Abstract
In vivo, many proteins must interact with molecular chaperones to attain their native conformation. In the case of tubulin, newly synthesized alpha- and beta-subunits are partially folded by cytosolic chaperonin, a double-toroidal ATPase with homologs in all kingdoms of life and in most cellular compartments. alpha- and beta-tubulin folding intermediates are then brought together by tubulin-specific chaperone proteins (named cofactors A-E) in a cofactor-containing supercomplex with GTPase activity. Here we show that tubulin subunit exchange can only occur by passage through this supercomplex, thus defining it as a dimer-making machine. We also show that hydrolysis of GTP by beta-tubulin in the supercomplex acts as a switch for the release of native tubulin heterodimer. In this folding reaction and in the related reaction of tubulin-folding cofactors with native tubulin, the cofactors behave as GTPase-activating proteins, stimulating the GTP-binding protein beta-tubulin to hydrolyze its GTP.
Highlights
To gather more direct evidence that the GTP hydrolysis step in the tubulin heterodimerization reaction is performed by the -tubulin subunit and not by one of the cofactor proteins contained in the supercomplex, we looked at the reaction of cofactors with tubulin in the presence of ribose-modified forms of GTP; both dGTP and ddGTP support microtubule growth in vitro and are hydrolyzed as efficiently as GTP by the -tubulin subunit upon polymerization [30]
The simple and direct data presented here showing that ␣and -tubulin subunits do not exchange in the absence of tubulin-folding cofactors imply either that the subunits are very tightly associated in the heterodimer or that they denature as they dissociate
The latter explanation cannot be true, because this would imply that tubulin is highly unstable, which it is not, even at concentrations below the reported dissociation constant of the heterodimer, long thought to be in the micromolar range [21,22,23,24]
Summary
§ To whom corresponding should be addressed: Dept. of Biochemistry, New York University Medical Center, 550 First Ave., New York, NY 10016. We show that both in the folding reaction and in the related reaction of cofactors with native tubulin, the cofactors act as GTPase-activating proteins (GAPs), stimulating many-fold the negligible intrinsic GTPase activity of the tubulin to which they are bound
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