ATP Synthase from Saccharomyces cerevisiae: Location of the OSCP Subunit in the Peripheral Stalk Region

A biotinylation signal has been fused to the C terminus of the oligomycin sensitivity conferral protein (OSCP) of the ATP synthase complex from Saccharomyces cerevisiae. The signal is biotinylated in vivo and the biotinylated complex binds avidin in vitro. By electron microscopy of negatively stained particles of the ATP synthase–avidin complex, the bound avidin has been localised close to the F1 domain. The images were subjected to multi-reference alignment and classification. Because of the presence of a flexible linker between the OSCP and the biotinylation signal, the class-averages differ in the position of the avidin relative to the F1 domain. These positions lie on an arc, and its centre indicates the position of the C terminus of the OSCP on the surface of the F1 domain. Since the N-terminal region of the OSCP is known to interact with the N-terminal regions of α-subunits, which are on top of the F1 domain distal from the Fo membrane domain, the OSCP extends almost 10 nm along the surface of F1 down towards Fo where it interacts with the C terminus of the b subunit, which extends up from Fo. The labelling technique has also allowed a reliable 2D projection map to be developed for the intact ATP synthase from S. cerevisiae. The map reveals a marked asymmetry in the Fo part of the complex that can be attributed to subunits in the Fo domain.