title: TetL tetracycline efflux protein from Bacillus subtilis is a dimer in the membrane and in detergent solution.
abstract: The TetL antiporter from the Bacillus subtilis inner membrane is a tetracycline-divalent cation efflux protein that is energized by the electrochemical proton gradient across the membrane. In this study, we expressed tetL in Escherichia coli and investigated the oligomeric state of TetL in the membrane and in detergent solution. Evidence for an oligomeric state of TetL emerged from SDS-PAGE and Western blot analysis of membrane samples as well as purified protein samples from cells that expressed two differently tagged TetL species. Furthermore, no formation or restoration of TetL oligomers occurred upon detergent solubilization of the membrane. Rather, oligomeric forms established in vivo persisted after solubilization. Mass spectrometry of the purified protein showed the absence of proteolysis and posttranslational modifications. Analytical size-exclusion chromatography of the purified protein revealed a dimeric TetL in dodecyl-maltoside solution. In addition, TetL dimers were found in a number of other detergents and over a wide pH range. It is therefore likely that the oligomeric form of the protein in the membrane is also a dimer.
authors.affiliation: Skirball Institute of Biomolecular Medicine and Department of Cell Biology, New York University School of Medicine, 540 First Avenue, New York, New York 10016, USA.
authors.forename: Markus
authors.initials: M
authors.lastname: Safferling
authors.forename: Heather
authors.initials: H
authors.lastname: Griffith
authors.forename: Jie
authors.initials: J
authors.lastname: Jin
authors.forename: Josh
authors.initials: J
authors.lastname: Sharp
authors.forename: Magdia
authors.initials: M
authors.lastname: De Jesus
authors.forename: Caroline
authors.initials: C
authors.lastname: Ng
authors.forename: Terry A
authors.initials: TA
authors.lastname: Krulwich
authors.forename: Da-Neng
authors.initials: DN
authors.lastname: Wang
mesh_terms: D017920:Antiporters / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001412:Bacillus subtilis / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D003902:Detergents; D019281:Dimerization; D005816:Genetic Complementation Test; D005960:Glucosides; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D012996:Solutions; D013696:Temperature; D013753:Tetracycline Resistance / Q000235:genetics
chemical_list: D017920:Antiporters; D001426:Bacterial Proteins; D003902:Detergents; D005960:Glucosides; D008565:Membrane Proteins; D012996:Solutions; C482657:TetL protein, Bacillus subtilis; C040358:dodecyl maltoside