Recombinant SFD isoforms activate vacuolar proton pumps

Zhiming Zhou, Sheng Bin Peng, Bill P. Crider, Per Andersen, Xiao Song Xie, Dennis K. Stone

Research output: Contribution to journalArticle

16 Scopus citations

Abstract

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 57kDa (SFDα)1 and 50-kDa (SFDβ) isoforms, we expressed these proteins in Escherichia coli. We determined that purified recombinant proteins, rSFDα and rSFDβ, 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 SFDα isoform in its native state and that rSFDα and rSFDβ are equally effective in restoring ATPase and proton pumping activities to SFD- depleted enzyme. Finally, we found that SFDα and SFDβ structurally interact not only with V1, but also with V0, indicating that these activator subunits may play both structural and functional roles in coupling ATP hydrolysis to proton flow.

Original languageEnglish (US)
Pages (from-to)15913-15919
Number of pages7
JournalJournal of Biological Chemistry
Volume274
Issue number22
DOIs
StatePublished - May 28 1999

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Zhou, Z., Peng, S. B., Crider, B. P., Andersen, P., Xie, X. S., & Stone, D. K. (1999). Recombinant SFD isoforms activate vacuolar proton pumps. Journal of Biological Chemistry, 274(22), 15913-15919. https://doi.org/10.1074/jbc.274.22.15913