Accessibility of Cysteine Residues in a Cytoplasmic Loop of CitS of Klebsiella pneumoniae Is Controlled by the Catalytic State of the Transporter

Abstract

The citrate transporter CitS of Klebsiella pneumoniae is a secondary transporter that transports citrate in symport with two sodium ions and one proton. Treatment of CitS with the alkylating agent N-ethylmaleimide resulted in a complete loss of transport activity. Treatment of mutant proteins in which the five endogenous cysteine residues were mutated into serines in different combinations revealed that two cysteine residues located in the C-terminal cytoplasmic loop, Cys-398 and Cys-414, were responsible for the inactivation. Labeling with the membrane impermeable methanethiosulfonate derivatives MTSET and MTSES in right-side-out membrane vesicles showed that the cytoplasmic loop was accessible from the periplasmic side of the membrane. The membrane impermeable but more bulky maleimide AmdiS did not inactivate the transporter in right-side-out membrane vesicles. Inactivation by N-ethylmaleimide, MTSES, and MTSET was prevented by the presence of the co-ion Na(+). Protection was obtained upon binding 2 Na(+), which equals the transport stoichiometry. In the absence of Na(+), the substrate citrate had no effect on the inactivation by permeable or impermeable thiol reagents. In contrast, when subsaturating concentrations of Na(+) were present, citrate significantly reduced inactivation suggesting ordered binding of the substrate and co-ion; citrate is bound after Na(+). In the presence of the proton motive force, the reactivity of the Cys residues was increased significantly for the membrane permeable N-ethylmaleimide, while no difference was observed for the membrane impermeable thiol reagents. The results are discussed in the context of a model for the opening and closing of the translocation pore during turnover of the transporter.

Alternating Access and a Pore-Loop Structure in the Na+-Citrate Transporter CitS of Klebsiella pneumoniae

“…Previous studies of the SSSCC mutant of CitS and cysteine mutants of the homologous transporter CimH of Bacillus subtilis showed, surprisingly, that residues in region Xa were accessible for membrane-impermeable MTS derivatives when presented at the periplasmic side of the membrane (11,27). The MTS derivatives are similar in size to citrate (32), and it was suggested that access from the periplasmic side of the membrane might follow (part of) the substrate translocation pathway (also see Ref.…”

“…The rate of uptake in RSO membranes prepared from E. coli cells expressing CitS and energized by the artificial electron donor system ascorbate/ PMS increased sigmoidally with increasing Na ϩ concentration. Half of the maximal rate was obtained at a Na ϩ concentration of 3 mM (27) (Fig. 2, q).…”

“…Plasmid pBADCitS is based on vector pBAD24 (Invitrogen) and codes for the wild type CitS protein with an N-terminal His tag. Expression of the gene is under control of the arabinose promoter (27). Expression of CitS was induced by adding 0.1% arabinose when the absorbance of the culture measured at 660 nm (A 660 ) reached a value of 0.6.…”

“…Plasmid pHisCitS is based on the pBluescript vector (Stratagene) and was used as the template (7). All mutants were sequenced (Baseclear, Leiden, The Netherlands) to confirm the presence of the desired mutations and cloned into the pBAD24 vector as described before (27).…”

“…1). Recently, we showed that alkylation of the two cysteine residues by N-ethylmaleimide (NEM) resulted in loss of activity, in line with the importance of region Xa in catalysis (27). Most importantly, inactivation was also observed with the membrane-impermeable thiol reagent [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET) added at the periplasmic side of the membrane revealing an access pathway for the reagent between the periplasm and the residues, possibly by formation of a pore-loop structure by the cytoplasmic loop.…”