Reconstitution of the recombinant 70-kDa subunit of the clathrin-coated vesicle H+ ATPase.
Vacuolar-type proton pumps are complex heterooligomers. When dissociated into subcomplexes and subunits, the partial reactions of ATP hydrolysis and transmembranous 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 [alpha 32P]ATP and UV irradiation with an apparent Kd of 35 microM. When reconstituted with biochemically prepared 58-, 40-, and 33-kDa polypeptides, the recombinant 70-kDa subunit restores Ca(2+)-activated ATP hydrolysis to a specific activity of 0.6 mumol 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.
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235-9121.
S B
SB
Peng
Y
Y
Zhang
B P
BP
Crider
A E
AE
White
V A
VA
Fried
D K
DK
Stone
X S
XS
Xie
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D003001:Cloning, Molecular; D018699:Coated Vesicles / Q000201:enzymology*; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D006180:Proton-Translocating ATPases / Q000737:chemistry*; D011994:Recombinant Proteins
D017931:DNA Primers; D018076:DNA, Complementary; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D002118:Calcium