Characterization and in vitro polymerization of Tetrahymena tubulin

Tetrahymena tubulin was purified from the cell extract using DEAE-Sephadex A-50 ion-exchanger and ammonium sulfate precipitation. About 2.2% of the total protein in the 20,000 X g supernatant was recovered as DEAE-Sephadex-purified tubulin fraction. Applying the temperature-dependent polymerization-depolymerization method to this fraction in the presence of Tetrahymena outer fibers as a seed, almost pure tubulin was obtained. Tetrahymena tubulin dimer showed different behavior on SDS-polyacrylamide gels from porcine brain tubulin, and showed very low affinity for colchicine, amounting to about one-twentieth of the binding to porcine brain tubulin. The tubulin fraction failed to polymerize into microtubules by itself. Addition of a small amount of the ciliary outer fiber fragment induced polymerization as demonstrated by viscometric measurements, but the reconstituted microtubules were very unstable in the absence of glycerol. Microtubule-depolymerizing agents such as Ca2+ ions, low temperature, or colchicine all inhibited in vitro polymerization. Although Tetrahymena tubulin purified by the polymerization-depolymerization method could copolymerize with porcine brain microtubules, the DEAE-Sephadex-purified tubulin fraction suppressed the initial rate of porcine brain microtubule assembly in vitro. There seemed to be no differences between cytoplasmic tubulin and outer fiber tubulin in colchicine binding activity or SDS-gel electrophoretic behavior, or between the fine structure of both reconstituted microtubules observed by electron microscopy.     

A porcine brain protein (35 K protein) which bundles microtubules and its identification as glyceraldehyde 3-phosphate dehydrogenase

A protein which binds to both tubulin and tubulin polymer was isolated from porcine brains. This protein has a molecular weight of 35,000 on SDS-polyacrylamide gel electrophoresis (designated as 35 K protein). The 35 K protein was purified through several steps of purification including ammonium sulfate fractionation, Sephadex G-100 gel filtration column chromatography, microtubule protein-agarose gel affinity column chromatography and phosphocellulose column chromatography. The 35 K protein caused pronounced enhancement of the turbidity increase produced by tubulin polymerization in the presence of DMSO, but did not have the ability to initiate polymerization of pure tubulin in the absence of DMSO. It was demonstrated that 35 K protein co-sediments with tubulin polymer in a concentration-dependent manner. Electron microscopic observation revealed the formation of bundles of tubulin polymer. Since the effect of 35 K protein was coupled with tubulin polymerization, 35 K protein did not cause the turbidity increase under conditions where tubulin polymerization was inhibited by Ca2+ or colchicine. The 35 K protein adsorbed on tubulin-Sepharose 4B was eluted by the addition of 2 mM ATP. ATP was shown to inhibit the interaction of 35 K protein with tubulin dimer or polymer. The 35 K protein was finally identified as glyceraldehyde 3-phosphate dehydrogenase from properties such as mobility on SDS-polyacrylamide gel electrophoresis, cleavage pattern on limited proteolysis, ability to bind to tubulin, and so on.     

Resistance of Rosa microtubule polymerization to colchicine results from a low-affinity interaction of colchicine and tubulin

The inhibition of the polymerization of tubulin from cultured cells of rose (Rosa. sp. cv. Paul's scarlet) by colchicine and the binding of colchicine to tubulin were examined in vitro and compared with data obtained in parallel experiments with bovine brain tubulin. Turbidimetric measurements of taxol-induced polymerization of rose microtubules were found to be sensitive and semiquantitative at low tubulin concentrations, and to conform to some of the characteristics of a nucleation and condensation-polymerization mechanism for assembly of filamentous helical polymers. Colchicine inhibited the rapid phase of polymerization at 24°C with an apparent inhibition constant (K _i) of 1.4·10^-4 M for rose tubulin and an apparent K _i=8.8·10^-7 M for brain tubulin. The binding of [³H]colchicine to rose tubulin to form tubulin-colchicine complex was mildly temperature-dependent and slow, taking 2–3 h to reach equilibrium at 24°C, and was not affected by vinblastine sulfate. The binding of [³H]colchicine to rose tubulin was saturable and Scatchard analysis indicated a single class of low-affinity binding sites having an apparent affinity constant (K) of 9.7·10² M^-1 and an estimated molar binding stoichiometry (r) of 0.47 at 24°C. The values for brain tubulin were K=2.46·10⁶ M^-1 and r=0.45 at 37°C. The binding of [³H]colchicine to rose tubulin was inhibited by excess unlabeled colchicine, but not by podophyllotoxin or tropolone. The data demonstrate divergence of the colchicine-binding sites on plant and animal tubulins and indicate that the relative resistance of plant microtubule polymerization to colchicine results from a low-affinity interaction of colchicine and tubulin.Abbreviations MT microtubule - TC tubulin-colchicine complex     

