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

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 polypep...

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Bibliographic Details
Published in:The Journal of biological chemistry 1994-04, Vol.269 (15), p.11356-11360
Main Authors: Peng, S B, Zhang, Y, Tsai, S J, Xie, X S, Stone, D K
Format: Article
Language:English
Subjects:
Animals
Base Sequence
Blotting, Western
Brain - enzymology
Cattle
Cell Line
Clathrin - metabolism
Cloning, Molecular
Coated Pits, Cell-Membrane - enzymology
DNA Primers
DNA, Complementary - metabolism
Electrophoresis, Polyacrylamide Gel
Escherichia coli
Gene Library
Macromolecular Substances
Molecular Sequence Data
Moths
Proton-Translocating ATPases - biosynthesis
Proton-Translocating ATPases - isolation & purification
Proton-Translocating ATPases - metabolism
Recombinant Proteins - biosynthesis
Recombinant Proteins - isolation & purification
Recombinant Proteins - metabolism
Transfection
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Summary: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.
ISSN:0021-9258
1083-351X
DOI:10.1016/S0021-9258(19)78132-0