Tubulin is the endogenous inhibitor of the glyceraldehyde 3-phosphate dehydrogenase isoform that catalyzes membrane fusion: Implications for the coordinated regulation of glycolysis and membrane fusion
Tubulin is the endogenous inhibitor of the glyceraldehyde 3-phosphate dehydrogenase isoform that catalyzes membrane fusion: Implications for the coordinated regulation of glycolysis and membrane fusion
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DOI：
10.1073/pnas.222542999
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发表时间：
2002-10-29
影响因子：
11.1
通讯作者：
Gross, RW
中科院分区：
文献类型：
--
作者：
Glaser, PE;Han, XL;Gross, RW
先前我们证明了兔脑GAPDH的特定色谱可分辨异构体催化糖酵解通量或膜融合活性（但不是两者都催化）。此外，GAPDH的膜融合活性是潜伏的，直到通过阴离子交换色谱将其从内源性胞质抑制剂中分离出来。在这里，我们证明了细胞质抑制剂是不可透析的，热不稳定的，胰蛋白酶敏感的，从而确定它是细胞质蛋白成分。对GAPDH异构体催化膜融合的兔脑胞质蛋白抑制剂进行色谱纯化，鉴定出一个优势的55-kDa双链，其内部含有一个与a-微管蛋白相同的15-aa肽（残基65-79）。通过Western blot分析和真正的微管蛋白对gapdh催化的膜融合的抑制，证实了55-kDa双联体是微管蛋白。停流动力学分析表明，微管蛋白对gapdh催化的融合活性具有高亲和力、快速和直接的调节作用。由于gtp激活的Rab 2将GAPDH招募到即将进行融合的膜上[Tisdale， E. J.(2001)]。陈晓明，陈晓明，等（2001）.微管蛋白与GAPDH相互作用的研究进展[j]。[j] .化学，277,3334-3341]，本研究提出了一种协调机制，通过该机制，膜运输和细胞信号可以与糖酵解通量相结合。
Previously we demonstrated that specific chromatographically resolvable isoforms of rabbit brain GAPDH catalyze either glycolytic flux or membrane fusion activity (but not both). Moreover, GAPDH membrane fusion activity was latent until it was separated from an endogenous cytosolic inhibitor by anion-exchange chromatography. Herein we demonstrate that the cytosolic inhibitor is nondialyzable, heat-labile, and trypsin-sensitive, thereby identifying it as a cytosolic protein constituent. Chromatographic purification of the rabbit-brain cytosolic protein inhibitor of GAPDH isoform-catalyzed membrane fusion identified a predominant 55-kDa doublet that contained an internal 15-aa peptide identical to a sequence present in a-tubulin (residues 65-79). The identity of the 55-kDa doublet as tubulin was substantiated through Western blot analysis and inhibition of GAPDH-catalyzed membrane fusion by authentic tubulin. Stopped-flow kinetic analysis demonstrated the high-affinity, rapid, and direct modulation of GAPDH-catalyzed fusion activity by tubulin. Because GTP-activated Rab 2 recruits GAPDH to membranes about to undergo fusion [Tisdale, E. J. (2001) J. Biol. Chem. 276, 2480-2486] and protein kinase Ci/lambda phosphorylates GAPDH modulating its interactions with tubulin [Tisdale, E. J. (2001) J. Biol. Chem. 277, 3334-3341], the present study suggests a coordinated mechanism through which membrane trafficking and cellular signaling can be integrated with glycolytic flux.