STRUCTURE-FUNCTION-RELATIONSHIPS IN OXLT, THE OXALATE FORMATE TRANSPORTER OF OXALOBACTER-FORMIGENES - TOPOLOGICAL FEATURES OF TRANSMEMBRANEHELIX 11 AS VISUALIZED BY SITE-DIRECTED FLUORESCENT LABELING/

Analysis of hydropathy suggests that in OxlT, the oxalate/formate anti porter of Oxalobacter formigenes, lysine 355 is within transmembrane h elix no. 11. To test this idea, we used single-cysteine, histidine-tag ged OxlT variants to study the organization of a 30-residue segment (r esidues 344-373) containing this region. Topology was examined by prob ing the A345C and A370C proteins with Oregon Green maleimide carboxyli c acid, an impermeant and fluorescent thiol-reactive agent. Examinatio n of purified protein showed that only A370C was fluorescent after tre ating intact cells with the probe, while both proteins were modified i n tests with isolated membrane ghosts, In addition, labeling of A370C, but not A345C, was blocked when external cysteines were protected with the impermeant and nonfluorescent agent, methanethiosulfonate ethyltr imethylammonium. These findings confirm that A345 faces the cytoplasm, while 4370C faces the periplasm, A similar study focused on 13 single -cysteine variants positioned throughout the target segment. That work revealed a striking discontinuity in reactivity toward Oregon Green m aleimide; cysteines within a 10-residue central core (residues 351-360 ) were not labeled when membranes were probed, but were readily modifi ed after protein denaturation. We suggest this core resides within the lipid bilayer, unavailable to an impermeant reporter. Since this regi on includes position 355, we also suggest that lysine 355 lies within the OxlT hydrophobic sector, where it may facilitate the binding and t ranslocation of the anionic substrates, oxalate and formate.