Monoclonal antibodies localize the exchangeable GTP‐binding site in β‐ and not α‐tubulins

Hesse, Juliane; Miyata, Hiroshi; Isenberg, G.

doi:10.1016/0014-5793(85)80198-8

Cited by 20 publications

(5 citation statements)

References 24 publications

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“…This eliminates the second possibility, leaving as the only explanation that colchicine-like ligands bind to tubulin on the subunit that contains the exchangeable nucleotide site. That site is known to be located on the ß subunit (Geahlen & Haley, 1977;Hesse et al, 1985Hesse et al, ,1987. This means that the colchicine binding site must be located on the ß subunit.…”

Section: Discussionmentioning

confidence: 99%

Effect of colchicine analogs on the dissociation of .alpha..beta. tubulin into subunits: The locus of colchicine binding

Shearwin¹,

Timasheff²

1994

Biochemistry

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A combination of ligand binding and sedimentation equilibrium studies was used to characterize the thermodynamic linkages between alpha beta tubulin association, nucleotide binding, and the interaction of colchicine analogues with dimeric and dissociated tubulins. The strength of binding of allocolchicine to the tubulin dimer was identical (8 x 10(5) M-1) whether the exchangeable nucleotide site (E site) was occupied by GTP or GDP. This drug bound to dimeric (alpha beta) tubulin and to one of the monomeric subunits, and the binding affinity for the dissociated state was linked to occupancy of the exchangeable nucleotide site. When the exchangeable site was occupied by GTP, the drug bound with very similar affinities to the dimeric and dissociated states of the protein. For tubulin-GDP, the binding of the drug to the dissociated state was significantly weaker (6.3 x 10(4) M-1) than to the dimeric state, suggesting the existence of an E-site-related conformational change in the dissociated state. Podophyllotoxin, which contains the A-ring portion of colchicine, bound with equal affinity to the dimeric and dissociated forms of both tubulin-GTP and tubulin-GDP, indicating that it is the C-ring portion of colchicine that is linked to the E-site-related conformational change.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Discussionmentioning

confidence: 99%

Effect of colchicine analogs on the dissociation of .alpha..beta. tubulin into subunits: The locus of colchicine binding

Shearwin¹,

Timasheff²

1994

Biochemistry

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“…UV irradiation could rapidly displace the GDP from the exchangeable site, allowing free GTP to bind and stabilize the polymer before significant rapid shortening occurs. Because the exchangeable site is located on the/~ subunit of the tubulin dimer (18,27,35), this mechanism predicts that the ~ subunit is oriented towards the minus end of a microtubule and the ct subunit is oriented towards the plus end. Unfortunately, the orientation of the tubulin dimer within the microtubule lattice is not yet known, An alternative explanation is that loss of the GTP cap is not sufficient for rapid shortening.…”

Section: Discussionmentioning

confidence: 99%

Asymmetric behavior of severed microtubule ends after ultraviolet-microbeam irradiation of individual microtubules in vitro.

Walker

Inoué

Salmon

1989

The Journal of Cell Biology

128

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The molecular basis of microtubule dynamic instability is controversial, but is thought to be related to a "GTP cap." A key prediction of the GTP cap model is that the proposed labile GDP-tubulin core will rapidly dissociate if the GTP-tubulin cap is lost. We have tested this prediction by using a UV microbeam to cut the ends from elongating microtubules. Phosphocellulose-purified tubulin was assembled onto the plus and minus ends of sea urchin flagellar axoneme fragments at 21-22 degrees C. The assembly dynamics of individual microtubules were recorded in real time using video microscopy. When the tip of an elongating plus end microtubule was cut off, the severed plus end microtubule always rapidly shortened back to the axoneme at the normal plus end rate. However, when the distal tip of an elongating minus end microtubule was cut off, no rapid shortening occurred. Instead, the severed minus end resumed elongation at the normal minus end rate. Our results show that some form of "stabilizing cap," possibly a GTP cap, governs the transition (catastrophe) from elongation to rapid shortening at the plus end. At the minus end, a simple GTP cap is not sufficient to explain the observed behavior unless UV induces immediate recapping of minus, but not plus, ends. Another possibility is that a second step, perhaps a structural transformation, is required in addition to GTP cap loss for rapid shortening to occur. This transformation would be favored at plus, but not minus ends, to account for the asymmetric behavior of the ends.

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“…6. The non-exchangeable GTPn is on ca-tubulin, the exchangeable one, GTPe, on ,B-tubulin (Geahlen and Haley, 1977;Nath et al, 1985;Hesse et al, 1985); the latter becomes non-exchangeable and is hydrolysed during microtubule assembly. Lane 1, control of undigested PC-tubulin; lanes 2 and 3, limited digestion with trypsin (about 60% of cx-tubulin nicked); lanes 4 and 5, limited digestion with chymotrypsin (about 70% of i3-tubulin nicked); lane 6, marker of bovine serum albumin containing monomers, dimers and trimers.…”

Section: Cross-linking Of Tubulin Monomers and Their Domainsmentioning

confidence: 99%

Tubulin domains responsible for assembly of dimers and protofilaments.

Kirchner¹,

Mandelkow²

1985

The EMBO Journal

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The protein domains responsible for the dimerization and polymerization of tubulin have been determined using chemical cross‐linking and limited proteolysis. The intra‐dimer bond is formed by the N‐terminal domain of alpha‐tubulin and the C‐terminal domain of beta‐tubulin. Conversely, the inter‐dimer bond along protofilaments is formed by the N‐terminal domain of beta‐tubulin (carrying the exchangeable GTP) and the C‐terminal domain of alpha‐tubulin. The domains of proteolytically cleaved tubulin remain tightly associated in solution. Apart from the monomer, tubulin shows three levels of assembly: the dimer, oligomer and polymer. Several oligomeric species can be visualized by electron microscopy of rotary shadowed phosphocellulose‐tubulin, h.p.l.c. and non‐denaturing gel electrophoresis. Tubulin's capacity to form the higher level aggregates is not destroyed by enzymatic nicking.

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Monoclonal antibodies localize the exchangeable GTP‐binding site in β‐ and not α‐tubulins

Cited by 20 publications

References 24 publications

Effect of colchicine analogs on the dissociation of .alpha..beta. tubulin into subunits: The locus of colchicine binding

Effect of colchicine analogs on the dissociation of .alpha..beta. tubulin into subunits: The locus of colchicine binding

Asymmetric behavior of severed microtubule ends after ultraviolet-microbeam irradiation of individual microtubules in vitro.

Tubulin domains responsible for assembly of dimers and protofilaments.

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