Transmembrane topography of subunit a in the Escherichia coli F1F0 ATP synthase.
Subunit a is the least understood of the three subunits that compose the F0 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 information on the folding of subunit a. The extent of labeling of the substituted Cys residues by fluorescein-5'-maleimide was determined. The localization of reactive Cys residues was inferred from differences in the extent of labeling in inside out and right side out membrane vesicles. 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 localized. The localization of two cytoplasmic Cys residues was confirmed by using 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid to block fluorescein-5'-maleimide labeling. From the localization of the Cys residues, a model for the topography is proposed that consists of five transmembrane segments with the NH2 terminus periplasmic and the COOH terminus cytoplasmic. The positions of second site suppressors, including several isolated here to the nonfunctional E219C and H245C substitutions, provide support for the topographical model proposed.
Valiyaveetil
F I
FI
Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA.
Fillingame
R H
RH
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology*; D005973:Glutamine / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics; D015183:Restriction Mapping; D012694:Serine / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013439:Sulfhydryl Reagents / Q000378:metabolism; D013499:Surface Properties
D013439:Sulfhydryl Reagents; D005973:Glutamine; D012694:Serine; D006639:Histidine; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D003545:Cysteine