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.
Highlights
Many microbial genomes encode integral membrane transporters, capable of mediating amino acid export [1]
An absolute requirement for a pair of conserved acidic residues in an intramembrane solvent exposed region comprising TMS7 and an adjacent segment located between TMS7 and TMS8 for LysO function is identified
Toward determining the membrane topology of LysO, we employed two approaches, one involving determining accessibility of Cys residues introduced in LysO and the other involving use of a compartment specific reporter protein linked to N-terminal segments of LysO of varying lengths
Summary
Many microbial genomes encode integral membrane transporters, capable of mediating amino acid export [1]. During the course of studies on Cys accessibility, we noted that multiple Cys residues in particular those incorporated in the region in LysO spanning the amino acids 182–266 (182–266 region) could be modified by NEM and MTSES (Fig. S1 and Table 1); assigning a cytoplasmic or periplasmic location to them was not straightforward.
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