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.     

Distinct colchicine binding kinetics of bovine brain tubulin lacking the type III isotype of beta-tubulin

In mammalian brain, beta-tubulin occurs as a mixture of four isotypes designated as types I, II, III, and IV. It has been speculated in recent years that the different tubulin isotypes may confer functional diversity to microtubules. In an effort to investigate whether different tubulin isotypes differ in their functional properties we have studied the colchicine binding kinetics of bovine brain tubulin upon removal of the beta III isotype. We found that the removal of the beta III isotype alters the binding kinetics from biphasic to monophasic with the disappearance of the slow phase. The kinetics become biphasic with the reappearance of the slow phase when the beta III-depleted tubulin was mixed with the beta III fraction eluted from the affinity column with 0.5 M NaCl. The analysis of the kinetic data reveals that the tubulin dimers containing beta III bind colchicine at an on-rate constant of 35 M-1 s-1 while those lacking beta III bind at 182 M-1 s-1. Our results strongly suggest that the beta-subunit plays a very important role in the interaction of tubulin with colchicine.     

A monoclonal antibody against the type II isotype of beta-tubulin. Preparation of isotypically altered tubulin

Mammalian brain tubulin consists of several isotypes of alpha and beta subunits that separate on polyacrylamide gels into three electrophoretic classes, designated alpha, beta 1, and beta 2. It has not been possible hitherto to resolve the different isotypes in a functional form. To this end, we have now isolated a monoclonal antibody, using as an immunogen a chemically synthesized peptide corresponding to the carboxyl-terminal sequence of the major tubulin isotype (type II) found in the beta 1-tubulin electrophoretic fraction. The antibody binds to beta 1 but not to alpha or beta 2. When pure tubulin from bovine brain is passed through an immunoaffinity column made from the anti-type II antibody, the tubulin that elutes in the unbound fraction is enriched greatly for the beta 2 electrophoretic variant. The tubulin that binds to the column appears to contain only alpha and beta 1, not beta 2. When these tubulin fractions are characterized by immunoblotting using the anti-type II antibody, the antibody binds only to the beta 1 band in the bound fraction, not to the beta 1 band in the unbound fraction. Using polyclonal antibodies generated against the carboxyl-termini of types I, III, and IV, we demonstrate that the beta 1 electrophoretic species is comprised of isotypes I, II, and IV, whereas the beta 2 variant is comprised exclusively of type III beta-tubulin. Further, we calculate that beta-tubulin in purified bovine brain tubulin is comprised of 3% type I, 58% type II, 25% type III, and 13% type IV tubulins.     

Biphasic kinetics of the colchicine-tubulin interaction: role of amino acids surrounding the A ring of bound colchicine molecule

Isotypes of vertebrate tubulin have variable amino acid sequences, which are clustered at their C-terminal ends. Isotypes bind colchicine at different on-rates and affinity constants. The kinetics of colchicine binding to purified (unfractionated) brain tubulin have been reported to be biphasic under pseudo-first-order conditions. Experiments with individual isotypes established that the presence of beta(III) in the purified tubulin is responsible for the biphasic kinetics. Because the isotypes mainly differ at the C termini, the colchicine-binding kinetics of unfractionated tubulin and the beta(III) isotype, cleaved at the C termini, have been tested under pseudo-first-order conditions. Removal of the C termini made no difference to the nature of the kinetics. Sequence alignment of different beta isotypes of tubulin showed that besides the C-terminal region, there are differences in the main body as well. To establish whether these differences lie at the colchicine-binding site or not, homology modeling of all beta-tubulin isotypes was done. We found that the isotypes differed from each other in the amino acids located near the A ring of colchicine at the colchicine-binding site on beta tubulin. While the beta(III) isotype has two hydrophilic residues (serine(242) and threonine(317)), both beta(II) and beta(IV) have two hydrophobic residues (leucine(242) and alanine(317)). beta(II) has isoleucine at position 318, while beta(III) and beta(IV) have valine at that position. Thus, these alterations in the nature of the amino acids surrounding the colchicine site could be responsible for the different colchicine-binding kinetics of the different isotypes of tubulin.     

Kinetics of colchicine binding to purified beta-tubulin isotypes from bovine brain.

