STRUCTURE-FUNCTION-RELATIONSHIPS IN OXLT, THE OXALATE FORMATE TRANSPORTER OF OXALOBACTER-FORMIGENES - TOPOLOGICAL FEATURES OF TRANSMEMBRANEHELIX 11 AS VISUALIZED BY SITE-DIRECTED FLUORESCENT LABELING/
Dx. Fu et Pc. Maloney, STRUCTURE-FUNCTION-RELATIONSHIPS IN OXLT, THE OXALATE FORMATE TRANSPORTER OF OXALOBACTER-FORMIGENES - TOPOLOGICAL FEATURES OF TRANSMEMBRANEHELIX 11 AS VISUALIZED BY SITE-DIRECTED FLUORESCENT LABELING/, The Journal of biological chemistry, 273(28), 1998, pp. 17962-17967
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.