Regulation of tubulin and actin synthesis and accumulation during Blastocladiella emersonii development

Actin and alpha and beta-tubulin have been identified in Blastocladiella emersonii by two-dimensional gel electrophoresis and Western blotting. The kinetics of synthesis of these proteins were compared by pulse-labeling experiments with [35S]methionine and with the accumulation of their corresponding mRNAs, translated in a cell-free system. Large increases occur in the rates of actin and alpha- and beta-tubulin biosynthesis during sporulation and there is an accumulation of the corresponding mRNAs. In parallel to the increased synthesis, these cytoskeletal proteins accumulate during the late stage of sporulation.     

Gamma-tubulin: the microtubule organizer?

Microtubules are composed predominantly of two related proteins: alpha- and beta-tubulin. These proteins form the tubulin heterodimer, which is the basic building block of microtubules. Surprisingly, recent molecular genetic studies have revealed the existence of gamma-tubulin, a new member of the tubulin family. Like alpha- and beta-tubulin, gamma-tubulin is essential for microtubule function but, unlike alpha- and beta-tubulin, it is not a component of microtubules. Rather, it is located at microtubule-organizing centres and may function in the nucleation of microtubule assembly and establishment of microtubule polarity.     

Multiple alpha- and beta-tubulin genes in Chlamydomonas and regulation of tubulin mRNA levels after deflagellation

We constructed and characterized recombinant cDNA clones containing alpha- and beta-tubulin DNA sequences. The inserted DNA was determined to code for alpha- and beta-tubulin by positive hybridization-selection. The selected mRNA was translated in vitro, and the translation products were shown to be alpha- or beta-tubulin by comigration with flagellar alpha- and beta-tubulin on one- and two-dimensional gels and by immunoprecipitation with antibodies specific for alpha- and beta-tubulin. Hybridization of the cloned tubulin probes with Chlamydomonas DNA indicated that there are at least two genes each for alpha- and beta-tubulin in this organism. No evidence of cross-hybridization between alpha- and beta-tubulin DNA sequences was found. Because previous experiments had shown that tubulin synthesis was stimulated in response to flagellar amputation, the tubulin clones were used to analyze the levels of tubulin sequences in RNA from cells before and after deflagellation. Hybridization of the tubulin cDNA probes with total or polyadenylated RNA indicated that tubulin sequences in RNA increased within 8 min following deflagellation, reached maximal levels by 50 min and began to decrease by 80 min after deflagellation. One hybridization band was detected with use of the beta-tubulin probe, but RNA in two size classes hybridized to the alpha-tubulin probe.     

Review: postchaperonin tubulin folding cofactors and their role in microtubule dynamics

The microtubule cytoskeleton consists of a highly organized network of microtubule polymers bound to their accessory proteins: microtubule-associated proteins, molecular motors, and microtubule-organizing proteins. The microtubule subunits are heterodimers composed of one alpha-tubulin polypeptide and one beta-tubulin polypeptide that should undergo a complex folding processing before they achieve a quaternary structure that will allow their incorporation into the polymer. Due to the extremely high protein concentration that exists at the cell cytoplasm, there are alpha- and beta-tubulin interacting proteins that prevent the unwanted interaction of these polypeptides with the surrounding protein pool during folding, thus allowing microtubule dynamics. Several years ago, the development of a nondenaturing electrophoretic technique made it possible to identify different tubulin intermediate complexes during tubulin biogenesis in vitro. By these means, the cytosolic chaperonin containing TCP-1 (CCT or TriC) and prefoldin have been demonstrated to intervene through tubulin and actin folding. Various other cofactors also identified along the alpha- and beta-tubulin postchaperonin folding route are now known to have additional roles in tubulin biogenesis such as participating in the synthesis, transport, and storage of alpha- and beta-tubulin. The future characterization of the tubulin-binding sites to these proteins, and perhaps other still unknown proteins, will help in the development of chemicals that could interfere with tubulin folding and thus modulating microtubule dynamics. In this paper, current knowledge of the above postchaperonin folding cofactors, which are in fact chaperones involved in tubulin heterodimer quaternary structure achievement, will be reviewed.     

