Preparation of a monoclonal antibody specific for the class IV isotype of beta-tubulin. Purification and assembly of alpha beta II, alpha beta III, and alpha beta IV tubulin dimers from bovine brain.

Tubulin, the 100-kDa subunit protein of microtubules, is a heterodimer of two 50-kDa subunits, alpha and beta. Both alpha and beta subunits exist as numerous isotypic forms. There are four isotypes of beta-tubulin in bovine brain tubulin preparations; their designations and relative abundances in these preparations are as follows: beta I, 3%; beta II, 58%; beta III, 25%; and beta IV, 13%. We have previously reported the preparation of monoclonal antibodies specific for beta II and beta III (Banerjee, A., Roach, M. C., Wall, K. A., Lopata, M. A., Cleveland, D. W., and Luduena, R. F. (1988) J. Biol. Chem. 263, 3029-3034; Banerjee, A., Roach, M. C., Trcka, P., and Luduena, R. F. (1990) J. Biol. Chem. 265, 1794-1799). We here report the preparation of a monoclonal antibody specific for beta IV. By using this antibody together with those specific for beta II and beta III, we have prepared isotypically pure tubulin dimers with the composition alpha beta II, alpha beta III, and alpha beta IV. We have found that, in the presence of microtubule-associated proteins, all three dimers assemble into microtubules considerably faster and to a greater extent than does unfractionated tubulin. More assembly was noted with alpha beta II and alpha beta III than with alpha beta IV. When assembly is measured in the presence of taxol (10 microM), little difference is seen among the isotypically purified dimers or between them and unfractionated tubulin. These results indicate that the assembly properties of a tubulin preparation are influenced by its isotypic composition and raise the possibility that the structural differences among tubulin isotypes may have functional significance.     

Regulation of tubulin synthesis during the cell cycle in the synchronous plasmodia of Physarum polycephalum

Regulation of alpha- and beta-tubulin isotype synthesis during the cell cycle has been studied in the myxomycete Physarum polycephalum, by subjecting synchronous plasmodia to temperature shifts and pharmacological perturbations. Temperature shifts interfered with the regulation of tubulin synthesis. Inhibition of DNA synthesis prevents tubulin degradation after completion of the cell cycle (Ducommun and Wright, Eur. J. Cell Biol., 50:48-55, 1989) but did not perturb the initiation of tubulin synthesis. The constant increase of tubulin synthesis in the presence of tubulin-sequestering drugs and the decrease of tubulin synthesis during a treatment with aphidicolin in late G2 phase suggest the existence of an autoregulatory mechanism of tubulin synthesis. Moreover, the microtubule poison methyl benzimidazole carbamate dissociated synthesis of the alpha 1-tubulin isotype from the generally strictly coordinated synthesis of all tubulin isotypes during the transient interruption of mitosis. These observations show that a microtubular poison can perturb regulation of the synthesis of specific isotubulins.     

Coordination of posttranslational modifications of bovine brain alpha-tubulin. Polyglycylation of delta2 tubulin

Microtubules participate in a large number of intracellular events including cell division, intracellular transport and secretion, axonal transport, and maintenance of cell morphology. They are composed of tubulin, a heterodimeric protein, consisting of two similar polypeptides alpha and beta. In mammalian cells, both alpha- and beta-tubulin occur as seven to eight different genetic variants, which also undergo numerous posttranslational modifications that include tyrosination-detyrosination and deglutamylation, phosphorylation, acetylation, polyglutamylation, and polyglycylation. Tyrosination-detyrosination is one of the major posttranslational modifications in which the C-terminal tyrosine residue in alpha-tubulin is added or removed reversibly. Although this modification does not alter the assembly activity of tubulin in vitro, these two forms of tubulin have been found to be distributed differently in vivo and are also correlated with microtubule stability (Gunderson, G. G., Kalnoski, M. H., and Bulinski, J. C. (1984) Cell 38, 779-789). Thus, the question arises as to whether these two forms of tubulin differ in any other modifications. In an effort to answer this question, the tyrosinated and the nontyrosinated forms of the alpha1/2 isoform have been purified from brain tubulin by immunoaffinity chromatography. matrix-assisted laser desorption/ionization-time of flight mass spectrometric analysis of the C-terminal peptide revealed that the tyrosinated form is polyglutamylated with one to four Glu residues, while the Delta2 tubulin is polyglycylated with one to three Gly residues. These results indicate that posttranslational modifications of tubulin are correlated with each other and that polyglutamylation and polyglycylation of tubulin may have important roles in regulating microtubule assembly, stability, and function in vivo.     

