The topology of the proton translocating F0 component of the ATP synthase from E. coli K12: studies with proteases.

The accessibility of the three F0 subunits a, b and c from the Escherichia coli K12 ATP synthase to various proteases was studied in F1-depleted inverted membrane vesicles. Subunit b was very sensitive to all applied proteases. Chymotrypsin produced a defined fragment of mol. wt. 15,000 which remained tightly bound to the membrane. The cleavage site was located at the C-terminal region of subunit b. Larger amounts of proteases were necessary to attack subunit a (mol. wt. 30,000). There was no detectable cleavage of subunit c. It is suggested that the major hydrophilic part of subunit b extends from the membrane into the cytoplasm and is in contact with the F1 sector. The F1 sector was found to afford some protection against proteolysis of the b subunit in vitro and in vivo. Protease digestion had no influence on the electro-impelled H+ conduction via F0 but ATP-dependent H+ translocation could not be reconstituted upon binding of F1. A possible role for subunit b as a linker between catalytic events on the F1 component and the proton pathway across the membrane is discussed.