Crystal Structures of Tubulin Acetyltransferase Reveal a Conserved Catalytic Core and the Plasticity of the Essential N Terminus

Tubulin acetyltransferase (TAT) acetylates Lys-40 of α-tubulin in the microtubule lumen. TAT is inefficient, and its activity is enhanced when tubulin is incorporated in microtubules. Acetylation is associated with stable microtubules and regulates the binding of microtubule motors and associated proteins. TAT is important in neuronal polarity and mechanosensation, and decreased tubulin acetylation levels are associated with axonal transport defects and neurodegeneration. We present the first structure of TAT in complex with acetyl-CoA (Ac-CoA) at 2.7 Å resolution. The structure reveals a conserved stable catalytic core shared with other GCN5 superfamily acetyltransferases consisting of a central β-sheet flanked by α-helices and a C-terminal β-hairpin unique to TAT. Structure-guided mutagenesis establishes the molecular determinants for Ac-CoA and tubulin substrate recognition. The wild-type TAT construct is a monomer in solution. We identify a metastable interface between the conserved core and N-terminal domain that modulates the oligomerization of TAT in solution and is essential for activity. The 2.45 Å resolution structure of an inactive TAT construct with an active site point mutation near this interface reveals a domain-swapped dimer in which the functionally essential N terminus shows evidence of marked structural plasticity. The sequence segment corresponding to this structurally plastic region in TAT has been implicated in substrate recognition in other GCN5 superfamily acetyltransferases. Our structures provide a rational platform for the mechanistic dissection of TAT activity and the design of TAT inhibitors with therapeutic potential in neuronal regeneration.     

Autopalmitoylation of tubulin

Pure rat brain tubulin is readily palmitoylated in vitro using [3H]palmitoyl CoA but no added enzymes. A maximum of approximately six palmitic acids are added per dimer in 2-3 h at 36-37 degrees C under native conditions. Both alpha and beta tubulin are labeled, and 63-73% of the label was hydroxylamine-labile, presumed thioesters. Labeling increases with increasing pH and temperature, and with low concentrations of guanidine HCl or KCl (but not with urea) to a maximum of approximately 13 palmitates/dimer. High SDS and guanidine HCl concentrations are inhibitory. At no time could all 20 cysteine residues of the dimer be palmitoylated. Polymerization to microtubules, or use of tubulin S, markedly decreases the accessibility of the palmitoylation sites. Palmitoylation increases the electrophoretic mobility of a portion of alpha tubulin toward the beta band. Palmitoylated tubulin binds a colchicine analogue normally, but during three warm/cold polymerization/depolymerization cycles there is a progressive loss of palmitoylated tubulin, indicating decreased polymerization competence. We postulate that local electrostatic factors are major regulators of reactivity of tubulin cysteine residues toward palmitoyl CoA, and that the negative charges surrounding a number of the cysteines are sensitive to negative charges on palmitoyl CoA.     

Microtubules and protein secretion in rat lacrimal glands. Inhibitory effect of the tubulin . colchicine complex isolated from lacrimal glands upon brain tubulin polymerization. Identification of the complex by gel electrophoresis.

The specific inhibitory effect of colchicine upon protein secretion by lacrimal glands could be related to the formation of a complex between colchicine and tubulin from the soluble fraction of the gland. By gel electrophoresis under nondissociating conditions, it is shown that this complex is similar to the colchicine . tubulin complex from brain. The complex isolated from lacrimal glands is highly inhibitory upon brain tubulin assembly since as low as 0.07 microM complex impedes the polymerization of 8 microM tubulin by 50%, compared to 3 microM for free colchicine. Therefore, a small percentage of complexed tubulin (0.9%) is enough for polymerization to be blocked. In lacrimal glands the complex might prevent the polymerization of tubulin, and colchicine shift the tubulin in equilibrium microtubules equilibrium to microtubules disassembly. The disorganization of the labile microtubular system could lead to a modification of the transport of the secretory granules and to a perturbation of secretion.     

