An analysis of suppressor mutations suggests that the two halves of the lactose permease function in a symmetrical manner

Nanette J. Pazdernik, Shane M. Cain, Robert J Brooker

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

A conserved motif, GXXX(D/E)(R/K)XG[X](R/K)(R/K), is located in loop 2/3 and loop 8/9 in the lactose permease, and also in hundreds of evolutionarily related transporters. The importance of conserved residues in loop 8/9 was previously investigated (Pazdernik, N.J., Jessen-Marshall, A. E., and Brooker, R. J. (1997) J. Bacteriol. 179, 735-741). Although this loop was tolerant of many substitutions, a few mutations in the first position of the motif were shown to dramatically decrease lactose transport. In the current study, a mutant at the first position in the motif having very low lactose transport, Leu280, was used as a parental strain to isolate second-site revertants that restore function. A total of 23 independent mutants were sequenced and found to have a second amino acid substitution at several locations (G46C, G46S, F49L, A50T, L212Q, L216Q, S233P, C333G, F354C, G370C, G370S, and G370V). A kinetic analysis revealed that the first-site mutation, Leu280, had a slightly better affinity for lactose compared with the wild- type strain, but its V(max) for lactose transport was over 30-fold lower. The primary effect of the second-site mutations was to increase the V(max) for lactose transport, in some cases, to levels that were near the wild-type value. When comparing this study to second-site mutations obtained from loop 2/3 defective strains, a striking observation was made. Mutations in three regions of the protein, codons 45-50, 234-241, and 366-370, were able to restore functionality to both loop 2/3 and loop 8/9 defects. These results are discussed within the context of a C1/C2 alternating conformation model in which lactose translocation occurs by a conformational change at the interface between the two halves of the protein.

Original languageEnglish (US)
Pages (from-to)26110-26116
Number of pages7
JournalJournal of Biological Chemistry
Volume272
Issue number42
DOIs
StatePublished - Oct 17 1997

Fingerprint

Genetic Suppression
Lactose
Mutation
Substitution reactions
Amino Acid Substitution
Codon
Conformations
lactose permease
Proteins
Observation
Amino Acids
Defects
Kinetics

Cite this

An analysis of suppressor mutations suggests that the two halves of the lactose permease function in a symmetrical manner. / Pazdernik, Nanette J.; Cain, Shane M.; Brooker, Robert J.

In: Journal of Biological Chemistry, Vol. 272, No. 42, 17.10.1997, p. 26110-26116.

Research output: Contribution to journalArticle

@article{3410cb6201a542148e320e9c1e11f233,
title = "An analysis of suppressor mutations suggests that the two halves of the lactose permease function in a symmetrical manner",
abstract = "A conserved motif, GXXX(D/E)(R/K)XG[X](R/K)(R/K), is located in loop 2/3 and loop 8/9 in the lactose permease, and also in hundreds of evolutionarily related transporters. The importance of conserved residues in loop 8/9 was previously investigated (Pazdernik, N.J., Jessen-Marshall, A. E., and Brooker, R. J. (1997) J. Bacteriol. 179, 735-741). Although this loop was tolerant of many substitutions, a few mutations in the first position of the motif were shown to dramatically decrease lactose transport. In the current study, a mutant at the first position in the motif having very low lactose transport, Leu280, was used as a parental strain to isolate second-site revertants that restore function. A total of 23 independent mutants were sequenced and found to have a second amino acid substitution at several locations (G46C, G46S, F49L, A50T, L212Q, L216Q, S233P, C333G, F354C, G370C, G370S, and G370V). A kinetic analysis revealed that the first-site mutation, Leu280, had a slightly better affinity for lactose compared with the wild- type strain, but its V(max) for lactose transport was over 30-fold lower. The primary effect of the second-site mutations was to increase the V(max) for lactose transport, in some cases, to levels that were near the wild-type value. When comparing this study to second-site mutations obtained from loop 2/3 defective strains, a striking observation was made. Mutations in three regions of the protein, codons 45-50, 234-241, and 366-370, were able to restore functionality to both loop 2/3 and loop 8/9 defects. These results are discussed within the context of a C1/C2 alternating conformation model in which lactose translocation occurs by a conformational change at the interface between the two halves of the protein.",
author = "Pazdernik, {Nanette J.} and Cain, {Shane M.} and Brooker, {Robert J}",
year = "1997",
month = "10",
day = "17",
doi = "10.1074/jbc.272.42.26110",
language = "English (US)",
volume = "272",
pages = "26110--26116",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "42",

}

TY - JOUR

T1 - An analysis of suppressor mutations suggests that the two halves of the lactose permease function in a symmetrical manner

AU - Pazdernik, Nanette J.

