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.
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 alpha-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 Ciota/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.
Glaser
Paul E
PE
Division of Bioorganic Chemistry and Molecular Pharmacology, Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA.
Han
Xianlin
X
Gross
Richard W
RW
D000818:Animals; D002384:Catalysis; D006019:Glycolysis; D007527:Isoenzymes / Q000037:antagonists & inhibitors; D008561:Membrane Fusion; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors*; D011817:Rabbits; D014404:Tubulin / Q000302:isolation & purification / Q000378:metabolism*
D007527:Isoenzymes; D014404:Tubulin; C055686:glyceraldehyde 3-phosphate phosphatase; D010744:Phosphoric Monoester Hydrolases