Reconstitution of the recombinant 70-kDa subunit of the clathrin-coated vesicle H+ ATPase

Sheng Bin Peng, Ying Zhang, Bill P. Crider, Allen E. White, Victor A. Fried, Dennis K. Stone, Xiao Song Xie

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Vacuolar-type proton pumps are complex hetero-oligomers. When dissociated into subcomplexes and subunits, the partial reactions of ATP hydrolysis and trans-membranous proton flow can be assigned to isolated domains. Data suggest that the molecular site of ATP hydrolysis resides within the 70-kDa subunit but that ATPase activity likely requires at least three additional subunits of 58, 40, and 33 kDa (Xie, X.-S., and Stone, D. K. (1988) J. Biol. Chem. 263, 9859-9867). We have now cloned and sequenced the 70-kDa subunit from bovine brain and have expressed the protein in insect Sf9 (Spodoptera frugiperda) cells with a recombinant baculovirus. When purified, the protein has no significant ATPase activity but can be photoaffinity labeled with [α32P]ATP and UV irradiation with an apparent K(d) of 35 μM. When reconstituted with biochemically prepared 58-, 40-, and 33-kDa polypeptides, the recombinant 70-kDa subunit restores Ca2+-activated ATP hydrolysis to a specific activity of 0.6 μmol P(i) · mg protein-1 · min-1, thus demonstrating that ATP hydrolysis in vacuolar-type proton pumps is dependent upon both the 70-kDa subunit as well as multi-subunit interactions.

Original languageEnglish (US)
Pages (from-to)27778-27782
Number of pages5
JournalJournal of Biological Chemistry
Volume269
Issue number44
StatePublished - Nov 4 1994

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Clathrin-Coated Vesicles
Clathrin
Proton-Translocating ATPases
Adenosine Triphosphate
Hydrolysis
Proton Pumps
Adenosine Triphosphatases
Insect Proteins
Spodoptera
Proteins
Baculoviridae
Oligomers
Protons
Brain
Irradiation
Peptides

ASJC Scopus subject areas

  • Biochemistry

Cite this

Peng, S. B., Zhang, Y., Crider, B. P., White, A. E., Fried, V. A., Stone, D. K., & Xie, X. S. (1994). Reconstitution of the recombinant 70-kDa subunit of the clathrin-coated vesicle H+ ATPase. Journal of Biological Chemistry, 269(44), 27778-27782.

Reconstitution of the recombinant 70-kDa subunit of the clathrin-coated vesicle H+ ATPase. / Peng, Sheng Bin; Zhang, Ying; Crider, Bill P.; White, Allen E.; Fried, Victor A.; Stone, Dennis K.; Xie, Xiao Song.

In: Journal of Biological Chemistry, Vol. 269, No. 44, 04.11.1994, p. 27778-27782.

Research output: Contribution to journalArticle

Peng, SB, Zhang, Y, Crider, BP, White, AE, Fried, VA, Stone, DK & Xie, XS 1994, 'Reconstitution of the recombinant 70-kDa subunit of the clathrin-coated vesicle H+ ATPase', Journal of Biological Chemistry, vol. 269, no. 44, pp. 27778-27782.
Peng SB, Zhang Y, Crider BP, White AE, Fried VA, Stone DK et al. Reconstitution of the recombinant 70-kDa subunit of the clathrin-coated vesicle H+ ATPase. Journal of Biological Chemistry. 1994 Nov 4;269(44):27778-27782.
Peng, Sheng Bin ; Zhang, Ying ; Crider, Bill P. ; White, Allen E. ; Fried, Victor A. ; Stone, Dennis K. ; Xie, Xiao Song. / Reconstitution of the recombinant 70-kDa subunit of the clathrin-coated vesicle H+ ATPase. In: Journal of Biological Chemistry. 1994 ; Vol. 269, No. 44. pp. 27778-27782.
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