ArticleJournal of bacteriology2001
Topology of OxlT, the oxalate transporter of Oxalobacter formigenes, determined by site-directed fluorescence labeling.
Article in Journal of bacteriology, 2001. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 14 papers.
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Who cites it
14 citing papers in PubMed, 32 citations in OpenAlex.
- Article
- The Structure and Function of OxlT, the Oxalate Transporter of Oxalobacter formigenes.The Journal of membrane biology · 2015Review
- Site-directed fluorescence labeling reveals a revised N-terminal membrane topology and functional periplasmic residues in the Escherichia coli cell division protein FtsK.The Journal of biological chemistry · 2014Article
- Article
- Functional and structural characterization of polysaccharide co-polymerase proteins required for polymer export in ATP-binding cassette transporter-dependent capsule biosynthesis pathways.The Journal of biological chemistry · 2011Article
- Death of the TonB Shuttle Hypothesis.Frontiers in microbiology · 2011Article
- Topological analysis of a haloacid permease of a Burkholderia sp. bacterium with a PhoA-LacZ reporter.BMC microbiology · 2009Article
- Structural and functional importance of transmembrane domain 3 (TM3) in the aspartate:alanine antiporter AspT: topology and function of the residues of TM3 and oligomerization of AspT.Journal of bacteriology · 2009Article
- Topology of AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, determined by site-directed fluorescence labeling.Journal of bacteriology · 2007Article
- Analysis of substrate-binding elements in OxlT, the oxalate:formate antiporter of Oxalobacter formigenes.Biochemistry · 2006Article
- Experimental tests of a homology model for OxlT, the oxalate transporter of Oxalobacter formigenes.Proceedings of the National Academy of Sciences of the United States of America · 2005Article
- Transmembrane protein topology mapping by the substituted cysteine accessibility method (SCAM(TM)): application to lipid-specific membrane protein topogenesis.Methods (San Diego, Calif.) · 2005Article
- Cytoplasmic domains of the reduced folate carrier are essential for trafficking, but not function.The Biochemical journal · 2002Article
- Projection structure and molecular architecture of OxlT, a bacterial membrane transporter.The EMBO journal · 2001Article
Corrections and comments
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Authors and funding
4 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
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 Ni2+ 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.
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