The interaction of cystamine with bovine brain tubulin

Microtubule assembly in vitro is sensitive to a variety of non-physiological sulfhydryl-oxidizing agents, but the physiological significance of this phenomenon is unknown, since no physiological sulfhydryl-oxidizing agent has been shown to affect microtubule assembly in vitro. We have accordingly investigated the interaction of tubulin with cystamine. We have found that millimolar concentrations of cystamine inhibit microtubule assembly and induce an abnormal form of tubulin polymerization. Cystamine-induced polymerization does not occur at cold temperature. Formation of the polymer requires reaction of cystamine with two sulfhydryls which become available at 37 degrees C. In addition, cystamine reacts with about three sulfhydryls at 0 degrees C without inducing polymerization. This latter set of sulfhydryls appear to include one or both of the previously defined beta s sulfhydryls whose reaction with N, N'-ethylene-bis(iodoacetamide) is markedly inhibited by GTP, maytansine and vinblastine [Roach, M. C. & Luduena, R. F. (1984) J. Biol. Chem. 259, 12063-12071]. Cystamine's specific manner of interacting with tubulin suggests that it may mimic an endogenous sulfhydryl-directed regulator of microtubule assembly.     

Taxol binds to polymerized tubulin in vitro

Taxol, a natural plant product that enhances the rate and extent of microtubule assembly in vitro and stabilizes microtubules in vitro and in cells, was labeled with tritium by catalytic exchange with (3)H(2)O. The binding of [(3)H]taxol to microtubule protein was studied by a sedimentation assay. Microtubules assembled in the presence of [(3)H]taxol bind drug specifically with an apparent binding constant, K(app), of 8.7 x 19(-7) M and binding saturates with a calculated maximal binding ration, B(max), of 0.6 mol taxol bound/mol tubulin dimer. [(3)H]Taxol also binds and assembles phosphocellulose-purified tubulin, and we suggest that taxol stabilizes interactions between dimers that lead to microtubule polymer formation. With both microtubule protein and phosphocellulose- purified tubulin, binding saturation occurs at approximate stoichiometry with the tubulin dimmer concentration. Under assembly conditions, podophyllotoxin and vinblastine inhibit the binding of [(3)H]taxol to microtubule protein in a complex manner which we believe reflects a competition between these drugs, not for a single binding site, but for different forms (dimer and polymer) of tubulin. Steady-state microtubules assembled with GTP or with 5’-guanylyl-α,β-methylene diphosphonate (GPCPP), a GTP analog reported to inhibit microtubule treadmilling (I.V. Sandoval and K. Weber. 1980. J. Biol. Chem. 255:6966-6974), bind [(3)H]taxol with approximately the same stoichiometry as microtubules assembled in the presence of [(3)H]taxol. Such data indicate that a taxol binding site exists on the intact microtubule. Unlabeled taxol competitively displaces [(3)H]taxol from microtubules, while podophyllotoxin, vinblastine, and CaCl(2) do not. Podophyllotoxin and vinblastine, however, reduce the mass of sedimented taxol-stabilized microtubules, but the specific activity of bound [(3)H]taxol in the pellet remains constant. We conclude that taxol binds specifically and reversibly to a polymerized form of tubulin with a stoichiometry approaching unity.     