Translational control of mRNA expression during the early mitogenic response in Swiss mouse 3T3 cells: identification of specific proteins

Addition of serum or epidermal growth factor to quiescent Swiss mouse 3T3 cells in culture leads to a number of specific changes in the pattern of protein synthesis. Earlier experiments with actinomycin D suggested that the altered expression of these proteins was controlled at either the pretranslational or translational level. Here we have identified and further characterized the regulation of mRNA expression for ten of these proteins, including protein synthesis elongation factor eEF-1 alpha, poly A binding protein, vimentin, the multiple forms of the actin protein family, and alpha- and beta-tubulin. Using an in vitro translation system, we determined the change in the level of mRNA encoding for each of these proteins after serum stimulation. The results showed that the amount of mRNA coding for eEF-1 alpha, poly A binding protein, vimentin, and alpha- and beta-tubulin remains unchanged during this time, whereas that of the actin family increases. Thus, with the exception of the actin family, the results argue that the expression of all the proteins identified is regulated at the translational level. The importance of this latter group of proteins in cell growth and the abundance of their cognate mRNAs should prove them useful tools in elucidating the mechanisms involved in the activation of translationally repressed mRNA during the mitogenic response.     

A dual effect on protein synthesis and degradation modulates the tubulin level in rice cells treated with oryzalin

The effect of the anti-microtubular drug oryzalin on growth and morphology of cultured rice (Oryza sativa L., cv. Roncarolo) cells was evaluated with specific reference to mechanisms that control intracellular tubulin levels. The addition of oryzalin caused a great reduction in the level of both alpha- and beta-tubulin polypeptides, as detected by Western blot analysis. However, no appreciable decrease was observed in the population of total or isotype-specific alpha- and beta-tubulin mRNAs. Only within the first 24 h of the oryzalin treatment, when the level of both alpha- and beta-tubulin polypeptides was still undiminished, was a consistent reduction in the amount of total beta-tubulin mRNA observed. Pulse-chase experiments performed on rice cells grown in the presence of 1 microM oryzalin revealed the presence of two distinct mechanisms that negatively control alpha- and beta-tubulin polypeptide levels. (i) There was an immediate effect on protein synthesis, which resulted in a reduction in the level of newly synthesized tubulin. (ii) There was a delayed response characterized by a substantial degradation of both alpha- and beta-tubulin monomers; this degradation occurred after 24 h of herbicide treatment. The possible involvement of Ca2+ in the degradation of the unincorporated tubulin monomers is also documented and discussed.     

Dominant effects of tubulin overexpression in Saccharomyces cerevisiae.

The consequences of altering the levels of alpha- and beta-tubulin in Saccharomyces cerevisiae were examined by constructing fusions of the structural genes encoding the tubulins to strong galactose-inducible promoters. Overexpression of beta-tubulin (TUB2) was lethal: cells arrested in the G2 stage of the cell cycle exhibited an increased frequency of chromosome loss, were devoid of microtubules, and accumulated beta-tubulin in a novel structure. Overexpression of the major alpha-tubulin gene (TUB1) was not lethal and did not affect chromosome segregation. The rate of alpha-tubulin mRNA and protein synthesis was increased, but the protein did not accumulate. Overexpression of both alpha- and beta-tubulin together resulted in arrested cell division, and cells accumulated excess tubules that contained both alpha- and beta-tubulin. Transient overexpression of both tubulins resulted in a high frequency of chromosome loss. These data suggest that strong selective pressure exists to prevent excess accumulation of microtubules or beta-tubulin and suggest a model by which this goal may be achieved by selective degradation of unassembled alpha-tubulin. Furthermore, the phenotype of beta-tubulin overexpression is similar to the phenotype of a beta-tubulin deficiency. These results add to a number of recent studies demonstrating that mutant phenotypes generated by overexpression can be informative about the function of the gene product.     

Dinitroaniline herbicide-resistant transgenic tobacco plants generated by co-overexpression of a mutant alpha-tubulin and a beta-tubulin.

