Topology of OxlT, the Oxalate Transporter of Oxalobacter formigenes , Determined by Site-Directed Fluorescence Labeling

Abstract

The topology of OxlT, the oxalate:formate exchange protein of Oxalobacter formigenes, was established by site-directed fluorescence labeling, a simple strategy that generates topological information in the context of the intact protein. Accessibility of cysteine to the fluorescent thiol-directed probe Oregon green maleimide (OGM) was examined for a panel of 34 single-cysteine variants, each generated in a His 9 -tagged cysteine-less host. The reaction with OGM was readily scored by examining the fluorescence profile after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of material purified by Ni 2؉ -linked affinity chromatography. A position was assigned an external location if its single-cysteine derivative reacted with OGM added to intact cells; a position was designated internal if OGM labeling required cell lysis. We also showed that labeling of external, but not internal, positions was blocked by prior exposure of cells to the impermeable and nonfluorescent thiol-specific agent ethyltrimethylammonium methanethiosulfonate. Of the 34 positions examined in this way, 29 were assigned unambiguously to either an internal or external location; 5 positions could not be assigned, since the target cysteine failed to react with OGM. There was no evidence of false-positive assignment. Our findings document a simple and rapid method for establishing the topology of a membrane protein and show that OxlT has 12 transmembrane segments, confirming inferences from hydropathy analysis.The gram-negative bacterium Oxalobacter formigenes sustains a proton motive force by utilizing a "virtual" proton pump based on the transport and metabolism of oxalate. An electric potential (negative inside) arises from action of the antiporter, OxlT, which links inward transport of divalent oxalate to the outward flow of monovalent formate, the product of oxalate decarboxylation. The net inflow of a single negative charge is then phenomenologically linked to generation of a pH gradient (alkaline inside), because decarboxylation of oxalate consumes a single cytosolic proton. Together, these elements comprise the proton motive force used to drive ATP synthesis in this obligate anaerobe (3,14,20,32). Virtual pumps of equivalent construction have now been observed in a number of microorganisms (14,22,25).It is evident that OxlT occupies a central position in the cell biology of O. formigenes and that study of this transporter is relevant to several aspects of microbial physiology. Added interest in OxlT stems from recent work (8,9,26) suggesting that this protein may also serve as a useful model for biochemical study of other transporters. Accordingly, OxlT may contribute to an understanding of membrane transport at both a functional level and a mechanistic level.Hydropathy analysis of the OxlT amino acid sequence, along with other considerations, suggests the presence of 12 transmembrane segments (TM1 to TM12) (1), consistent with the presumed structure of most other members of the major facilitator superfamily (MFS) (30), the superfami...

Experimental tests of a homology model for OxlT, the oxalate transporter of Oxalobacter formigenes

“…After introduction of small (one-to tworesidue) gaps in the OxlT sequence at loops 8-9 and 9-10, to maximize agreement with the OxlT vs. GlpT CLUSTALW alignment for TM9 and TM10, we submitted the alignment to the Swiss-Model server. This yielded a first-generation model in general agreement with existing experimental data, including OxlT topology as probed by site-directed fluorescence labeling (22) and the locations of several residues (Lys-355, Ser-359, Gln-63) expected to lie on the permeation pathway (19,20,23).…”

“…1A) with the model derived earlier from analysis of hydropathy (2), which had successfully guided much experimental work (19)(20)(21)(22)(23). Both models make comparable predictions as to the relative positions of residues critical to the study of OxlT.…”

“…The topology initially suggested for OxlT was based, in part, on work in which cysteine substitution mutants were probed with OGM, an impermeant and fluorescent thiolactive agent (20,22,23). In that work, a collection of functional single-cysteine derivatives was evaluated for OGM accessibility, and reactive cysteines were assigned either a periplasmic or cytoplasmic location based on responses found by using intact cells (revealing positions facing the periplasm), or membrane preparations (in which both periplasmic and cytoplasmic surfaces are exposed).…”

“…In all cases, the presence of an OGM-reactive cysteine was confirmed by parallel tests of denatured protein (data not shown; see ref. 22). …”

“…Membranes suspended in 20 mM potassium phosphate (pH 8) were incubated with 40 M Oregon green maleimide (OGM) for 10 min at 23°C before SDS͞PAGE analysis of purified OxlT, as described (19,22,23). Fluorescence of SDS͞PAGE gels was recorded by using a Fuji LAS 1000 system, whereas protein content was revealed by Coomassie brilliant blue staining.…”