The F subunit of Thermus thermophilus V1-ATPase promotes ATPase activity but is not necessary for rotation

V-1-ATPase from the thermophilic bacterium Thermus thermophilus is a molecular rotary motor with a subunit composition of A(3)B(3)DF, and its central rotor is composed of the D and F subunits. To determine the role of the F subunit, we generated an A(3)B(3)D subcomplex and compared it with A(3)B(3)DF. The ATP hydrolyzing activity of A(3)B(3)D (V-max = 20 s(-1)) was lower than that of A(3)B(3)DF (V-max = 31 s(-1)) and was more susceptible to MgADP inhibition during ATP hydrolysis. A3B3D was able to bind the F subunit to form A(3)B(3)DF. The C-terminally truncated F(Delta85-106) subunit was also bound to A(3)B(3)D, but the F(Delta69-106) subunit was not, indicating the importance of residues 69 - 84 of the F subunit for association with A(3)B(3)D. The ATPase activity of A(3)B(3)DF((Delta85-106)) (V-max = 24 s(-1)) was intermediate between that of A(3)B(3)D and A(3)B(3)DF. A single molecule experiment showed the rotation of the D subunit in A(3)B(3)D, implying that the F subunit is a dispensable component for rotation itself. Thus, the F subunit binds peripherally to the D subunit, but promotes V-1-ATPase catalysis.