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 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.
Peng
S B
SB
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235-9121.
Zhang
Y
Y
Tsai
S J
SJ
Xie
X S
XS
Stone
D K
DK
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology; D002417:Cattle; D002460:Cell Line; D002966:Clathrin / Q000378:metabolism; D003001:Cloning, Molecular; D003034:Coated Pits, Cell-Membrane / Q000201:enzymology*; D017931:DNA Primers; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D015723:Gene Library; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009036:Moths; D006180:Proton-Translocating ATPases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D002966:Clathrin; D017931:DNA Primers; D018076:DNA, Complementary; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D006180:Proton-Translocating ATPases