Ionic and nucleotide requirements for microtubule polymerization in vitro.

The ionic and nucleotide requirements for the in vitro polymerization of microtubules from purified brain tubulin have been characterized by viscometry. Protein was purified by successive cycles of a temperature dependent assembly-diassembly scheme. Maximal polymerization occurred at a concentration of 0.1 M Pipes (piperazine-N,N'-bis(2-ethanesulfonic acid)); increasing ionic strength by addition of NaCl to samples prepared in lower buffer concentrations did not result in an equivalent level of polymerization. Both Na-+ and K-+ inhibited microtubule formation at levels greater than 240 mM, withmaximal assembly occurring at physiological concentrations of 150 mM. Maximal extent of assembly occurred at pH 6.8 and optimal rate at pH 6.6. Inhibition of polymerization was half-maximal at added calcium concentrations of 1.0 mM and magnesium concentrations of 10.0 mM. EGTA (ethylene glycol bis(beta-aminoethyl ether)tetraacetic acid), which chelates Ca-2+, had no effect on polymerization over a concentration range of 0.01-10.0 mM. In contrast, EDTA (ethylenediaminetetraacetic acid), which chelates both Mg-2+ and Ca-2+, inhibited assemble half-maximally at 0.25 mM and totally at 2.0 mM. As determined from experiments using Mg-2+-EDTA buffers, magnesium was required for polymerization. Magnesium promoted the maximal extent of assembly at substoichiometric levels relative to tubulin, but was maximal for both rate and extent at stoichiometric concentrations. Elemental analyses indicated that approximately 1 mol of magnesium was tightly bound/mol of tubulin dimer. Viscosity development was dependent upon hydrolyzable nucleoside triphosphate, and stoichiometric levels of GTP were sufficient for maximal polymerization. The effect of magnesium in increasing the rate of GTP-dependent polymerization suggests that a Mg-2+-GTP complex is the substrate required for a step in assembly.     

Kinetic analysis of tubulin assembly in the presence of the microtubule-associated protein TOGp

The microtubule-associated protein TOGp, which belongs to a widely distributed protein family from yeasts to humans, is highly expressed in human tumors and brain tissue. From purified components we have determined the effect of TOGp on thermally induced tubulin association in vitro in the presence of 1 mm GTP and 3.4 m glycerol. Physicochemical parameters describing the mechanism of tubulin polymerization were deduced from the kinetic curves by application of the classical theoretical models of tubulin assembly. We have calculated from the polymerization time curves a range of parameters characteristic of nucleation, elongation, or steady state phase. In addition, the tubulin subunits turnover at microtubule ends was deduced from tubulin GTPase activity. For comparison, parallel experiments were conducted with colchicine and taxol, two drugs active on microtubules and with tau, a structural microtubule-associated protein from brain tissue. TOGp, which decreases the nucleus size and the tenth time of the reaction (the time required to produce 10% of the final amount of polymer), shortens the nucleation phase of microtubule assembly. In addition, TOGp favors microtubule formation by increasing the apparent first order rate constant of elongation. Moreover, TOGp increases the total amount of polymer by decreasing the tubulin critical concentration and by inhibiting depolymerization during the steady state of the reaction.     

Polymerization of tubulin in the presence of colchicine or podophyllotoxin. Formation of a ribbon structure induced by guanylyl-5'-methylene diphosphonate