Polymerization of tubulin in vivo: direct evidence for assembly onto microtubule ends and from centrosomes

Microtubule assembly in vivo was studied by hapten-mediated immunocytochemistry. Tubulin was derivatized with dichlorotriazinylaminofluorescein (DTAF) and microinjected into living, interphase mammalian cells. Sites of incorporation were determined at the level of individual microtubules by double-label immunofluorescence. The haptenized tubulin was localized by an anti-fluorescein antibody and a second antibody conjugated with fluorescein. Total microtubules were identified by anti-tubulin and a secondary antibody conjugated with rhodamine. Contrary to recent studies (Salmon, E. D., et al., 1984, J. Cell Biol., 99:2165-2174; Saxton, W. M., et al., 1984, J. Cell Biol., 99:2175-2186) which suggest that tubulin incorporates all along the length of microtubules in vivo, we found that microtubule assembly in interphase cells was in vivo, as in vitro, an end-mediated process. Microtubules that radiated out toward the cell periphery incorporated the DTAF-tubulin solely at their distal, that is, their plus ends. We also found that a proportion of the microtubules connected to the centrosomes incorporated the DTAF-tubulin along their entire length, which suggests that the centrosome can nucleate the formation of new microtubules.     

Removal of the projection domain of microtubule-associated protein 2 alters its interaction with tubulin

Microtubule-associated proteins (MAPs) can promote microtubule assemblyin vitro. One of these MAPs (MAP2) consists of a short promoter domain which binds to the microtubule and promotes assembly and a long projection domain which projects out from the microtubule and may interact wth other cytoskeletal elements. We have previously shown that MAP2 and another MAP, tau, differ in their interactions with tubulin in that tau, but not MAP2, promotes extensive aggregation of tubulin into spiral clusters in the presence of vinblastine and that microtubules formed with MAP2 are more resistant than those formed with tau to the antimitotic drug maytansine [Luduena, R. F.,et al. (1984),J. Biol. Chem. 259, 12890–12898; Fellous, A.,et al. (1985),Cancer Res. 45, 5004–5010]. Here we have used chymotryptic digestion to remove the projection domain of MAP2 and examined the interaction of the digested MAP2 (ctMAP2) with tubulin in the presence of vinblastine and maytansine. We have found that ctMAP2 behaves very much like tau, but not like undigested MAP2, in the presence of vinblastine, in that ctMAP2 causes tubulin to polymerize into large clusters of spirals. In contrast, microtubule assembly in the presence of ctMAP2 is much more resistant to maytansine inhibition than is assembly in the presence of tau or undigested MAP2. Our results suggest that the projection domain of MAP2 may play a role in the interaction of tubulin with MAP2 during microtubule assembly.Abbreviations MAPs microtubule-associated proteins - ctMAP2 MAP2 digested with-chymotrypsin - nMAP2 untreated MAP2 - PMSF phenylmethylsulfonyl fluoride - GMPCPP guanosine-5-(,-methylene)triphosphate     

Purification and characterization of oocyte cytoplasmic tubulin and meiotic spindle tubulin of the surf clam Spisula solidissima

