Topology of OxlT, the oxalate transporter of Oxalobacter formigenes, determined by site-directed fluorescence labeling

The topology of OxlT, the oxalate:formate exchange protein of Oxalobacter f ormigenes, was established by site-directed fluorescence labeling, a simple strategy that generates topological information in the context of the inta ct protein. Accessibility of cysteine to the fluorescent thiol-directed pro be Oregon green maleimide (OGM) was examined for a panel of 34 single-cyste ine variants, each generated in a His(9)-tagged cysteine-less host. The rea ction with OGM was readily scored by examining the fluorescence profile aft er sodium dodecyl sulfate-polyacrylamide gel electrophoresis of material Pu rified by Ni2+-linked affinity chromatography, A position was assigned an e xternal location if its single-cysteine derivative reacted with OGM added t o intact cells; a position was designated internal if OGM labeling required cell lysis. We also showed that labeling of external, but not internal, po sitions was blocked by prior exposure of cells to the impermeable and nonfl uorescent thiol-specific agent ethyltrimethylammonium methanethiosulfonate. Of the 34 positions examined in this way, 29 were assigned unambiguously t o either an internal or external location; 5 positions could not be assigne d, since the target cysteine failed to react with OGM, There was no evidenc e of false-positive assignment. Our findings document a simple and rapid me thod for establishing the topology of a membrane protein and show that OxlT has 12 transmembrane segments, confirming inferences from hydropathy analy sis.