| The kinetics of assembly and disassembly of the vacuolar H{dollar}sp+{dollar}-ATPase, or V-ATPase, have been examined in cultured mammalian cells. The data indicate that V-ATPases are dynamic structures undergoing cycles of assembly and disassembly prior to degradation. Immunoprecipitation of metabolically labeled intact V-ATPases demonstrated that different subunits have different half-lives in the complex. Using a combination of metabolic labeling to label V-ATPases synthesized during a defined period, and transient expression of V-ATPase subunits specifically recognized by antibodies, we have observed directly the incorporation of previously assembled subunits into newly synthesized enzymes. Extracellular acidification causes a reversible loss of some V-ATPase subunits from isolated membrane fractions suggesting that assembly and disassembly are regulated processes which may play an important role controlling activity and targeting of V-ATPases. Immunoprecipitation of intact V-ATPases and V-ATPase subcomplexes indicates that the cytosolic domain of the enzyme dissociates intact from the transmembrane portion of the enzyme and that under normal conditions, cells have an excess of these cytosolic domains unassembled free in the cytosol. This is the first demonstration of an ion pump that undergoes disassembly and reassembly. |