Assembly-competent tubulin was purified from the cytoplasm of unfertilized and parthogenetically activated oocytes, and from isolated meiotic spindles of the surf clam, Spisula solidissima. At 22 degrees C or 37 degrees C, Spisula tubulin assembled into 48-51-nm macrotubules during the first cycle of polymerization and 25-nm microtubules during the third and subsequent cycles of assembly. Macrotubules were formed from sheets of 26-27 protofilaments helically arranged at a 36 degree angle relative to the long axis of the polymer and were composed of alpha and beta tubulins and several other proteins ranging in molecular weight from 30,000 to 270,000. Third cycle microtubules contained 14-15 protofilaments in cross-section and were composed of greater than 95% alpha and beta tubulins. After three cycles of polymerization at 37 degrees C, unfertilized and activated oocyte tubulin self-assembled into microtubules at a critical concentration (Ccr) of 0.09 mg/ml. At the physiological temperature of 22 degrees C, unfertilized oocyte tubulin assembled into microtubules at a Ccr of 0.36 mg/ml, activated oocyte tubulin assembled at a Ccr of 0.42 mg/ml, and isolated meiotic spindle tubulin assembled at a Ccr of 0.33 mg/ml. The isoelectric points of tubulin from both unfertilized oocytes and isolated meiotic spindles were 5.8 for alpha tubulin and 5.6 for beta tubulin. In addition, one dimensional peptide maps of oocyte and spindle alpha and beta tubulins were very similar, if not identical. These results indicate that unfertilized oocyte tubulin and tubulin isolated from the first meiotic spindle are indistinguishable on the basis of assembly properties, isoelectric focusing, and one dimensional peptide mapping. These results suggest that the transition of tubulin from the quiescent oocyte state to that competent to form spindle microtubules in vivo does not require special modification of tubulin but may involve changes in the availability of microtubule organizing centers or assembly-promoting microtubule-associated proteins.     

Different reactivities of brain and erythrocyte tubulins toward a sulfhydryl group-directed reagent that inhibits microtubule assembly

There is considerable evidence that tubulin exists in multiple isotypes, differing in amino acid sequence and tissue distribution. Little is known, however, about the functional significance of these isotypes. Chicken erythrocyte beta-tubulin has been shown by peptide mapping to differ significantly from chicken brain beta-tubulin (Murphy, D. B., and Wallis, K. T. (1983) J. Biol. Chem. 258, 7870-7875). We now find that when the two tubulins, in their native states, are incubated with N,N'-ethylenebis(iodoacetamide) (EBI), a bifunctional sulfhydryl-directed reagent, microtubule assembly by brain tubulin is much more sensitive to inhibition by EBI than is erythrocyte tubulin assembly. The resistance of erythrocyte microtubule assembly to inhibition by EBI is correlated with a low reactivity of erythrocyte tubulin with [14C]EBI. This difference is most marked in the beta subunit which reacts 15 and 17% as well, respectively, with [14C]EBI as do the beta 1 and beta 2 subunits of brain tubulin. Also, erythrocyte beta reacts about 33% as well as does brain beta with iodo[14C]acetamide. These results suggest that a reactive sulfhydryl group, whose oxidation prevents microtubule assembly, is present in brain tubulin but absent or inaccessible in erythrocyte tubulin. Since purified erythrocyte tubulin self-aggregates much more readily than does brain tubulin, it is conceivable that erythrocyte and brain tubulin may differ in that the latter may have its assembly subject to a complex regulation, while erythrocyte tubulin assembly may be regulated by a simpler mechanism.     

Nucleotide release from tubulin and nucleoside-5'-diphosphate kinase action in microtubule assembly.

ATP and UTP support microtubule assembly through the action of brain nucleoside-5'-diphosphate kinase on GDP. Penningroth and Kirschner (1977) J. Mol. Biol. 115, 643-673) have proposed that microtubule assembly may occur by either of two mechanisms: indirectly, through nucleoside-5'-diphosphate kinase-catalyzed phosphorylation of uncomplexed GDP and directly by nucleoside-5'-diphosphate kinase-mediated transphosphorylation of tubulin-bound GDP at low tubulin concentrations. We find the rates of GDP and GTP release (0.68 and 0.32 min-1, respectively) are sufficiently fast relative to assembly to permit GDP release, phosphorylation, and GTP binding as the sole mechanism of nucleoside-5'-diphosphate kinase action in microtubule assembly. Computer simulation studies accord with the conclusion that GDP release is rapid relative to microtubule assembly. The specific activity of the nucleoside-5'-diphosphate kinase is 1.7 nmol/min/mg of microtubular protein under the conditions studied. Pulse-chase experiments with tubulin . [14C]GDP complex and the rapidity of GDP phosphorylation by the kinase are in agreement with this scheme. Finally, it was observed that the extent and rate of microtubule assembly depends upon the [ATP]/[ADP] ratio.     

