The membrane topology of proton-pumping Escherichia coli transhydrogenase determined by cysteine labeling

The membrane topology of proton-pumping nicotinamide-nucleotide transhydrog enase from Escherichia coli was determined by site-specific chemical labeli ng, A His-tagged cysteine-free transhydrogenase was used to introduce uniqu e cysteines in positions corresponding to potential membrane loops. The cys teines mere 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 w as measured as fluorescence on UV-illuminated acrylamide gels. The differen ce in labeling between intact cells and inside-out vesicles was used to dis criminate between a periplasmic and a cytosolic location of the residues. T he membrane region was found to be composed of 13 helices (four in the alph a-subunit and nine in the beta-subunit), with the C terminus of the alpha-s ubunit and the N terminus of the beta-subunit facing the cytosolic and peri plasmic 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 transmem brane segments of transhydrogenase have been experimentally determined and provides an explanation for the different topologies of the mitochondrial a nd E. coli transhydrogenases.