Tubulin, the constituent protein of microtubules, is an alpha beta heterodimer; both alpha and beta exist in several isotypic forms whose functional significance is not precisely known. The antimitotic alkaloid colchicine binds to mammalian brain tubulin in a biphasic manner under pseudo-first-order conditions in the presence of a large excess of colchicine (Garland, D. L. (1978) Biochemistry 17, 4266-4272). We have studied the kinetics of colchicine binding to purified beta-tubulin isotypes and find that each of the purified beta-tubulin isotypes binds colchicine in a monophasic manner. The apparent on-rate constants for the binding of colchicine to alpha beta II-, alpha beta III-, and alpha beta IV-tubulin dimers are respectively 132 +/- 5, 30 +/- 2, and 236 +/- 7 M-1 s-1. When the isotypes are mixed, the kinetics become biphasic. Scatchard analysis revealed that the isotypes differ significantly in their affinity constants (Ka) for binding colchicine. The affinity constants are 0.24 x 10(6), 0.12 x 10(6), and 3.31 x 10(6) M-1, respectively, for alpha beta II-, alpha beta III-, and alpha beta IV-tubulin dimers. Our results are in agreement with the hypothesis that the beta-subunit of tubulin plays a major role in the interaction of colchicine with tubulin. Our binding data raise the possibility that the tubulin isotypes might play important regulatory roles by interacting differently with other non-tubulin proteins in vivo, which in turn, may regulate microtubule-based functions in living cells.     

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.     

Human apolipoprotein E isoprotein subclasses are genetically determined.

In a recent communication, we showed that human very low density lipoprotein (VLDL) apolipoprotein E (Apo E) from different individuals appears upon two-dimensional gel electrophoretic analysis in either one of two complex patterns. These have been designated class alpha and class beta. Mixing of VLDL from different subjects revealed that not all alpha or beta apo E patterns were the same. In this manner, we identified three subclasses of class alpha (alpha II, alpha III, and alpha IV) and three subclasses of class beta (beta II, beta III, and beta IV). We report here the results of family studies that reveal that the subclasses (alpha II, alph III, and alpha IV and beta II, beta III, and beta IV) of apo E are determined at a single genetic locus with three common alleles, epsilon II, epsilon III, and epsilon IV. The class beta phenotypes (beta II, beta III, and beta IV) represent homozygosity for two identical apo E alleles (epsilon). In contrast, class alpha phenotypes (alpha II, alpha III, and alpha IV) represent heterozygosity for two different apo E alleles. The apo E subclasses and their corresponding genotypes are as follows: beta II = epsilon II/epsilon II; beta III = epsilon III; beta IV = epsilon IV/epsilon IV; alpha II = epsilon II/epsilon III; alpha III = epsilon III/epsilon IV; and alpha IV = epsilon II/epsilon IV. To estimate the frequencies of the apo E alleles in the general population, apo E subclasses were then investigated in 61 unrelated volunteers and the results were: beta II = 1 (2%), beta III = 30 (49%), alpha II = 9 (15%, alpha III = 13 (31%), and alpha IV = 2 (3%). Utilizing the frequencies of these phenotypes, the gene frequencies were calculated to be epsilon II = 11%, epsilon III = 72%, and epsilon IV = 17%. In addition, apo E subclasses were studied in a clinic for individuals with plasma lipid disorders and the apo E subclass beta IV was found to be associated with type III hyperlipoproteinemia. There was no association of any apo E subclass with type II, type IV, or type VI hyperlipoproteinemia or plasma HDL cholesterol levels. This study explains the genetic basis for the common variation in a human plasma protein, apo E. Since the apo E subclass beta IV is associated with type III hyperlipoproteinemia, a disease characterized by xanthomatosis and premature atherosclerosis, understanding the genetic basis of the apo E subclasses should provide insight into the genetics of type III hyperlipoproteinemia.     