A common amino acid sequence in 190-kDa microtubule-associated protein and tau for the promotion of microtubule assembly

Previously we reported that chymotryptic fragments of bovine adrenal 190-kDa microtubule-associated proteins (27-kDa fragment) and bovine brain tau (14-kDa fragment) contained microtubule-binding domain (Aizawa, H., Murofushi, H., Kotani, Hisanaga, S., Hirokawa, N., and Sakai, H. (1987) J. Biol. Chem. 262, 3782-3787; Aizawa, H., Kawasaki, H., Murofushi, H., Kotani, S., Suzuki, K., and Sakai, H. (1988) J. Biol. Chem. 263, 7703-7707). In order to study the structure of microtubule-binding domain of the two microtubule-associated proteins, we analyzed the amino acid sequence of the 27-kDa fragment and compared the sequence with that of the 14-kDa fragment. This revealed that 190-kDa microtubule-associated protein and tau contained at least one common sequence of 20 amino acid residues in their microtubule-binding domains. A synthetic polypeptide corresponding to the common sequence (Lys-Asn-Val-Arg-Ser-Lys-Val-Gly-Ser-Thr-Glu-Asn-Ile-Lys- His-Gln-Pro-Gly-Gly-Gly-Arg-Ala-Lys) was bound to microtubules competitively with the 190-kDa MAP. The apparent dissociation constant (KD) for the binding of the polypeptide to microtubules was estimated to be 1.8 x 10(-4) M, and the maximum binding reached 1.2 mol of the synthetic polypeptide/mol of tubulin dimer. This synthetic polypeptide increased the rate and extent of tubulin polymerization and decreased the critical concentration of tubulin for polymerization. The polypeptide-induced tubulin polymers were morphologically normal microtubules and were disassembled by cold treatment. The common sequence (termed assembly-promoting sequence) was thus identified as the active site of 190-kDa microtubule-associated protein and tau for the promotion of microtubule assembly. The reconstitution system of microtubules with this synthetic polypeptide with assembly-promoting sequence may be useful to elucidate detailed molecular mechanism of the promotion of microtubule assembly by microtubule-associated proteins.     

Differential interaction of tubulin isotypes with the antimitotic compound IKP-104

The tubulin molecule is a heterodimer composed of two polypeptide chains, designated alpha and beta; both alpha and beta exist in numerous isotypic forms, which differ in their assembly and drug binding properties. 2-(4-Fluorophenyl)-1-(2-chloro-3, 5-dimethoxyphenyl)-3-methyl-6-phenyl-4(1H)-pyridinone (IKP-104) is an antimitotic compound which inhibits polymerization and induces depolymerization of microtubules [Mizuhashi, F., et al. (1992) Jpn. J. Cancer Res. 83, 211]. Since the previous work was undertaken with isotypically unfractionated tubulin, we have investigated the interactions of IKP-104 with the isotypically purified tubulin dimers (alpha beta(II), alpha beta(III), and alpha beta(IV)). We find that IKP-104 binds to alpha beta(II) and alpha beta(III) at two classes of binding sites. However, affinities for each class of site are much weaker for alpha beta(III) than for alpha beta(II). Interestingly, the low-affinity site on alpha beta(IV) was not detectable. Its high-affinity site was weaker than those of either alpha beta(II) or alpha beta(III). In a pattern consistent with these results, IKP-104 inhibited assembly better with alpha beta(II) than with the other two dimers. Higher concentrations of IKP-104 induced formation of spiral aggregates from alpha beta(II) and alpha beta(III) but not from alpha beta(IV). Our results suggest that the interaction of IKP-104 with tubulin isotypes is very complex: alpha beta(II) and alpha beta(III) differ quantitatively in their interaction with IKP-104, and alpha beta(IV)'s interaction differs both quantitatively and qualitatively from those of the other two dimers.     

