Mutations affecting assembly of beta-tubulin localize to a region near the carboxyl terminus

The generation of Chinese hamster ovary cell lines that express assembly defective forms of beta-tubulin were isolated using selections based on reversion of conditional lethal or drug resistance phenotypes. Two such cell lines, D2 and 6H3, were chosen for further characterization because they contain beta-tubulin polypeptides that exhibit decreases in apparent molecular weight on two-dimensional gel electrophoresis. Analysis of the nucleic acid from these cell lines using both Southern and Northern procedures suggests a deletion in one of the beta-tubulin genes in each cell line. Localization of the missing sequence in D2 was first determined by tryptic peptide mapping by high performance liquid chromatography. Subsequently, the assignment was confirmed by constructing appropriate subclones of a wild type Chinese hamster ovary beta-tubulin cDNA for Southern analysis to demonstrate a failure to recognize characteristic hybridization patterns of the mutant tubulin gene. In the other revertant, 6H3, the deletion was detected on a Northern blot by differential hybridization of a 3' fragment of the cDNA to the beta-tubulin messages. The results indicate that D2 has an internal deletion whose approximate limits extend from amino acid residues 250 through 345. Cell line 6H3 has a deletion that begins near amino acid residue 330 and extends into the 3'-untranslated region of the gene.     

The alpha- and beta-tubulin folding pathways

The alpha-beta tubulin heterodimer is the subunit from which microtubules are assembled. The pathway leading to correctly folded alpha- and beta-tubulins is unusually complex: it involves cycles of ATP-dependent interaction of newly synthesized tubulin subunits with cytosolic chaperonin, resulting in the production of quasi-native folding intermediates, which must then be acted upon by additional protein cofactors. These cofactors form a supercomplex containing both alpha- and beta-tubulin polypeptides, from which native heterodimer is released in a GTP-dependent reaction. Here, we discuss the current state of our understanding of the function of cytosolic chaperonin and cofactors in tubulin folding.     

Mutations affecting assembly and stability of tubulin: evidence for a nonessential beta-tubulin in CHO cells.

Eight strains of Chinese hamster ovary (CHO) cells having an assembly-defective beta-tubulin were found among revertants of strain Cmd 4, a mutant with a conditional lethal mutation in a beta-tubulin gene (F. Cabral, M. E. Sobel, and M. M. Gottesman, Cell 20:29-36, 1980). The altered beta-tubulins in these strains have electrophoretically silent alterations or, in some cases, an increase or a decrease in apparent molecular weight based on their migration in two-dimensional gels. The identity of these variant proteins as beta-tubulin was confirmed by peptide mapping, which also revealed the loss of distinct methionine-containing peptides in the assembly-defective beta-tubulins of lower apparent molecular weight. The altered mobility of these beta-tubulin polypeptides was not the result of a posttranslational modification, since the altered species could be labeled in very short incubations with [35S]methionine and were found among in vitro-translated polypeptides by using purified mRNA. In at least one strain, an altered DNA restriction fragment could be demonstrated, suggesting that an alteration occurred in one of the structural genes for beta-tubulin. Assembly-defective beta-tubulin was unstable and turned over with a half-life of only 1 to 2 h in exponentially growing cells. This rapid degradation of a tubulin gene product resulted in approximately 30% lower steady-state levels of both alpha- and beta-tubulin yet did not affect the growth rate of the cells or the distribution of the microtubules as judged by immunofluorescence microscopy. These results argue that CHO cells possess a beta-tubulin gene product that is not essential for survival.     

Mutations affecting gonadotropin secretion and action

A number of mutations are known to disturb the development and function of the hypothalamic-pituitary-gonadal axis. They affect hypothalamic-pituitary-gonadal function at multiple levels, from the migration of gonadotropin releasing hormone neurons to the hypothalamus right through to gonadotropin action in the ovary and testis. Most of the mutations are inactivating, causing various forms of hypogonadism. Exceptions are the activating mutations of the luteinizing hormone receptor, causing male-limited gonadotropin-independent precocious puberty. The human mutations and genetically modified animal models have clarified the molecular pathogenesis of hypogonadism and such disorders can now be diagnosed using molecular biological techniques, enabling selection of specific treatments and appropriate counselling of patients and their families.     