AU - Cain, Shane M.

AU - Brooker, Robert J

PY - 1997/10/17

Y1 - 1997/10/17

N2 - A conserved motif, GXXX(D/E)(R/K)XG[X](R/K)(R/K), is located in loop 2/3 and loop 8/9 in the lactose permease, and also in hundreds of evolutionarily related transporters. The importance of conserved residues in loop 8/9 was previously investigated (Pazdernik, N.J., Jessen-Marshall, A. E., and Brooker, R. J. (1997) J. Bacteriol. 179, 735-741). Although this loop was tolerant of many substitutions, a few mutations in the first position of the motif were shown to dramatically decrease lactose transport. In the current study, a mutant at the first position in the motif having very low lactose transport, Leu280, was used as a parental strain to isolate second-site revertants that restore function. A total of 23 independent mutants were sequenced and found to have a second amino acid substitution at several locations (G46C, G46S, F49L, A50T, L212Q, L216Q, S233P, C333G, F354C, G370C, G370S, and G370V). A kinetic analysis revealed that the first-site mutation, Leu280, had a slightly better affinity for lactose compared with the wild- type strain, but its V(max) for lactose transport was over 30-fold lower. The primary effect of the second-site mutations was to increase the V(max) for lactose transport, in some cases, to levels that were near the wild-type value. When comparing this study to second-site mutations obtained from loop 2/3 defective strains, a striking observation was made. Mutations in three regions of the protein, codons 45-50, 234-241, and 366-370, were able to restore functionality to both loop 2/3 and loop 8/9 defects. These results are discussed within the context of a C1/C2 alternating conformation model in which lactose translocation occurs by a conformational change at the interface between the two halves of the protein.

AB - A conserved motif, GXXX(D/E)(R/K)XG[X](R/K)(R/K), is located in loop 2/3 and loop 8/9 in the lactose permease, and also in hundreds of evolutionarily related transporters. The importance of conserved residues in loop 8/9 was previously investigated (Pazdernik, N.J., Jessen-Marshall, A. E., and Brooker, R. J. (1997) J. Bacteriol. 179, 735-741). Although this loop was tolerant of many substitutions, a few mutations in the first position of the motif were shown to dramatically decrease lactose transport. In the current study, a mutant at the first position in the motif having very low lactose transport, Leu280, was used as a parental strain to isolate second-site revertants that restore function. A total of 23 independent mutants were sequenced and found to have a second amino acid substitution at several locations (G46C, G46S, F49L, A50T, L212Q, L216Q, S233P, C333G, F354C, G370C, G370S, and G370V). A kinetic analysis revealed that the first-site mutation, Leu280, had a slightly better affinity for lactose compared with the wild- type strain, but its V(max) for lactose transport was over 30-fold lower. The primary effect of the second-site mutations was to increase the V(max) for lactose transport, in some cases, to levels that were near the wild-type value. When comparing this study to second-site mutations obtained from loop 2/3 defective strains, a striking observation was made. Mutations in three regions of the protein, codons 45-50, 234-241, and 366-370, were able to restore functionality to both loop 2/3 and loop 8/9 defects. These results are discussed within the context of a C1/C2 alternating conformation model in which lactose translocation occurs by a conformational change at the interface between the two halves of the protein.

UR - http://www.scopus.com/inward/record.url?scp=0030724004&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030724004&partnerID=8YFLogxK

U2 - 10.1074/jbc.272.42.26110

DO - 10.1074/jbc.272.42.26110

M3 - Article

C2 - 9334175

AN - SCOPUS:0030724004

VL - 272

SP - 26110

EP - 26116

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 42

ER -