GTP-dependent in vitro polymerization of rat brain microtubular protein is inhibited to 50% by substoichiometric concentrations of the antimitotic drugs colchicine (0.12 mol/mol of tubulin) and podophyllotoxin (0.14 mol/mol of tubulin). Substitution of pp(CH₂)pG² for GTP, however, results in an extensive microtubular protein polymerization at such concentrations. In the presence of pp(CH₂)pG, suprastoichiometric concentrations of podophyllotoxin (19 mol/mol of tubulin) are required to inhibit the polymerization process by 50%. Colchicine is very ineffective since 3 × 10⁵ moles/mole of tubulin are required to give a 50% inhibition. Electron microscopical analysis shows that the polymers formed by microtubular protein in the presence of suprastoichiometric concentrations of drugs are not the normal short microtubules typical of pp(CH₂)pG-driven polymerization, but are ribbons with three or four protofilaments. The colchicine content of the harvested ribbons has been measured directly and found to be approximately 0.8 moles colchicine/mole of tubulin. Treatment of microtubular protein with substoichiometric concentrations of drugs results in an increase in the number of protofilaments forming the ribbons. Many of the ribbons can close into morphologically normal microtubules when microtubular protein is treated with only 0.05 moles of either colchicine or podophyllotoxin per mole of tubulin.     

Initiation of brain tubulin assembly by a high molecular weight flagellar protein factor

A protein factor found within the flagella of Chlamydomonas and sea urchin sperm is capable of stimulating the initiation of calf and chick brain tubulin dimer assembly in vitro.     

Inhibition of tubulin assembly by antileprosy drug dapsone

The effect of dapsone on assembly-disassembly process of bovine brain tubulin was examined. The drug was found to readily bind tubulin dimer and that in its presence colchicine binding to tubulin was enhanced. Although dapsone associated with tubulin at a site other than the colchicine binding site, distinct inhibition of microtubule assembly was detected.     

Assembly of GMPCPP-Bound Tubulin into Helical Ribbons and Tubes and Effect of Colchicine

Microtubule assembly and disassembly is a complex structural process that doesnot proceed by simple addition and subtraction of individual subunits to and from ahelical polymer, as would be the case for actin and other helical assemblies. Thedynamic process of microtubule growth and shrinking involves short-lasting polymerforms that differ substantially from the microtubule itself and constitute crucial assemblyand disassembly intermediates. Structural characterization thus depends on thestabilization of these brief intermediates and their preservation as polymericassemblies. This paper gives experimental details on the polymerization of GMPCPPtubulininto low-temperature stable polymers that we propose to correspond to the earlystages in microtubule assembly and includes new data on the effect of colchicine onGMPCPP-tubulin polymerization. Finally, we include our thoughts on the possiblebiological meaning of tubulin polymerization versatility.     

Isolation and characterization of a unique 15 kilodalton trypanosome subpellicular microtubule-associated protein.

A protein of 15 kDa (p15) was isolated from Trypanosoma brucei subpellicular microtubules by tubulin affinity chromatography. The protein bound tubulin specifically both in its native form and after SDS-PAGE in tubulin overlay experiments. p15 promoted both the in vitro polymerization of purified calf brain tubulin and the bundling of preformed mammalian microtubules. Immunolabeling identified p15 at multiple sites along microtubule polymers comprising calf brain tubulin and p15 as well as on the subpellicular microtubules of cryosectioned trypanosomes. Antibodies directed against p15 did not cross react with mammalian microtubules. It is suggested that p15 is a trypanosome-specific microtubule-associated protein (MAP) that contributes to the unique organization of the subpellicular microtubules.     

In vitro polymerization of flagellar and ciliary outer fiber tubulin into microtubules.

About 10--20% of the total protein in the outer fiber fraction was solubilized by sonication in a solution containing 5 mM MES, 0.5 mM MgSO4, 1.0 mM EGTA, 1.0 mM GTP, and 0 or 50 mM KC1 at pH 6.7. The sonicated extract was shown by analytical centrifugation to consist largely of a 6 S component (tubulin dimer), having a molecular weight of 103,000, as determined by gel filtration, and possessing a colchicine-binding activity of 0.8 mole per tubulin dimer. The tubulin fraction failed to polymerize into microtubules by itself. Addition of a small amount of the ciliary outer fiber fragments or reconstituted short brain microtubules, however, induced polymerization, as demonstrated by viscosity of flow birefringence changes as well as light or electron microscopic observations. The growth of heterogeneous microtubules upon mixing outer fiber tubulin with DEAE-dextran-decorated brain microtubules was observed by electron microscopy. Microtubules were reconstituted from outer fiber tubulin without addition of any nuclei fraction when a concentrated tubulin fraction was warmed at 35degree. A few doublet-like microtubules or pairs of parallel singlet microtubules that were closely aligned longitudinally could be observed among many singlet microtubules. Unlike other fiber microtubules, the reconstituted polymers were depolymerized by exposure to Ca2+ ions, high or low ionic strength, colchicine, low temperature or SH reagents. No microtubules were assembled under these conditions.     