Translation of beta-tubulin mRNA in vitro generates multiple molecular forms

We describe the in vitro expression and characterization of the isolated beta-tubulin subunit in rabbit reticulocyte lysates and compare its assembly and chromatographic properties with that of the isolated alpha-subunit and the tubulin heterodimer. The beta-tubulin polypeptides, derived from a single chicken beta-tubulin cDNA, were found in three distinct molecular forms: a multimeric or lysate-associated form, beta I (Mr approximately 180,000); the free beta-subunit beta II (Mr approximately 55,000); and the hybrid heterodimer alpha(rabbit) beta(chick), beta III (Mr approximately 80,000-100,000). The hybrid heterodimers were 100% assembly competent, whereas beta-tubulin in the "associated" beta I and the monomeric beta II forms displayed only approximately 70 +/- 15 and 25 +/- 10% competence, respectively, in coassembly assays with bovine brain tubulin. This reduced functionality was not a consequence of diminished beta-subunit stability or protein denaturation. By comparing the elution positions of the three beta forms, the monomeric alpha-subunit, and tubulin dimer purified from bovine brain, we demonstrate that anion-exchange columns (Mono-Q) interact preferentially with the alpha-subunit and chromatograph tubulin dimer on the basis of alpha-subunit isotype. The rate of exchange of the free beta-subunit into bovine tubulin dimer was followed chromatographically. The exchange was slow at 4 degrees C and rapid at 37 degrees C where it is essentially complete in 40 min in the presence of 2.5 mg/ml bovine microtubule protein. Exogenous GTP, a potent effector of microtubule assembly, binds exchangeably to beta II and enhances the recovery of this form from the Mono-Q column, suggesting that GTP binding may occur at identical sites in the isolated beta-subunit and in the tubulin heterodimer.     

A discrete repeated sequence defines a tubulin binding domain on microtubule-associated protein tau

The protein domain responsible for the interaction of tau with tubulin has been identified. Biophysical studies indicated that the synthetic peptide Val187-Gly204 (VRSKIG-STENLKHQPGGG) from the repetitive sequence on tau binds to two sites on the tubulin heterodimer and to one site on each of the microtubule-associated protein-interacting C-terminal tubulin peptides alpha(430-441) and beta(422-434). The binding data showed a relatively stronger interaction of Val187-Gly204 with beta(422-434) as compared to that with alpha(430-441). The interaction of this tau peptide with either alpha or beta tubulin peptides appears to be associated with conformational changes in both the tau and the tubulin peptides. The beta tubulin peptide also appears to induce a structural change of tau fragment Val218-Gly235. Interestingly, tau peptides Val187-Gly204 and Val218-Gly235 induced tubulin self-assembly in a cold-reversible fashion, and incorporated into the assembled polymers. The specificity of the interaction of the tau peptide was supported by the competition of tau protein for the interaction with the tubulin polymer. In addition, the tau peptide appears to contain the principal antigenic determinant(s) recognized by anti-idiotypic antibodies that react with the tubulin binding domains on microtubule-associated proteins. The present findings together with the demonstration of the presence of multiple sites for the binding of the alpha(430-441) and beta(422-434) tubulin fragments to tau, and the existence of repetitive sequences on tau, strongly support the hypothesis that the region of tau defined by the repetitive sequences is involved in its interaction with tubulin.     

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)     

Differential sorting of beta tubulin isotypes into colchicine-stable microtubules during neuronal and muscle differentiation of embryonal carcinoma cells.

Pluripotent P19 embryonal carcinoma (EC) cells were differentiated along the neuronal and muscle pathways. Comparisons of class I, II, III, and IV beta tubulin isotypes in total and colchicine-stable microtubule (MT) arrays from uncommitted EC, neuronal, and muscle cells were made by immunoblotting and by indirect immunofluorescence microscopy. In undifferentiated EC cells the relative amounts of these four isotypes are the same in both the total and stable MT populations. Subcellular sorting of beta tubulin isotypes was demonstrated in both neuronal and muscle differentiated cells. During neuronal differentiation, class II beta tubulin is preferentially incorporated into the colchicine-stable MTs while class III beta tubulin is preferentially found in the colchicine-labile MTs. The subcellular sorting of class II into stable MTs correlates with the increased staining of MAP 1B, and with the expression of MAP 2C and tau. Although muscle differentiated cells express class II beta tubulin, stable MTs in these cells do not preferentially incorporate this isotype but instead show increased incorporation of class IV beta tubulin. Muscle cells do not show high levels of MAP 1B and do not express MAP 2C or tau. These results are consistent with the hypothesis that a subcellular sorting of tubulin isotypes is the result of a complex interaction between tubulin isotypes and MT-associated proteins.     

