Membrane topology of subunit a of the F1F0 ATP synthase as determined by labeling of unique cysteine residues.
The membrane topology of the a subunit of the F1F0 ATP synthase from Escherichia coli has been probed by surface labeling using 3-(N-maleimidylpropionyl) biocytin. Subunit a has no naturally occurring cysteine residues, allowing unique cysteines to be introduced at the following positions: 8, 24, 27, 69, 89, 128, 131, 172, 176, 196, 238, 241, and 277 (following the COOH-terminal 271 and a hexahistidine tag). None of the single mutations affected the function of the enzyme, as judged by growth on succinate minimal medium. Membrane vesicles with an exposed cytoplasmic surface were prepared using a French pressure cell. Before labeling, the membranes were incubated with or without a highly charged sulfhydryl reagent, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid. After labeling with the less polar biotin maleimide, the samples were solubilized with octyl glucoside/cholate and the subunit a was purified via the oligohistidine at its COOH terminus using immobilized nickel chromatography. The purified samples were electrophoresed and transferred to nitrocellulose for detection by avidin conjugated to alkaline phosphatase. Results indicated cytoplasmic accessibility for residues 69, 172, 176, and 277 and periplasmic accessibility for residues 8, 24, 27, and 131. On the basis of these and earlier results, a transmembrane topology for the subunit a is proposed.
Long
J C
JC
Department of Biological Sciences, Southern Methodist University, Dallas, Texas 75275, USA.
Wang
S
S
Vik
S B
SB
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D003545:Cysteine / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics; D015183:Restriction Mapping; D013439:Sulfhydryl Reagents / Q000378:metabolism; D013499:Surface Properties
D013439:Sulfhydryl Reagents; D006180:Proton-Translocating ATPases; D003545:Cysteine