Inhibition of tubulin self-assembly and tubulin-colchicine GTPase activity by guanosine 5'-(gamma-fluorotriphosphate)

The inhibitory effects of guanosine 5'-(gamma-fluorotriphosphate) [GTP(gamma F)] on both the polymerization and the colchicine-dependent GTPase activity of calf brain tubulin have been studied. The results demonstrate that this analogue of GTP, with a fluorine atom on the gamma-phosphate, is a reversible competitive dead-end inhibitor of the colchicine-induced GTPase activity with a K1 value of (1.8 +/- 0.6) X 10(-4) M. GTP(gamma F) did not promote assembly of tubulin from which the E-site guanine nucleotide had been removed. It binds to the exchangeable nucleotide site competitively with respect to GTP, diminishing both the rate and extent of tubulin polymerization. Treatment in terms of the Oosawa-Kasai model of the inhibitory effect of GTP(gamma F) on the assembly led to a value of Kdis = 1.1 X 10(-6) M for the complex GTP(gamma F)-tubulin. This analogue does not bind to the postulated third site. The growing of tubulin polymers at 37 degrees C was arrested by GTP(gamma F), and only limited depolymerization was induced by the addition of this analogue after assembly in the presence of GTP. This result confirms that the E-site is blocked in the polymer and that this analogue can bind only to the ends of the polymers. Sedimentation velocity and circular dichroism studies showed that the conformation of the tubulin-GTP(gamma F) complex is not identical with that of tubulin-GTP. This is caused by the replacement of the hydroxyl group in the gamma-phosphate by the fluorine group, which have 2.20- and 1.35-A van der Waals radii, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microinjection of fluorescent tubulin into dividing sea urchin cells

To follow the dynamics of microtubule (MT) assembly and disassembly during mitosis in living cells, tubulin has been covalently modified with the fluorochrome 5-(4,6-dichlorotriazin-2-yl)aminofluorescein and microinjected into fertilized eggs of the sea urchin Lytechinus variegatus. The changing distribution of the fluorescent protein probe is visualized in a fluorescence microscope coupled to an image intensification video system. Cells that have been injected with fluorescent tubulin show fluorescent linear polymers that assemble very rapidly and radiate from the spindle poles, coincident with the position of the astral fibers. No fluorescent polymer is apparent in other areas of the cytoplasm. When fluorescent tubulin is injected near the completion of anaphase, little incorporation of fluorescent tubulin into polymer is apparent, suggesting that new polymerization does not occur past a critical point in anaphase. These results demonstrate that MT polymerization is very rapid in vivo and that the assembly is both temporally and spatially regulated within the injected cells. Furthermore, the microinjected tubulin is stable within the sea urchin cytoplasm for at least 1 h since it can be reutilized in successive daughter cell spindles. Control experiments indicate that the observed fluorescence is dependent on MT assembly. The fluorescence is greatly diminished upon treatment of the cells with cold or colchicine agents known to cause the depolymerization of assembled MT. In addition, cells injected with fluorescent bovine serum albumin or assembly-incompetent fluorescent tubulin do not exhibit fluorescence localized in the spindle but rather appear diffusely fluorescent throughout the cytoplasm.     

Linkages between the effects of taxol, colchicine, and GTP on tubulin polymerization

