TetL tetracycline efflux protein from Bacillus subtilis is a dimer in the membrane and in detergent solution.
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.
Safferling
Markus
M
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.
Griffith
Heather
H
Jin
Jie
J
Sharp
Josh
J
De Jesus
Magdia
M
Ng
Caroline
C
Krulwich
Terry A
TA
Wang
Da-Neng
DN
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
D017920:Antiporters; D001426:Bacterial Proteins; D003902:Detergents; D005960:Glucosides; D008565:Membrane Proteins; D012996:Solutions; C482657:TetL protein, Bacillus subtilis; C040358:dodecyl maltoside