Mutations in alpha- and beta-tubulin affect spindle formation in Chinese hamster ovary cells

Two Chinese hamster ovary cell lines with mutated beta-tubulins (Grs-2 and Cmd-4) and one that has a mutation in alpha-tubulin (Tax-1) are temperature sensitive for growth at 40.5 degrees C. To determine the functional defect in these mutant cells at the nonpermissive temperature, they were characterized with respect to cell cycle parameters and microtubule organization and function after relatively short periods at 40.5 degrees C. At the nonpermissive temperature all the mutants had normal appearing cytoplasmic microtubules. Premature chromosome condensation analysis failed to show any discrete step in the interphase cell cycle in which these mutants are arrested. These cells, however, show several defects at the nonpermissive temperature that appear related to the function of microtubules during mitosis. Time-lapse studies showed that mitosis was lengthened in the three mutant lines at 40.5 degrees C as compared with the wild-type cells at this temperature, resulting in a higher proportion of cells in mitosis after temperature shift. There was also a large increase in multinucleated cells in mutant populations after incubation at the nonpermissive temperature. Immunofluorescent studies using a monoclonal anti--alpha-tubulin antibody showed that the mutant cells had a high proportion of abnormal spindles at the nonpermissive temperature. The two altered beta-tubulins and the altered alpha-tubulin all were found to cause a similar phenotype at the high temperature that results in mitotic delay, defective cytokinesis, multinucleation, and ultimately, cell death. We conclude that spindle formation is the limiting microtubule function in these mutant cell lines at the nonpermissive temperature and that these cell lines will be of value for the study of the precise role of tubulin in mammalian spindle formation.     

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.     

Mutations affecting sodium channels in Drosophila

Ion channels play key roles in the generation and propagation of nerve impulses by mediating ionic fluxes across excitable membranes. Elucidation of the structure and function of these proteins is a major goal in understanding the molecular mechanisms of membrane excitability. Much work has focused on sodium channels, whose activity is of primary importance in producing nerve impulses. Despite the recent cloning and sequencing of a sodium channel structural gene, many unanswered questions remain. To address some of these question genetically, Drosophila mutants with altered sodium channels are being isolated and subjected to multidisciplinary analyses. Ultimately, these can provide novel approaches for understanding the function and regulation of sodium channels at a molecular level.     

Physical evidence for cotranslational regulation of beta-tubulin mRNA degradation.

Tubulin synthesis is controlled by an autoregulatory mechanism through which an increase in the intracellular concentration of tubulin subunits leads to specific degradation of tubulin mRNAs. The sequence necessary and sufficient for the selective degradation of a beta-tubulin mRNA in response to changes in the level of free tubulin subunits resides within the first 13 translated nucleotides that encode the amino-terminal sequence of beta-tubulin, Met-Arg-Glu-Ile (MREI). Previous results have suggested that the sequence responsible for autoregulation resides in the nascent peptide rather than in the mRNA per se, raising the possibility that the regulation of the stability of tubulin mRNA is mediated through binding of tubulin or some other cellular factor to the nascent amino-terminal tubulin peptide. We now show that this putative cotranslational interaction is not mediated by tubulin alone, as no meaningful binding is detectable between tubulin subunits and the amino-terminal beta-tubulin polypeptide. However, microinjection of a monoclonal antibody that binds to the beta-tubulin nascent peptide selectively disrupts the regulation of beta-tubulin, but not alpha-tubulin, synthesis. This finding provides direct evidence for cotranslational degradation of beta-tubulin mRNA mediated through binding of one or more cellular factors to the beta-tubulin nascent peptide.     

Mutations affecting meiosis in Podospora anserina

Forty mutants affecting meiosis and two affecting caryogamy were isolated in Podospora anserina. Growth and mitosis of these mutants were normal. Eighteen of them were studied by both light and electron microscopy so as to determine precisely how the behaviour of the chromosomes and/or the structure of the kinetic apparatus were altered.In the caryogamy-deficient mutants, the formation of crosiers is normal, but the nuclei of the apical cells never fuse. Three genes affect prophase I. For two of them the mutants are blocked before pachytene, the third being blocked between pachytene and diplotene. The other mutations concern the kinetic apparatus (centrosomal plaques, spindle) and the distribution of the nuclei in the spores.Some of these mutations are pleiotropic and also affect spore pigmentation, recombination frequency, and mutation rate.     