A comparative study has been carried out of the effects of taxol on the polymerizations into microtubules of microtubule-associated protein-free tubulin, prepared by the modified Weisenberg procedure, and of the tubulin-colchicine complex into large aggregates. Taxol enhances, to a much greater extent, the stability of microtubules than that of the tubulin-colchicine polymers so that, with highly purified tubulin, assembly into microtubules takes place at 10 degrees C, even in the absence of exogenous GTP. The polymerization of tubulin-colchicine requires both heat and GTP, and the process is reversed by cooling. These results indicate that in both systems polymerization is linked to interactions with taxol and GTP, the interplay of linkage free energies imparting the observed polymer stabilities. In the case of microtubule formation, the linkage free energy provided by taxol binding is approximately -3.0 kcal/mol of alpha-beta-tubulin dimer, whereas this quantity is reduced to approximately -0.5 kcal/mol in tubulin-colchicine, indicating the expenditure of much more binding free energy in the latter case for overcoming unfavorable factors, such as steric hindrance and geometric strain. The difference in the effect of GTP on the two polymerization processes reflects the respective abilities of the bindings of taxol to the two states of tubulin to overcome the loss of the linkage free energy of GTP binding. Analysis of the linkages leads to the conclusions that taxol need not change qualitatively the mechanism of microtubule assembly and that tubulin with the E-site unoccupied by nucleotide should have the capacity to form microtubules, the reaction being extremely weak.     

Aging of tubulin at neutral pH: stabilization by colchicine and its analogues.

The effect of colchicine and its analogues, allocolchicine, 2,3,4-trimethoxy-4'-carbomethoxy-1,1'biphenyl, 2,3,4,4'-tetramethoxy-1,1'-biphenyl, 2,3,4-trimethoxy-4'-acetyl-1,1'-biphenyl, and tropolone methyl ether, on the aging process of tubulin has been examined. In contrast to the vinca alkaloid drugs which accelerate the formation of the paucidisperse 9 S polymers by a factor of 3.5, the colchicine class of ligands stabilize alpha,beta-tubulin. Less than 10% of the protein is transformed into the aggregates after 50 h of incubation in the presence of 1 x 10(-3) M colchicine, as compared to nearly 70-75% transformation in its absence. These results are supported by fluorescence examination of the retention of colchicine binding ability, as well as circular dichroism spectroscopy. In the presence of colchicine, the rate determining step is a conformational change, just as in its absence. The colchicine analogues which bind to tubulin in a rapidly reversible equilibrium were almost as effective in tubulin stabilization. Addition of vincristine to the system reduced the stability of the tubulin-colchicine complex. Furthermore, vincristine was found to have the same effects on the fresh complex as it does on pure tubulin; i.e., it induced the isodesmic linear polymerization and inhibited assembly into the microtubule-mimicking large polymers. This inhibition, however, was stoichiometric, whereas it is substoichiometric in the case of microtubules.     

Non-hydrolysable GTP-gamma-S stabilizes the FtsZ polymer in a GDP-bound state

FtsZ, a tubulin homologue, forms a cytokinetic ring at the site of cell division in prokaryotes. The ring is thought to consist of polymers that assemble in a strictly GTP-dependent way. GTP, but not guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S), has been shown to induce polymerization of FtsZ, whereas in vitro Ca2+ is known to inhibit the GTP hydrolysis activity of FtsZ. We have studied FtsZ dynamics at limiting GTP concentrations in the presence of 10 mM Ca2+. GTP and its non-hydrolysable analogue GTP-gamma-S bind FtsZ with similar affinity, whereas the non-hydrolysable analogue guanylyl-imidodiphosphate (GMP-PNP) is a poor substrate. Preformed FtsZ polymers can be stabilized by GTP-gamma-S and are destabilized by GDP. As more than 95% of the nucleotide associated with the FtsZ polymer is in the GDP form, it is concluded that GTP hydrolysis by itself does not trigger FtsZ polymer disassembly. Strikingly, GTP-gamma-S exchanges only a small portion of the FtsZ polymer-bound GDP. These data suggest that FtsZ polymers are stabilized by a small fraction of GTP-containing FtsZ subunits. These subunits may be located either throughout the polymer or at the polymer ends, forming a GTP cap similar to tubulin.     

B-ring of colchicine and its role in taxol-induced tubulin polymerization

Taxol-induced assembly of purified tubulin is not inhibited by the colchicine analogue 2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone. Colchicine analogues having intact A, C and B-rings (without NH-CO-CH3) such as desacetamidocolchicine have also been found to be inactive. It has been observed that these two colchicine analogues are incorporated into polymers when incubated in the presence of taxol. Furthermore, preformed taxol-induced polymers of tubulin have been found to bind these two colchicine analogues. These results suggest that colchicine-binding domains on the tubulin molecule are mostly (if not completely) exposed in the taxol-induced polymers.