Roles of colchicine rings B and C in the binding process to tubulin

The interactions of tubulin with colchicine analogues in which the tropolone methyl ether ring had been transformed into a p-carbomethoxybenzene have been characterized. The analogues were allocolchicine (ALLO) and 2,3,4-trimethoxy-4'-carbomethoxy-1,1'-biphenyl (TCB), the first being transformed colchicine and the second transformed colchicine with ring B eliminated. The binding of both analogues has been shown to be specific for the colchicine binding site on tubulin by competition with colchicine and podophyllotoxin. Both analogues bind reversibly to tubulin with the generation of ligand fluorescence. The binding of ALLO is slow, the fluorescence reaching a steady state in the same time span as colchicine; that of TCB is rapid. The displacement of ALLO by podophyllotoxin proceeds with a half-life of ca. 40 min. Binding isotherms generated from gel filtration and fluorescence measurements have shown that both analogues bind to tubulin with a stoichiometry of 1 mol of analogue/mol of alpha-beta tubulin. The equilibrium binding constants at 25 degrees C have been found to be (9.2 +/- 2.5) x 10(5) M-1 for ALLO and (1.0 +/- 0.2) X 10(5) M-1 for TCB. Binding of both analogues was accompanied by quenching of protein fluorescence, perturbation of the far-ultraviolet circular dichroism of tubulin, and induction of the tubulin GTPase activity, similarly to colchicine binding. Both inhibited microtubule assembly in vitro, ALLO substoichiometrically, and both induced the abnormal cooperative polymerization of tubulin, which is characteristic of the tubulin-colchicine complex. Analysis in terms of the simple bifunctional ligand binding mechanism developed for colchicine [Andreu, J.M., & Timasheff, S.N. (1982) Biochemistry 21, 534-543] and comparison with the binding of the colchicine two-ring analogue, 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one [Andreu, J. M., Gorbunoff, M. J., Lee, J. C., & Timasheff, S. N. (1984) Biochemistry 23, 1742-1752], have shown that transformation of the tropolone methyl ether part of colchicine into p-carbomethoxybenzene weakens the standard free energy of binding to tubulin by 1.4 +/- 0.1 kcal/mol, while elimination of ring B weakens it by 1.0 +/- 0.1 kcal/mol. The roles of rings C and B of colchicine in the thermodynamic and kinetic mechanisms of binding to tubulin were analyzed in terms of these findings.     

Identification of tubulin isoforms in the plasmodium of Physarum polycephalum by in vitro microtubule assembly

The tubulins of the plasmodium of Physarum polycephalum have been identified by in vitro microtubule assembly from partially purified extracts of asynchronous microplasmodia and late G2 macroplasmodia. The plasmodial tubulin group comprised of 2 alpha tubulins (app. m.w. 51000 daltons) and 2 beta tubulins (app. m.w. 58000 daltons and 55000 daltons) and appeared to be identical with a group of polypeptides which are synthesized periodically in late G2. Two of the plasmodial tubulin subunits (one alpha and one beta) were identical to the Physarum amoebal tubulin alpha and beta subunits as characterised by 2D gel positions.     

Increased microtubule assembly in bovine brain tubulin lacking the type III isotype of beta-tubulin

Tubulin, the major constituent protein of microtubules, is a heterodimer of alpha and beta subunits. Both alpha and beta exist in multiple isotypic forms. It is not clear if different isotypes perform different functions. In order to approach this question, we have made a monoclonal antibody specific for the beta III isotype of tubulin. This particular isotype is neuron-specific and appears to be phosphorylated near the C terminus. We have used immunoaffinity depletion chromatography to prepare tubulin lacking the beta III subunit. We find that removal of the beta III isotype results in a tubulin mixture able to assemble much more rapidly than is unfractionated tubulin when reconstituted with either of the two microtubule-associated proteins (MAPs), tau or MAP 2. Our results suggest that the different isotypes of tubulin differ from each other in their ability to polymerize into microtubules. We have also found that the anti-beta III antibody can stimulate microtubule assembly when reconstituted with tubulin and either tau or MAP 2. When reconstituted with tubulin lacking the beta III isotype, the antibody causes the tubulin to polymerize into a polymer that is a microtubule in the presence of MAP 2 and a ribbon in the presence of tau.     

