The membrane topology of proton-pumping Escherichia coli transhydrogenase determined by cysteine labeling.
The membrane topology of proton-pumping nicotinamide-nucleotide transhydrogenase from Escherichia coli was determined by site-specific chemical labeling. A His-tagged cysteine-free transhydrogenase was used to introduce unique cysteines in positions corresponding to potential membrane loops. The cysteines were reacted with fluorescent reagents, fluorescein 5-maleimide or 2-[(4'-maleimidyl)anilino]naphthalene-6-sulfonic acid, in both intact cells and inside-out vesicles. Labeled transhydrogenase was purified with a small-scale procedure using a metal affinity resin, and the amount of labeling was measured as fluorescence on UV-illuminated acrylamide gels. The difference in labeling between intact cells and inside-out vesicles was used to discriminate between a periplasmic and a cytosolic location of the residues. The membrane region was found to be composed of 13 helices (four in the alpha-subunit and nine in the beta-subunit), with the C terminus of the alpha-subunit and the N terminus of the beta-subunit facing the cytosolic and periplasmic sides, respectively. These results differ from previous models with regard to both number of helices and the relative location and orientation of certain helices. This study constitutes the first in which all transmembrane segments of transhydrogenase have been experimentally determined and provides an explanation for the different topologies of the mitochondrial and E. coli transhydrogenases.
Meuller
J
J
Department of Chemistry, Division of Biochemistry and Biophysics, Göteborg University and Chalmers University of Technology, Medicinaregatan 9C, P. O. Box 462, 40530 Göteborg, Sweden.
Rydström
J
J
D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D003545:Cysteine / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D008969:Molecular Sequence Data; D009250:NADP Transhydrogenases / Q000378:metabolism*; D010069:Oxadiazoles / Q000378:metabolism; D017494:Proton Pumps / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000378:metabolism; D014466:Ultraviolet Rays
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D010069:Oxadiazoles; D017494:Proton Pumps; D013439:Sulfhydryl Reagents; C035573:4-(N-(iodoacetoxy)ethyl-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole; C021310:2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid; D009250:NADP Transhydrogenases; D003545:Cysteine