J Biol Chem, Vol. 274, Issue 22, 15913-15919, May 28, 1999

Recombinant SFD Isoforms Activate Vacuolar Proton Pumps

From the Division of Molecular Transport, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75235.

The vacuolar proton pump of clathrin-coated vesicles is composed of two general sectors, a cytosolic, ATP hydrolytic domain (V₁) and an intramembranous proton channel, V₀. V₁ is comprised of 8-9 subunits including polypeptides of 50 and 57 kDa, termed SFD (Sub Fifty-eight-kDa Doublet). Although SFD is essential to the activation of ATPase and proton pumping activities catalyzed by holoenzyme, its constituent polypeptides have not been separated to determine their respective roles in ATPase functions. Recent molecular characterization of these subunits revealed that they are isoforms that arise through an alternative splicing mechanism (Zhou, Z., 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)¹ and 50-kDa (SFD) isoforms, we expressed these proteins in Escherichia coli. We determined that purified recombinant proteins, rSFD and rSFD, when reassembled with SFD-depleted holoenzyme, are functionally interchangeable in restoration of ATPase and proton pumping activities. In addition, we determined that the V-pump of chromaffin granules has only the SFD isoform in its native state and that rSFD and rSFD are equally effective in restoring ATPase and proton pumping activities to SFD-depleted enzyme. Finally, we found that SFD and SFD structurally interact not only with V₁, but also withV₀, indicating that these activator subunits may play both structural and functional roles in coupling ATP hydrolysis to proton flow.