Mutations affecting retina development in Medaka

In a large scale mutagenesis screen of Medaka we identified 60 recessive zygotic mutations that affect retina development. Based on the onset and type of phenotypic abnormalities, the mutants were grouped into five categories: the first includes 11 mutants that are affected in neural plate and optic vesicle formation. The second group comprises 15 mutants that are impaired in optic vesicle growth. The third group includes 18 mutants that are affected in optic cup development. The fourth group contains 13 mutants with defects in retinal differentiation. 12 of these have smaller eyes, whereas one mutation results in enlarged eyes. The fifth group consists of three mutants with defects in retinal pigmentation. The collection of mutants will be used to address the molecular genetic mechanisms underlying vertebrate eye formation.     

Mutations affecting transport and stability of lysosomal enzymes

The biosynthesis, post-translational processing and receptor-mediated transport of lysosomal enzymes will be briefly summarized. Mutations affecting the transport or the stability of a lysosomal enzyme but not its catalytic properties can result in a lysosomal storage disorder. Mutations causing a loss of catalytic activity may in addition affect transport or stability. Such mutations should not be classified as transport or stability mutations. Prototypes for transport and stability mutations are I-cell disease and late onset forms of metachromatic leukodystrophy.     

Mutations at leucine 215 of beta-tubulin affect paclitaxel sensitivity by two distinct mechanisms

Paclitaxel resistance mutations in Chinese hamster ovary cells frequently alter a cluster of leucine residues in the H6-H7 loop region of beta-tubulin. To gain further insight into the role of this region in microtubule assembly and drug resistance, site-directed mutagenesis was used to systematically change amino acid L215. The mutated genes were cloned into a tetracycline-regulated expression vector and transfected into wild-type cells. Most of the mutations destabilized microtubule assembly, causing a decreased fraction of tubulin to appear in the microtubule cytoskeleton. In each case, the decreased level of assembly was associated with paclitaxel resistance and increased colcemid sensitivity. In two cases, however, the alteration did not significantly perturb the level of assembled tubulin or confer resistance to paclitaxel. One of these, L215V, produced little or no detectable phenotype, while the other, L215I, conferred increased sensitivity to paclitaxel. The increased drug sensitivity did not extend to epothilone A, a drug that binds to the same site and has a mechanism of action similar to that of paclitaxel, or colcemid, a drug with an opposing mechanism of action and a distinct binding site. Moreover, L215I conferred enhanced paclitaxel sensitivity at very low levels of expression, and sensitivity was not further enhanced in cells with higher levels of expression, implying that paclitaxel acts substoichiometrically. These properties, along with the proximity of L215 to the drug binding site, suggests that the L215I substitution may enhance the binding or effectiveness of paclitaxel. Our studies confirm the importance of the H6-H7 loop of beta-tubulin in microtubule assembly and resistance to antimitotic drugs. They also identify the first mammalian mutation shown to specifically increase sensitivity to paclitaxel.     

A novel step in beta-tubulin folding is important for heterodimer formation in Saccharomyces cerevisiae

Undimerized beta-tubulin is toxic in the yeast S. cerevisiae. It can arise if levels of beta-tubulin and alpha-tubulin are unbalanced or if the tubulin heterodimer dissociates. We are using the toxicity of beta-tubulin to understand early steps in microtubule morphogenesis. We find that deletion of PLP1 suppresses toxic beta-tubulin formed by disparate levels of alpha- and beta-tubulin. That suppression occurs either when alpha-tubulin is modestly underexpressed relative to beta-tubulin or when beta-tubulin is inducibly and strongly overexpressed. Plp1p does not affect tubulin expression. Instead, a significant proportion of the undimerized beta-tubulin in plp1Delta cells is less toxic than that in wild-type cells. It is also less able to combine with alpha-tubulin to form a heterodimer. As a result, plp1Delta cells have lower levels of heterodimer. Importantly, plp1Delta cells that also lack Pac10, a component of the GimC/PFD complex, are even less affected by free beta-tubulin. Our results suggest that Plp1p defines a novel early step in beta-tubulin folding.     