Dinitroaniline herbicides are used for the selective control of weeds in arable crops. Dinitroaniline herbicide resistance in the invasive weed goosegrass was previously shown to stem from a spontaneous mutation in an alpha-tubulin gene. We transformed and regenerated tobacco plants with an alpha/beta-tubulin double gene construct containing the mutant alpha-tubulin gene and showed that expression of this construct confers a stably inherited dinitroaniline-resistant phenotype in tobacco. In all transformed lines, the transgene alpha- and beta-tubulins increased the cytoplasmic pool of tubulin approximately 1.5-fold while repressing endogenous alpha- and beta-tubulin synthesis by up to 45% in some tissues. Transgene alpha- and beta-tubulin were overexpressed in every plant tissue analyzed and comprised approximately 66% of the total tubulin in these tissues. Immunolocalization studies revealed that transgene alpha- and beta-tubulins were incorporated into all four microtubule arrays, indicating that they are functional. The majority of the alpha/beta-tubulin pools are encoded by the transgenes, which implies that the mutant alpha-tubulin and the beta-tubulin can perform the majority, if not all, of the roles of microtubules in both juvenile and adult tobacco plants.     

Microtubule polyglutamylation in Drosophila melanogaster brain and testis.

The presence of glutamylated tubulin, a widespread posttranslational modification of alpha- and beta-tubulin, has been investigated in Drosophila melanogaster using the specific monoclonal antibody GT335. We show here that this modification is strongly detected in brain and testis whereas other tissues analyzed did not appear to contain any glutamylated isoforms. Neuronal microtubules are glutamylated on alpha-tubulin only whereas sperm flagella showed a strong modification of both alpha- and beta-tubulin. These results argue for an essential role for glutamylation in differentiation processes that require microtubule stabilization.     

Changes in the level of tubulin subunits during development of cotton (Gossypium hirsutum) fiber

The levels of tubulin protein in developing cotton ( Gossypium hirsutum L. cv. Stoneville 825) fibers were measured from 8 to 28 days post-anthesis using commercially available monoclonal antibodies against alpha- and beta-tubulin. As the monoclonal antibodies against alpha- and beta-tubulin were prepared from yeast tubulin and chick brain tubulin, respectively, indirect immunofluorescence microscopy was used to establish that the two monoclonal antibodies recognized microtubule structures in cotton fibers. Western blots of electrophoretically separated proteins in crude extracts of cotton roots and fibers showed that single polypeptides with the expected apparent molecular weight for tubulin subunits were recognized by the antisera. An enzyme-linked immunosorbent assay was used to quantify tubulin levels. From 10 to 20 days post-anthesis the level of tubulin protein increases approximately three-fold. After 20 days post-anthesis, the amount of tubulin relative to total fiber protein reaches a plateau or decreases slightly. The rapid rise in tubulin is correlated with the elongation of the fiber and an increase in cellulose synthesis.     

Two cofactors and cytoplasmic chaperonin are required for the folding of alpha- and beta-tubulin.

Though the chaperonins that mediate folding in prokaryotes, mitochondria, and chloroplasts have been relatively well characterized, the folding of proteins in the eukaryotic cytosol is much less well understood. We recently identified a cytoplasmic chaperonin as an 800-kDa multisubunit toroid which forms a binary complex with unfolded actin; the correctly folded polypeptide is released upon incubation with Mg-ATP (Y. Gao, J. O. Thomas, R. L. Chow, G.-H. Lee, and N. J. Cowan, Cell 69:1043-1050, 1992). Here we show that the same chaperonin also forms a binary complex with unfolded alpha- or beta-tubulin; however, there is no detectable release of the correctly folded product, irrespective of the concentration of added Mg-ATP and Mg-GTP or the presence of added carrier tubulin heterodimers with which newly folded alpha- or beta-tubulin polypeptides might exchange. Rather, two additional protein cofactors are required for the generation of properly folded alpha- or beta-tubulin, which is then competent for exchange into preexisting alpha/beta-tubulin heterodimers. We show that actin and tubulins compete efficiently with one another for association with cytoplasmic chaperonin complexes. These data imply that actin and alpha- and beta-tubulin interact with the same site(s) on chaperonin complexes.     

