Topological analyses of the L-lysine exporter LysO reveal a critical role for a conserved pair of intramembrane solvent-exposed acidic residues |
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Authors: | Swati Dubey Puja Majumder Aravind Penmatsa Abhijit A Sardesai |
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Institution: | 1.Laboratory of Bacterial Genetics, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, India;2.Graduate Studies, Manipal Academy of Higher Education, Manipal, India;3.Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India |
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Abstract: | LysO, a prototypical member of the LysO family, mediates export of L-lysine (Lys) and resistance to the toxic Lys antimetabolite, L-thialysine (Thl) in Escherichia coli. Here, we have addressed unknown aspects of LysO function pertaining to its membrane topology and the mechanism by which it mediates Lys/Thl export. Using substituted cysteine (Cys) accessibility, here we delineated the membrane topology of LysO. Our studies support a model in which both the N- and C-termini of LysO are present at the periplasmic face of the membrane with a transmembrane (TM) domain comprising eight TM segments (TMSs) between them. In addition, a feature of intramembrane solvent exposure in LysO is inferred with the identification of membrane-located solvent-exposed Cys residues. Isosteric substitutions of a pair of conserved acidic residues, one E233, located in the solvent-exposed TMS7 and the other D261, in a solvent-exposed intramembrane segment located between TMS7 and TMS8, abolished LysO function in vivo. Thl, but not Lys, elicited proton release in inside-out membrane vesicles, a process requiring the presence of both E233 and D261. We postulate that Thl may be exported in antiport with H+ and that Lys may be a low-affinity export substrate. Our findings are compatible with a physiological scenario wherein in vivo LysO exports the naturally occurring antimetabolite Thl with higher affinity over the essential cellular metabolite Lys, thus affording protection from Thl toxicity and limiting wasteful export of Lys. |
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Keywords: | membrane protein amino acid transport Escherichia coli (E coli) membrane transport bacterial metabolism lysine export proton-coupled antiport |
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