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

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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

Fingerprint

Clathrin-Coated Vesicles
Clathrin
Proton-Translocating ATPases
Adenosine Triphosphatases
Sf9 Cells
Proton Pumps
Spodoptera
Peptides
Molecular mass
Escherichia coli
Adenosine Triphosphate
Calcium
maleimide

ASJC Scopus subject areas

  • Biochemistry

Cite this

Peng, S. B., Zhang, Y., Tsai, S. J., Xie, X. S., & Stone, D. K. (1994). Reconstitution of recombinant 33-kDa subunit of the clathrin-coated vesicle H+-ATPase. Journal of Biological Chemistry, 269(15), 11356-11360.

Reconstitution of recombinant 33-kDa subunit of the clathrin-coated vesicle H+-ATPase. / Peng, Sheng Bin; Zhang, Ying; Tsai, Sue Jean; Xie, Xiao Song; Stone, Dennis K.

In: Journal of Biological Chemistry, Vol. 269, No. 15, 15.04.1994, p. 11356-11360.

Research output: Contribution to journalArticle

Peng, SB, Zhang, Y, Tsai, SJ, Xie, XS & Stone, DK 1994, 'Reconstitution of recombinant 33-kDa subunit of the clathrin-coated vesicle H+-ATPase', Journal of Biological Chemistry, vol. 269, no. 15, pp. 11356-11360.
Peng, Sheng Bin ; Zhang, Ying ; Tsai, Sue Jean ; Xie, Xiao Song ; Stone, Dennis K. / Reconstitution of recombinant 33-kDa subunit of the clathrin-coated vesicle H+-ATPase. In: Journal of Biological Chemistry. 1994 ; Vol. 269, No. 15. pp. 11356-11360.
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