title: 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.
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.
authors.affiliation: Department of Genetics, Cell Biology, and Development, BioProcess Technology Institute, University of Minnesota, St. Paul 55108, USA.
authors.forename: N J
authors.initials: NJ
authors.lastname: Pazdernik
authors.forename: E A
authors.initials: EA
authors.lastname: Matzke
authors.forename: A E
authors.initials: AE
authors.lastname: Jessen-Marshall
authors.forename: R J
authors.initials: RJ
authors.lastname: Brooker
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001692:Biological Transport; D002462:Cell Membrane / Q000201:enzymology; D017124:Conserved Sequence; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D005697:Galactosides / Q000378:metabolism; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D027981:Symporters
chemical_list: D029968:Escherichia coli Proteins; D005697:Galactosides; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011994:Recombinant Proteins; D027981:Symporters; C021499:lactose permease