PMID:10741430
| Citation |
Pazdernik, NJ, Matzke, EA, Jessen-Marshall, AE and Brooker, RJ (2000) Roles of charged residues in the conserved motif, G-X-X-X-D/E-R/K-X-G-[X]-R/K-R/K, of the lactose permease of Escherichia coli. J. Membr. Biol. 174:31-40 |
|---|---|
| Abstract |
The lactose permease is a polytopic membrane protein that has a duplicated conserved motif, GXXX(D/E)(R/K)XG[X](R/K)(R/K), located in cytoplasmic loops 2/3 and 8/9. In the current study, the roles of the basic residues and the acidic residue were investigated in greater detail. Neutral substitutions of two positive charges in loop 2/3 were tolerated, while a triple mutant resulted in a complete loss of expression. Neutral substitutions of a basic residue in loop 8/9 (i.e., K289I) also diminished protein stability. By comparison, neutral substitutions affecting the negative charge in loop 2/3 had normal levels of expression, but were defective in transport. A double mutant (D68T/N284D), in which the aspartate of loop 2/3 was moved to loop 8/9, did not have appreciable activity, indicating that the negative charge in the conserved motif could not be placed in loop 8/9 to recover lactose transport activity. An analysis of site-directed mutants in loop 7/8 and loop 8/9 indicated that an alteration in the charge distribution across transmembrane segment 8 was not sufficient to alleviate a defect caused by the loss of a negative charge in loop 2/3. To further explore this phenomenon, the double mutant, D68T/N284D, was used as a parental strain to isolate suppressor mutations which restored function. One mutant was obtained in which an acidic residue in loop 11/12 was changed to a basic residue (i.e., Glu374 --> Lys). Overall, the results of this study suggest that the basic residues in the conserved motif play a role in protein insertion and/or stability, and that the negative charge plays a role in conformational changes. |
| Links | |
| Keywords |
Amino Acid Sequence; Amino Acid Substitution; Biological Transport; Cell Membrane/enzymology; Conserved Sequence; Escherichia coli/enzymology; Escherichia coli Proteins; Galactosides/metabolism; Kinetics; Membrane Transport Proteins/chemistry; Membrane Transport Proteins/metabolism; Models, Molecular; Monosaccharide Transport Proteins; Mutagenesis, Site-Directed; Protein Structure, Secondary; Recombinant Proteins/chemistry; Recombinant Proteins/metabolism; Symporters |
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