Differential interaction of synthetic peptides from the carboxyl-terminal regulatory domain of tubulin with microtubule-associated proteins.

In previous studies we have demonstrated that a 4-kd tubulin fragment, including amino acid residues from Phe418 to Glu450 in alpha-subunit and Phe408-Ala445 of the beta-sequence, plays a major role in controlling tubulin interactions leading to microtubule assembly. The 4-kd carboxyl-terminal domain also constitutes an essential domain for the interaction of microtubule-associated proteins (MAPs). Removal of the 4-kd fragment facilitates tubulin self-association and renders the assembly MAP-independent. In order to define the substructure of the tubulin domain for MAP interaction, we have examined the binding of 3H-acetylated C-terminal peptides to MAP-2 and tau. Two synthetic peptides from the low-homology region within the 4-kd domain alpha (430-441) and beta (422-434) and the peptide, alpha (401-410) of the high-homology region adjacent to the 4-kd domain, were analyzed with respect to MAP interaction. The binding data showed a relatively strong interaction of MAP-2 with the beta (422-434) peptide and a weaker interaction of both MAPs components with alpha (430-441) tubulin peptide as analyzed by Airfuge ultracentrifugation and zone filtration chromatography. The homologous alpha (401-410) peptide did not bind to either MAP-2 or tau. Equilibrium dialysis experiments showed a co-operative binding of beta (422-434) peptide to multiple sites in tau. The alpha (430-441) peptide exhibited a stronger interaction for tau as compared with MAP-2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutations that encode partially functional beta 2 tubulin subunits have different effects on structurally different microtubule arrays

The testis-specific beta 2 tubulin of Drosophila is required for assembly and function of at least three architecturally different microtubule arrays (Kemphues et al., 1982). Two recessive male-sterile mutations in the B2t locus that encode partially functional, stable, variant forms of beta 2 tubulin cause defects in only certain microtubule-based processes during spermatogenesis. These mutations could thus identify aspects of beta tubulin primary structure critical for function only in specific microtubule arrays. In males carrying the B2t6 mutation, meiotic chromosome segregation and nuclear shaping are normal and flagellar axonemes are formed, but there is a subtle defect in axoneme structure; the outer doublet microtubules fill in with a central core normally seen only in the central pair and accessory microtubules. In homozygous B2t7 males, chromosome movement is usually normal during meiosis but cytokinesis often fails, cytoplasmic microtubules are assembled and nuclear shaping appears to be normal, but the flagellar axoneme lacks structural integrity. In contrast, the B2t8 allele affects a general property of tubulin, the ability to form normal side-to-side association of protofilaments (Fuller et al., 1987), and causes defects in meiosis, axoneme assembly and nuclear shaping. Certain combinations of these beta 2 tubulin mutations show interallelic complementation; in B2t6/B2t8 males functional sperm are produced and both variant subunits are incorporated into mature sperm, in the absence of wild-type beta 2 tubulin. Comparison of the phenotypes of the three partially functional beta 2 tubulin alleles reveals some aspects of tubulin primary structure more important for function in specific subsets of microtubule arrays, and other aspects required for the construction of microtubules in general.     