Secretion of heterologous proteins in Bacillus subtilis can be improved by engineering cell components affecting post-translocational protein folding and degradation

AIMS: To explore the potential to enhance secretion of heterologous proteins in Bacillus subtilis by engineering cell factors affecting extracytoplasmic protein folding and degradation. METHODS AND RESULTS: Bottleneck components affecting the extracytoplasmic phase of protein secretion were genetically engineered and their effects on the secretion of 11 industrially interesting heterologous proteins were studied by Western blotting and enzymatic assays. Overproduction of PrsA lipoprotein enhanced the secretion of alpha-amylase of Bacillus stearothermophilus (fourfold) and pneumolysin (1.5-fold). Increasing the net negative charge of the cell wall because of lack of the d-alanine substitution of anionic cell wall polymers enhanced the secretion of pneumolysin c. 1.5-fold. Decreasing the level of HtrA-type quality control proteases caused harmful effects on growth and did not enhance secretion. Pertussis toxin subunit, S1 was found to be a substrate for HtrA-type proteases and its secretion was dependent on these proteases. CONCLUSIONS: Secretion of heterologous proteins can be enhanced by engineering components involved in late stages of secretion in a protein-dependent manner. SIGNIFICANCE AND IMPACT OF THE STUDY: The study revealed both possibilities and limitations of modulating the post-translocational phase of secretion as a means to improve the yield of heterologous proteins.     

Mutations affecting nerve attachment of Caenorhabditis elegans

Using a pan-neuronal GFP marker, a morphological screen was performed to detect Caenorhabditis elegans larval lethal mutants with severely disorganized major nerve cords. We recovered and characterized 21 mutants that displayed displacement or detachment of the ventral nerve cord from the body wall (Ven: ventral cord abnormal). Six mutations defined three novel genetic loci: ven-1, ven-2, and ven-3. Fifteen mutations proved to be alleles of previously identified muscle attachment/positioning genes, mup-4, mua-1, mua-5, and mua-6. All the mutants also displayed muscle attachment/positioning defects characteristic of mua/mup mutants. The pan-neuronal GFP marker also revealed that mutants of other mua/mup loci, such as mup-1, mup-2, and mua-2, exhibited the Ven defect. The hypodermis, the excretory canal, and the gonad were morphologically abnormal in some of the mutants. The pleiotropic nature of the defects indicates that ven and mua/mup genes are required generally for the maintenance of attachment of tissues to the body wall in C. elegans.     

Mutations affecting pigment synthesis in Mycobacterium aurum

Pigmentation mutants of Mycobacterium aurum were isolated after chemical mutagenesis. Examination of the pigments extracted from these mutants indicated that at least 15 carotenoids were formed. beta-Carotene was not detected and the major carotene of M. aurum appeared to be leprotene. The possible biosynthetic pathway is discussed on the basis of these results.     

Mutations affecting gyrase in Haemophilus influenzae.

Mutants separately resistant to novobiocin, coumermycin, nalidixic acid, and oxolinic acid contained gyrase activity as measured in vitro that was resistant to the antibiotics, indicating that the mutations represented structural alterations of the enzyme. One Novr mutant contained an altered B subunit of the enzyme, as judged by the ability of a plasmid, pNov1, containing the mutation to complement a temperature-sensitive gyrase B mutation in Escherichia coli and to cause novobiocin resistance in that strain. Three other Novr mutations did not confer antibiotic resistance to the gyrase but appeared to increase the amount of active enzyme in the cell. One of these, novB1, could only act in cis, whereas a new mutation, novC, could act in trans. An RNA polymerase mutation partially substituted for the novB1 mutation, suggesting that novB1 may be a mutation in a promoter region for the B subunit gene. Growth responses of strains containing various combinations of mutations on plasmids or on the chromosome indicated that low-level resistance to novobiocin or coumermycin may have resulted from multiple copies of wild-type genes coding for the gyrase B subunit, whereas high-level resistance required a structural change in the gyrase B gene and was also dependent on alteration in a regulatory region. When there was mismatch at the novB locus, with the novB1 mutation either on a plasmid or the chromosome, and the corresponding wild-type gene present in trans, chromosome to plasmid recombination during transformation was much higher than when the genes matched, probably because plasmid to chromosome recombination, eliminating the plasmid, was inhibited by the mismatch.