Reconstitution of recombinant 33-kDa subunit of the clathrin-coated vesicle H+-ATPase

Sheng Bin Peng, Ying Zhang, Sue Jean Tsai, Xiao Song Xie, Dennis K. Stone

Research output: Contribution to journalArticle

20 Scopus citations

Abstract

Evidence suggests that the ATP hydrolytic sector of the clathrin-coated vesicle proton-translocating ATPase is composed of four subunits of molecular masses of 70, 58, 40, and 33 kDa (Xie, X.S., and Stone, D. K. (1988) J. Biol. Chem. 263, 9859-9867). We have now expressed recombinant 33-kDa polypeptide in Escherichia coli and in Spodoptera frugiperda (Sf9) cells. This subunit, renatured and purified from both sources, lacks intrinsic ATPase activity. Co-reconstitution of these recombinant 33-kDa polypeptides and recombinant 40-kDa subunit to a biochemically prepared 70-58-kDa subcomplex results in a 6-fold stimulation of calcium-activated, N-ethyl-maleimide-sensitive ATPase activity, documenting the essential role of the 33- and 40-kDa components in vacuolar type proton pump function and furthering the aim of reconstitution of a purely recombinant hydrolytic core.

Original languageEnglish (US)
Pages (from-to)11356-11360
Number of pages5
JournalJournal of Biological Chemistry
Volume269
Issue number15
StatePublished - Apr 15 1994

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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