Activation of the plasma membrane H+-ATPase of Saccharomyces cerevisiae by glucose is mediated by dissociation of the H+-ATPase-acetylated tubulin complex
Activation of the plasma membrane H+-ATPase of Saccharomyces cerevisiae by glucose is mediated by dissociation of the H+-ATPase-acetylated tubulin complex
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DOI：
10.1111/j.1742-4658.2005.04959.x
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发表时间：
2005-11-01
期刊：
影响因子：
5.4
通讯作者：
Casale, CH
中科院分区：
文献类型：
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作者：
Campetelli, AN;Previtali, G;Casale, CH
在酵母中，质膜H+- atp酶被d -葡萄糖激活。我们发现，在缺乏葡萄糖的情况下，这种酶与乙酰化的微管蛋白形成复合物。乙酰化的微管蛋白通常表现出亲水性，但当与H+- atp酶络合时表现为疏水化合物，因此分裂成洗涤相。当细胞用葡萄糖处理时，H+- atp酶-微管蛋白复合物被破坏，有两个结果，即(a)质膜中乙酰化的微管蛋白水平随着葡萄糖浓度的变化而降低；(b) H+- atp酶活性随着葡萄糖浓度的变化而增加，通过atp水解能力和H+泵送活性来测量。2-脱氧-d -葡萄糖的加入抑制了上述葡萄糖诱导的现象，表明葡萄糖转运体参与其中。总微管蛋白在细胞内均匀分布，而乙酰化微管蛋白则集中在质膜附近。利用抗（乙酰化）微管蛋白和抗（H+- atp酶）免疫球蛋白进行的免疫沉淀实验结果表明，在葡萄糖饥饿细胞的细胞膜上，H+- atp酶和乙酰化微管蛋白之间存在物理相互作用。当细胞用1mm葡萄糖预处理时，这种相互作用被破坏。双免疫荧光共聚焦显微镜观察显示，H+- atp酶和乙酰化微管蛋白部分共定位于葡萄糖饥饿细胞的外周，分别以膜外侧和膜内侧为主。当细胞用1mm葡萄糖预处理时，未观察到共定位，这加强了葡萄糖处理产生H+- atp酶-微管蛋白复合物解离的观点。利用酵母分离膜和纯化的大鼠脑微管蛋白进行的生化实验表明，乙酰化的微管蛋白抑制H+-ATP酶活性，并伴随H+-ATP酶-微管蛋白复合物的增加。
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.