Proteolysis of tubulin and the substructure of the tubulin dimer

The alpha and beta subunits of tubulin each have a single highly reactive site for a variety of proteases that divides each subunit into two unequal regions. The position of cleavage is not the same for alpha and beta, since alpha is consistently cleaved into about 38- and 14-kDa pieces, while beta is cleaved into about 34- and 21-kDa pieces. The larger fragment is amino-terminal in both subunits as shown: by size reduction of the smaller fragment by subtilisin (which cleaves at the extreme carboxyl-terminal end), but no change in size of the larger fragment; by the charge/mass ratios of the proteolytic fragments; and by sequence analysis which locates trypsin cleavage after residue 339 (alpha) and chymotrypsin cleavage after residue 281 (beta). Since this cleavage pattern of the alpha and beta subunits is found for very different proteases, we suggest that it is determined by structural features of the tubulin molecule. The two pieces of each subunit remain associated following cleavage. While both cleavage sites are exposed in the free dimer, assembly of dimers into microtubules or sheets protects the internal site against cleavage. By contrast, the carboxyl-terminal subtilisin-sensitive sites remain exposed. Based on these results we propose a model for the substructure of the tubulin dimer that accommodates internal cleavage in the dimer but not the polymer, access to the COOH termini in both forms, and the orientation of the dimer in the polymer.     

Tubulin is an inherent component of mitochondrial membranes that interacts with the voltage-dependent anion channel

We have previously reported that anti-tubulin agents induce the release of cytochrome c from isolated mitochondria. In this study, we show that tubulin is present in mitochondria isolated from different human cancerous and non-cancerous cell lines. The absence of polymerized microtubules and cytosolic proteins was checked to ensure that this tubulin is an inherent component of the mitochondria. In addition, a salt wash did not release the tubulin from the mitochondria. By using electron microscopy, we then showed that tubulin is localized in the mitochondrial membranes. As compared with cellular tubulin, mitochondrial tubulin is enriched in acetylated and tyrosinated alpha-tubulin and is also enriched in the class III beta-tubulin isotype but contains very little of the class IV beta-tubulin isotype. The mitochondrial tubulin is likely to be organized in alpha/beta dimers and represents 2.2 +/- 0.5% of total cellular tubulin. Lastly, we showed by immunoprecipitation experiments that the mitochondrial tubulin is specifically associated with the voltage-dependent anion channel, the main component of the permeability transition pore. Thus, tubulin is an inherent component of mitochondrial membranes, and it could play a role in apoptosis via interaction with the permeability transition pore.     

Intracellular competition for component chains determines class II MHC cell surface phenotype

Mixed isotype (E alpha A beta and A alpha E beta) dimers are not found on Ia+ hematopoietic cells, although some pairs (e.g., E alpha A beta d) reach the membrane of transfected cells expressing only the two relevant class II genes. To examine the basis for this difference in potential versus actual Ia molecule expression, we utilized an L cell transfection model more closely resembling the normal condition of multiple class II alpha and beta chain synthesis within a single cell, such that competition among alpha and beta chains could occur. The surface expression of individual Ia dimers was compared with the available class II chains in such cells. Our data indicate that 3- to 5-fold preferences in assembly or transport of the predominant A alpha A beta and E alpha E beta species preclude expression of the mixed isotype E alpha A beta pair under physiologic conditions of balanced chain synthesis, but that asymmetric chain synthesis can lead to the expression of such mixed dimers on the cell surface in biologically significant amounts.     

Stereoselectivity of the guanyl-exchangeable nucleotide-binding site of tubulin probed by guanosine 5'-O-(2-thiotriphosphate) diastereoisomers