Tubulin folding cofactors as GTPase-activating proteins. GTP hydrolysis and the assembly of the alpha/beta-tubulin heterodimer.

In vivo, many proteins must interact with molecular chaperones to attain their native conformation. In the case of tubulin, newly synthesized alpha- and beta-subunits are partially folded by cytosolic chaperonin, a double-toroidal ATPase with homologs in all kingdoms of life and in most cellular compartments. alpha- and beta-tubulin folding intermediates are then brought together by tubulin-specific chaperone proteins (named cofactors A-E) in a cofactor-containing supercomplex with GTPase activity. Here we show that tubulin subunit exchange can only occur by passage through this supercomplex, thus defining it as a dimer-making machine. We also show that hydrolysis of GTP by beta-tubulin in the supercomplex acts as a switch for the release of native tubulin heterodimer. In this folding reaction and in the related reaction of tubulin-folding cofactors with native tubulin, the cofactors behave as GTPase-activating proteins, stimulating the GTP-binding protein beta-tubulin to hydrolyze its GTP.     

mRNAs for alpha- and beta-tubulin and flagellar calmodulin are among those coordinately regulated when Naegleria gruberi amebae differentiate into flagellates

Three of four mRNAs that are specific to the differentiation of Naegleria gruberi amebae into flagellates (Mar, J., J. H. Lee, D. Shea, and C. J. Walsh, 1986, J. Cell Biol., 102:353-361) have been identified as coding for flagellar proteins. The products of these mRNAs, which are coordinately regulated during the differentiation, were identified by in vitro translation of hybrid-selected RNA followed by two-dimensional gel electrophoresis and antibody binding. Six cross-hybridizing clones complementary to a 1.7-kb RNA (class II) all selected mRNA that was translated into two alpha-tubulins. The principal in vitro product, alpha-1, comigrated with a cytoplasmic alpha-tubulin, while the minor product with a more acidic pI, alpha-2, comigrated with flagellar alpha-tubulin. While Naegleria flagellar alpha-tubulin was found to be acetylated based on its reaction with a monoclonal antibody specific to this form, we suggest that alpha-2 is not likely to arise due to acetylation in vitro but probably represents the product of a second alpha-tubulin gene. The class III clone, also complementary to a 1.7-kb RNA, selected beta-tubulin mRNA. In the course of this work it was found, using monoclonal antibodies to the alpha- and beta-subunits of tubulin, that Naegleria alpha-tubulin migrated faster than beta-tubulin on SDS-PAGE. The class IV clone, which hybridizes with a 0.5-kb RNA, selected an mRNA that was translated into a heat stable calcium-binding protein, flagellar calmodulin.     

Identification of three coated vesicle components as alpha- and beta- tubulin linked to a phosphorylated 50,000-dalton polypeptide

Coated vesicles are involved in the intracellular transport of membrane proteins between a variety of membrane compartments. The coats of bovine brain coated vesicles contain at least six polypeptides in addition to an 180,000-dalton polypeptide called clathrin. In this report we show that the 54,000- and 56,000-dalton coated vesicle polypeptides are alpha- and beta-tubulin, determined by immunoblotting and two-dimensional gel electrophoresis. An affinity-purified tubulin antiserum can precipitate coated vesicles. The tubulin polypeptides are tightly associated with a 50,000-dalton coated vesicle polypeptide, which is phosphorylated. The phosphorylated 50,000-dalton polypeptide appears to be related to brain microtubule-associated tau proteins since it can be specifically immunoprecipitated by an affinity-purified antiserum directed against these proteins. In addition, gel filtration experiments indicate that at least a fraction of the 50,000-dalton polypeptide may associate with the 100,000-dalton coated vesicle polypeptide. Since brain is a tissue rich in tubulins, liver coated vesicles were analyzed for the presence of alpha- and beta-tubulin. Like brain coated vesicles, liver coated vesicles also contain an endogenous kinase activity, which phosphorylates polypeptides of the same molecular weights and isoelectric points as the brain coated vesicle 50,000-dalton, tau-like polypeptide, and alpha- and beta-tubulin. The phosphorylated 50,000-dalton polypeptide may link the membrane and contents of coated vesicles with components of the cytoskeleton.