Interaction between chromium GTP and tubulin. Stereochemistry of GTP binding, GTP hydrolysis, and microtubule stabilization

Chromium GTP (CrGTP) has been used to probe the stereochemistry of metal-GTP binding to exchangeable site of tubulin and to examine the fate and role of nucleotide-bound metal ion in GTP hydrolysis associated with microtubule assembly. The absolute stereoconfiguration of the two pairs of diastereomers of beta,gamma-bidentate CrGTP has been determined by comparison of their visible circular dichroism spectra with those of the beta,gamma-CrATP isomers whose configurations have been established (Lin, I., and Dunaway-Mariano, D. (1988) J. Am. Chem. Soc. 110, 950-956). Tubulin binds metal-GTP preferentially in the delta pseudoaxial configuration. CrGTP-tubulin shows a high propensity to undergo tubulin-tubulin interactions with associated hydrolysis of CrGTP. Hydrolysis of CrGTP in microtubule assembly develops in two consecutive steps: cleavage of the gamma-phosphate followed by release of Pi and chromium. In contrast to other NTPases (actin, hexokinase) tubulin appears able to catalyze the dissociation of the stable chromium-phosphate bonds, which implies a highly nucleophilic environment of the binding site of the metal-triphosphate moiety of GTP. Microtubules assembled from CrGTP-tubulin are made of 90% GDP subunits, and their stability is linked to a 10% proportion of CrGDP-Pi subunits, scattered along the microtubule, from which Pi does not dissociate. The possibility is evoked that some tubulin variants do not catalyze release of Pi and metal ion efficiently, and their presence could affect microtubule dynamics.     

Linkage between ligand binding and control of tubulin conformation.

The effect of both antimitotic drugs and nucleotide analogues on the magnesium-induced self-association of purified tubulin into 42S double rings has been examined by sedimentation velocity. In the absence of magnesium, all complexes sedimented as the 5.8S species. The binding of colchicine to tubulin led to a small but consistent (-0.1 to -0.2 kcal/mol) enhancement in the self-association of tubulin alpha-beta dimers. In the absence of nucleotide at the exchangeable site, tubulin retained a weak ability (K2 = 7.5 x 10(3) M-1) to self-associate, which was unchanged by the addition of guanosine or GMP. Analogues with altered P-O-P bonds (GMPPCP, GMPPNP) did not support ring formation at the protein concentrations examined, although GMPPCP supported microtubule assembly. When the exchangeable site was occupied by nucleotides altered on the gamma-phosphate (GTP gamma S, GTP gamma F), rings were formed; tubulin-GTP gamma F formed rings to an extent slightly greater than did tubulin-GTP, and tubulin-GTP gamma S to about the same extent as tubulin-GDP. Both of these analogues are inhibitors of microtubule assembly. These results are consistent with a model [Melki, R., Carlier, M.-F., Pantaloni, D., & Timasheff, S. N. (1989) Biochemistry 28, 9143-9152] in which an equilibrium exists between straight (microtubule-forming) and curved (ring-forming) conformations of tubulin. Furthermore, the present results indicate that the "switch" which controls the nature of the final polymeric product via free energy linkages is the occupancy of the gamma-phosphate binding locus of the exchangeable site by a properly coordinated metal-nucleotide complex.     

Erythrocyte microtubule assembly in vitro. Tubulin oligomers limit the rate of microtubule self-assembly

Chicken erythrocyte tubulin containing a unique beta tubulin variant polymerizes with greater efficiency (lower critical concentration) but at a slower rate than chicken brain tubulin. In a previous study we demonstrated that the low net rate of assembly is partly due to the presence of large oligomers and rings which reduce the initial rate of subunit elongation on microtubule seeds (Murphy, D.B., and Wallis, K.T. (1985) J. Biol. Chem. 260, 12293-12301). In this study we show that erythrocyte tubulin oligomers also retard the rate of microtubule nucleation and the net rate of self-assembly. The inhibitory effect is most likely to be due to the increased stability of erythrocyte tubulin oligomers, including a novel polymer of coiled rings that forms during the rapid phase of microtubule polymerization. The slow rate of dissociation of rings and coils into dimers and small oligomers appears to limit both the nucleation and elongation steps in the self-assembly of erythrocyte microtubules.