The active site of the exchangeable nucleotide-binding site of tubulin was studied by using diastereoisomers A (Sp) and B (Rp) of guanosine 5'-O-(2-thiotriphosphate) (GTP beta S) where the phosphorus atom to which sulfur is attached is chiral. Turbidimetric measurements were used to follow kinetics, and electron microscopy was used to evaluate polymeric forms. Both isomers at 0.5 mM promoted the assembly of tubulin in buffer containing 0.1 M 2-(N-morpholino)ethanesulfonic acid, 30% glycerol, 3 mM MgCl2, and 1 mM EGTA, pH 6.6, 23-37 degrees C. GTP beta S(A) promoted assembly into microtubules, although a few bundles were also found by electron microscopy. However, GTP beta S(B) induced assembly of tubulin into bundles of sheets and microtubules. As expected, 0.5 mM GTP induced tubulin to assemble into microtubules, thin sheets, and a few bundles. Both GTP and GTP beta S(A) were hydrolyzed in the tubulin polymers. However, more than 95% of the bound GTP beta S(B) was not hydrolyzed. Higher concentrations of GTP beta S(B), i.e., 1 mM, also induced bundles of sheets and microtubules, with 86% of the thionucleotide bound as the triphosphate. The GTP beta S(B)-induced polymers were considerably more cold stable than the GTB beta S(A)-induced microtubules, which were more cold stable than GTP-induced polymers. Mg(II) (2-5 mM) had minimal effects on the structures induced by GTP beta S(A) or -(B) isomers in the tubulin assembly system. However, at 1 mM Mg(II), no assembly was found with GTP beta S(A) and tubulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Loss of assembly of the main basement membrane collagen, type IV, but not fibril-forming collagens and embryonic death in collagen prolyl 4-hydroxylase I null mice

Collagen prolyl 4-hydroxylases (C-P4Hs) catalyze the formation of the 4-hydroxyproline residues that are essential for the generation of triple helical collagen molecules. The vertebrate C-P4Hs I, II, and III are [alpha(I)]2beta2, [alpha(II)]2beta2, and [alpha(III)]2beta2 tetramers with identical beta subunits. We generated mice with targeted inactivation of the P4ha1 gene encoding the catalytic alpha subunit of C-P4H I to analyze its specific functions. The null mice died after E10.5, showing an overall developmental delay and a dilated endoplasmic reticulum in their cells. The capillary walls were frequently ruptured, but the capillary density remained unchanged. The C-P4H activity level in the null embryos and fibroblasts cultured from them was 20% of that in the wild type, being evidently due to the other two isoenzymes. Collagen IV immunofluorescence was almost absent in the basement membranes of the null embryos, and electron microscopy revealed disrupted basement membranes, while immunoelectron microscopy showed a lack of collagen IV in them. The amount of soluble collagen IV was increased in the null embryos and cultured null fibroblasts, indicating a lack of assembly of collagen IV molecules into insoluble structures, probably due to their underhydroxylation and hence abnormal conformation. In contrast, the null embryos had collagen I and III fibrils with a typical cross-striation pattern but slightly increased diameters, and the null fibroblasts secreted fibril-forming collagens, although less efficiently than wild-type cells. The primary cause of death of the null embryos was thus most likely an abnormal assembly of collagen IV.     

Structural model of a complex between the heterotrimeric G protein, Gsalpha, and tubulin

A number of studies have demonstrated interplay between the cytoskeleton and G protein signaling. Many of these studies have determined a specific interaction between tubulin, the building block of microtubules, and G proteins. The alpha subunits of some heterotrimeric G proteins, including Gsalpha, have been shown to interact strongly with tubulin. Binding of Galpha to tubulin results in increased dynamicity of microtubules due to activation of GTPase of tubulin. Tubulin also activates Gsalpha via a direct transfer of GTP between these molecules. Structural insight into the interaction between tubulin and Gsalpha was required, and was determined, in this report, through biochemical and molecular docking techniques. Solid phase peptide arrays suggested that a portion of the amino terminus, alpha2-beta4 (the region between switch II and switch III) and alpha3-beta5 (just distal to the switch III region) domains of Gsalpha are important for interaction with tubulin. Molecular docking studies revealed the best-fit models based on the biochemical data, showing an interface between the two molecules that includes the adenylyl cyclase/Gbetagamma interaction regions of Gsalpha and the exchangeable nucleotide-binding site of tubulin. These structural models explain the ability of tubulin to facilitate GTP exchange on Galpha and the ability of Galpha to activate tubulin GTPase.     

