Activation of the plasma membrane H+-ATPase of Saccharomyces cerevisiae by glucose is mediated by dissociation of the H+-ATPase-acetylated tubulin complex

摘要

In the yeast Saccharomyces cerevisiae, plasma membrane H+-ATPase is activated by D-glucose. We found that in the absence of glucose, this enzyme forms a complex with acetylated tubulin. Acetylated tubulin usually displays hydrophilic properties, but behaves as a hydrophobic compound when complexed with H+-ATPase, and therefore partitions into a detergent phase. When cells were treated with glucose, the H+-ATPase-tubulin complex was disrupted, with two consequences, namely (a) the level of acetylated tubulin in the plasma membrane decreased as a function of glucose concentration and (b) the H+-ATPase activity increased as a function of glucose concentration, as measured by both ATP-hydrolyzing capacity and H+-pumping activity. The addition of 2-deoxy-D-glucose inhibited the above glucose-induced phenomena, suggesting the involvement of glucose transporters. Whereas total tubulin is distributed uniformly throughout the cell, acetylated tubulin is concentrated near the plasma membrane. Results from immunoprecipitation experiments using anti-(acetylated tubulin) and anti-(H+-ATPase) immunoglobulins indicated a physical interaction between H+-ATPase and acetylated tubulin in the membranes of glucose-starved cells. When cells were pretreated with 1 mM glucose, this interaction was disrupted. Double immunofluorescence, observed by confocal microscopy, indicated that H+-ATPase and acetylated tubulin partially colocalize at the periphery of glucose-starved cells, with predominance at the outer and inner sides of the membrane, respectively. Colocalization was not observed when cells were pretreated with 1 mM glucose, reinforcing the idea that glucose treatment produces dissociation of the H+-ATPase-tubulin complex. Biochemical experiments using isolated membranes from yeast and purified tubulin from rat brain demonstrated inhibition of H+-ATPase activity by acetylated tubulin and concomitant increase of the H+-ATP ase-tubulin complex.