Dubey, S and Majumder, P and Penmatsa, A and Sardesai, AA (2021) Topological analyses of the L-lysine exporter LysO reveal a critical role for a conserved pair of intramembrane solvent-exposed acidic residues. In: Journal of Biological Chemistry, 279 (4).
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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.
| Item Type: | Journal Article |
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| Publication: | Journal of Biological Chemistry |
| Publisher: | American Society for Biochemistry and Molecular Biology Inc. |
| Additional Information: | The copyright for this article belongs to the Authors. |
| Keywords: | Escherichia coli; Membranes; Metabolites; Solvents; Topology, Acidic residues; C-terminus; In-vivo; Membrane topology; N terminus; Solvent exposure; Topological analysis; Trans-membrane domains; Transmembrane domain; Transmembrane segments, Amino acids, carrier protein; cysteine; lysine; lysine derivative; protein LysO; proton; solvent; thialysine; unclassified drug; amino acid transporter; Escherichia coli protein; YbjE protein, E coli, acidity; amino acid substitution; amino terminal sequence; Article; carboxy terminal sequence; controlled study; Escherichia coli K-12; membrane structure; membrane vesicle; nonhuman; protein function; proton transport; active transport; cell membrane; chemistry; genetics; metabolism; protein domain; structure activity relation, Amino Acid Transport Systems, Basic; Biological Transport, Active; Cell Membrane; Escherichia coli K12; Escherichia coli Proteins; Protein Domains; Structure-Activity Relationship |
| Department/Centre: | Division of Biological Sciences > Molecular Biophysics Unit |
| Date Deposited: | 15 May 2023 09:40 |
| Last Modified: | 15 May 2023 09:40 |
| URI: | https://eprints.iisc.ac.in/id/eprint/81639 |
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