Role of a 50-57-kDa polypeptide heterodimer in the function of the clathrin-coated vesicle proton pump.
The vacuolar-type proton-translocating ATPase of clathrin-coated vesicles is composed of an integral membrane proton channel (VB) and a peripheral catalytic sector (VC). Native enzyme can catalyze the hydrolysis of both MgATP and CaATP and support proton pumping when reconstituted into liposomes. In contrast, isolated VC catalyzes only Ca(2+)-activated ATP hydrolysis and cannot support proton pumping when reconstituted into liposomes (Xie, X.-S., and Stone, D. K. (1988) J. Biol. Chem. 263, 9859-9867). We now report that solubilized isolated VC can be reassembled with purified VB to restore properties of native enzyme, including Mg(2+)-activated ATP hydrolysis and proton-pumping capability. Investigation of this reassembly revealed that a heterodimer, composed of polypeptides of 50 and 57 kDa, stimulates Ca(2+)-activated ATPase activity of isolated VC 2-fold and Mg(2+)-activated ATPase activity catalyzed by the reassembled pump 9-fold. Moreover, this heterodimer stimulated proton transport by the reassembled pump > 20-fold. When separated from the proton pump, the dimer has no detectable kinase activity. Maximal stimulation occurs at a molar ratio of heterodimer to reassembled pump of 3, implying a structural, nonenzymatic mechanism. These data indicate that the 50-kDa and/or the 57-kDa polypeptide likely plays an essential and potentially regulatory role in the function of the proton-translocating ATPase of clathrin-coated vesicles.
Xie
X S
XS
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235-9121.
Crider
B P
BP
Ma
Y M
YM
Stone
D K
DK
D000255:Adenosine Triphosphate / Q000378:metabolism; D002966:Clathrin; D004789:Enzyme Activation; D006868:Hydrolysis / Q000187:drug effects; D016623:Ion Pumps; D008081:Liposomes / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D011487:Protein Conformation; D017494:Proton Pumps / Q000187:drug effects / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000187:drug effects / Q000378:metabolism*; D014617:Vacuoles / Q000201:enzymology*
D002966:Clathrin; D016623:Ion Pumps; D008081:Liposomes; D017494:Proton Pumps; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D008274:Magnesium