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

摘要

Previously we demonstrated that specific chromatographically resolvable isoforms of rabbit brain GAPDH catalyze either glyco-lytic 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 chro-matography. 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 α-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 C_ι/λ phosphor-ylates 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.