Recombinant SFD isoforms activate vacuolar proton pumps

The vacuolar proton pump of clathrin-coated vesicles is composed of two gen eral sectors, a cytosolic, ATP hydrolytic domain (V-1) and an intramembrano us proton channel, V-0. V-1 is comprised of 8-9 subunits including polypept ides of 50 and 57 kDa, termed SFD (Sub Fifty-eight-kDa Doublet). Although S FD is essential to the activation of ATPase and proton pumping activities c atalyzed by holoenzyme, its constituent polypeptides have not been separate d to determine their respective roles in ATPase functions. Recent molecular characterization of these subunits revealed that they are isoforms that ar ise through an alternative splicing mechanism (Zhou,., Peng, S.-B., Crider, B.P., Slaughter, C., Xie, X.S., and Stone, D.K. (1998) J. Biol. Chem. 273, 5878-5884). To determine the functional characteristics of the 57-kDa (SFD alpha)(1) an d 50-kDa (SFD beta) isoforms, we expressed these proteins in Escherichia co li. We determined that purified recombinant proteins, rSFD alpha and rSFD b eta, when reassembled with SFD-depleted holoenzyme, are functionally interc hangeable in restoration of ATPase and proton pumping activities. In additi on, we determined that the V-pump of chromaffin granules has only the SFDa isoform in its native state and that rSFD alpha and rSFD beta are equally e ffective in restoring ATPase and proton pumping activities to SFD-depleted enzyme. Finally, we found that SFD alpha and SFD beta structurally interact not only with V-1, but also with V-0, indicating that these activator subu nits may play both structural and functional roles in coupling ATP hydrolys is to proton flow.