Mutagenesis of SugE, a small multidrug resistance protein.
The small multidrug resistance protein family has two subclasses. In this study we used a mutation approach to see what is necessary to convert a SUG subgroup member into a quaternary ammonium compound (QAC) transporter. We chose four key residues (H24, M39, I43, and A44) conserved within SUGs but conserved differently within the QAC transporters. Altogether, seven mutants were generated in Citrobacter freundii SugE. Surprisingly, the mutated SugE demonstrated an increased sensitivity to representative QACs. Additionally, ethidium uptake is found to be more prominent in the hypersensitive mutants. We conducted orientation studies using topology reporter gene fusions which indicated that SugE and the QAC transporter EmrE both have their N- and C-termini in the cytoplasm as predicted. The results imply that SugE can be converted to a QAC transporter with only a single mutation. However, because hypersensitivity was observed, the SugE mutant proteins are behaving as importers rather than as exporters.
Son
Mike S
MS
Department of Biological Sciences, University of Calgary, 2500 University Dr. NW, Alta., T2N 1N4, Calgary, Canada.
Del Castilho
Colin
C
Duncalf
Karen A
KA
Carney
Dominic
D
Weiner
Joel H
JH
Turner
Raymond J
RJ
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D017920:Antiporters / Q000737:chemistry* / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D024881:Drug Resistance, Bacterial / Q000502:physiology; D004926:Escherichia coli / Q000166:cytology / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004996:Ethidium / Q000493:pharmacokinetics*; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010269:Paraquat / Q000494:pharmacology*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017385:Sequence Homology; D013329:Structure-Activity Relationship
D018435:ATP Binding Cassette Transporter, Subfamily B; D017920:Antiporters; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; C080403:sugE protein, E coli; C075436:EmrE protein, E coli; D004996:Ethidium; D010269:Paraquat