TRANSMEMBRANE TOPOGRAPHY OF SUBUNIT A IN THE ESCHERICHIA-COLI F1F0 ATP SYNTHASE

Subunit a is the least understood of the three subunits that compose t he F-0 sector in the Escherichia coli F0F1 ATP synthase. In this study , we have substituted Cys into predicted extramembranous loops of the protein and used chemical modification to obtain topographical informa tion on the folding of subunit a. The extent of labeling of the substi tuted Cys residues by fluorescein-5'-maleimide was determined. The loc alization of reactive Cys residues was inferred from differences in th e extent of labeling in inside out and right side out membrane vesicle s. The NH2-terminal segment of subunit a was localized to the outside (periplasmic) surface and the COOH terminus to the cytoplasmic surface by these procedures. Loop residues in two periplasmic extramembranous loops and in two cytoplasmic extramembranous loops were also localize d. The localization of two cytoplasmic Cys residues was confirmed by u sing 4-acetamido-4'-maleimidylstilbene-2,2 '-disulfonic acid to block fluorescein-5'-maleimide labeling. From the localization of the Cys re sidues, a model for the topography is proposed that consists of five t ransmembrane segments with the NH, terminus periplasmic and the COOH t erminus cytoplasmic. The positions of second site suppressors, includi ng several isolated here to the nonfunctional E219C and H245C substitu tions, provide support for the topographical model proposed.