/K-X-G-R/K-R/K in the loops that connect transmembrane segments 2 and 3, and transmembrane segments 8 and 9. In three previous studies (Jessen-Marshall, A.E., and Brooker, R.J. 1996. J. Biol. Chem. 271:1400-1404; Jessen-Marshall, A.E., Parker, N., and Brooker, R.J. 1997. J. Bacteriol. 179:2616-2622; and Pazdernik, N., Cain, S.M., and Brooker, R.J. 1997. J. Biol. Chem. 272:26110-26116), suppressor mutations at twenty different sites were identified which restore function to mutant permeases that have deleterious mutations in the conserved loop 2/3 or loop 8/9 motif. In the current study, several of these second-site suppressor mutations have been separated from the original mutation in the conserved motif. The loop 2/3 suppressors were then coupled to a loop 8/9 mutation (P280L) and the loop 8/9 suppressors were coupled to a loop 2/3 mutation (i.e., G64S) to determine if the suppressors could restore function only to a loop 2/3 mutation, a loop 8/9 mutation, or both. The single parent mutations changing the first position in loop 2/3 (i.e., G64S) and loop 8/9 (i.e., P280L) had less than 4{\%} lactose transport activity. Interestingly, most of the suppressors were very inhibitory when separated from the parent mutation. Two suppressors, A50T and G370V, restored substantial transport activity when individually coupled to the mutation in loop 2/3 and also when coupled to the corresponding mutation in loop 8/9. In other words, these suppressors could alleviate a defect imposed by mutations in either half of the permease. From a kinetic analysis, these suppressors were shown to exert their effects by increasing the V(max) values for lactose transport compared with the single G64S and P280L strains. These results are discussed within the context of our model in which the two halves of the lactose permease interact at a rotationally symmetrical interface, and that lactose transport is mediated by conformational changes at the interface.",
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