Baccatin III induces assembly of purified tubulin into long microtubules

Baccatin III is widely considered to be an inactive derivative of Taxol. We have reexamined its effect on in vitro assembly of tubulin under a variety of conditions. We found baccatin III to be active in all circumstances in which Taxol is active: it assembled GTP-tubulin, GDP-tubulin, and microtubule protein into normal microtubules and stabilized these polymers against cold-induced disassembly. The effect of baccatin III on in vitro microtubule assembly was quantitatively assessed through determination of critical concentrations, which can be used to obtain the apparent equilibrium constants for the addition of tubulin subunits to growing microtubules. The apparent equilibrium constants for the growth reaction for baccatin III-induced GTP-tubulin and GDP-tubulin assembly measured at 37 degrees C were 4.2-4.6-fold less than those measured for Taxol-induced GTP-tubulin and GDP-tubulin assembly. These data indicate that the entire Taxol side chain contributes only about -1 kcal/mol to the apparent standard free energy of microtubule growth at 37 degrees C regardless of the nature of the E site nucleotide. These data also support the idea that the majority of the interactions between Taxol and tubulin that affect this equilibrium occur between the baccatin portion of the molecule and the binding site. We have also observed a structural difference in microtubules formed using baccatin III and Taxol. Baccatin III-induced microtubules were routinely much longer than those assembled by Taxol, even when very high concentrations of baccatin III were employed. One interpretation of these data is that baccatin III and Taxol differ in their abilities to nucleate GTP-tubulin. This difference in activity may have bearing on the large disparity in cytotoxicity of the two molecules.     

Arabidopsis tubulin folding cofactor B interacts with alpha-tubulin in vivo

Microtubule biogenesis requires alphabeta tubulin dimers that are generated from alpha and beta tubulin following post-translational modification by several tubulin folding cofactors (TFCs). Here we report the isolation and characterization of Arabidopsis TFCB (AtTFCB). AtTFCB is expressed in all organs of Arabidopsis. The subcellular localization of AtTFCB is mainly cytosolic. AtTFCB-overexpressing cells have fewer microtubules compared with the controls. Multimode fluorescence resonance energy transfer (FRET) microscopy reveals a direct physical interaction of AtTFCB with alpha tubulin in living plant cells. We conclude that AtTFCB interacts with alpha tubulin in vivo and its overexpression reduces the number of microtubules.     

G beta gamma mediates the interplay between tubulin dimers and microtubules in the modulation of Gq signaling

Agonist stimulation causes tubulin association with the plasma membrane and activation of PLC beta 1 through direct interaction with, and transactivation of, G alpha q. Here we demonstrate that G beta gamma interaction with tubulin down-regulates this signaling pathway. Purified G beta gamma, alone or with phosphatidylinositol 4,5-bisphosphate (PIP2), inhibited carbachol-evoked membrane recruitment of tubulin and G alpha q transactivation by tubulin. Polymerization of microtubules elicited by G beta gamma overrode tubulin translocation to the membrane in response to carbachol stimulation. G beta gamma sequestration of tubulin reduced the inhibition of PLC beta 1 observed at high tubulin concentration. G beta 1 gamma 2 interacted preferentially with tubulin-GDP, whereas G alpha q was transactivated by tubulin-GTP. Prenylation of the gamma 2 polypeptide was required for G beta gamma/tubulin interaction. Both confocal microscopy and coimmunoprecipitation studies revealed the spatiotemporal pattern of G beta gamma/tubulin interaction during carbachol stimulation of neuroblastoma SK-N-SH cells. In resting cells G beta gamma localized predominantly at the cell membrane, whereas tubulin was found in well defined microtubules in the cytosol. Within 2 min of agonist exposure, a subset of tubulin translocated to the plasma membrane and colocalized with G beta. Fifteen min post-carbachol addition, tubulin and G beta colocalized in vesicle-like structures in the cytosol. G beta/tubulin colocalization increased after pretreatment of cells with the microtubule-depolymerizing agent, colchicine, and was inhibited by taxol. Taxol also inhibited carbachol-induced PIP2 hydrolysis. It is suggested that G beta gamma/tubulin interaction mediates internalization of membrane-associated tubulin at the offset of PLC beta 1 signaling. Newly cytosolic G beta gamma/tubulin complexes might promote microtubule polymerization attenuating further tubulin association with the plasma membrane. Thus G protein-coupled receptors might evoke G alpha and G beta gamma to orchestrate regulation of phospholipase signaling by tubulin dimers and control of cell shape by microtubules.