Sto1p, a fission yeast protein similar to tubulin folding cofactor E, plays an essential role in mitotic microtubule assembly

1999 ◽  
Vol 112 (12) ◽  
pp. 1979-1988 ◽  
Author(s):  
E.L. Grishchuk ◽  
J.R. McIntosh
Keyword(s):  
Fission Yeast ◽  
Microtubule Assembly ◽  
Wild Type ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Cold Sensitive ◽  
Alpha Beta ◽  
Sensitive Allele ◽  
Delta Cells

The proper functioning of microtubules depends crucially on the availability of polymerizable alpha/beta tubulin dimers. Their production occurs concomitant with the folding of the tubulin polypeptides and is accomplished in part by proteins known as Cofactors A through E. In the fission yeast, Schizosaccharomyces pombe, this tubulin folding pathway is essential. We have taken advantage of the excellent cytology available in S. pombe to examine the phenotypic consequences of a deletion of sto1(+), a gene that encodes a protein similar to Cofactor E, which is required for the folding of alpha-tubulin. The interphase microtubule cytoskeleton in sto1-delta cells is severely disrupted, and as cells enter mitosis their spindles fail to form. After a transient arrest with condensed chromosomes, the cells exit mitosis and resume DNA synthesis, whereupon they septate abnormally and die. Overexpression of Spo1p is toxic to cells carrying a cold-sensitive allele of the alpha- but not the beta-tubulin gene, consistent with the suggestion that this protein plays a role like that of Cofactor E. Unlike its presumptive partner Cofactor D (Alp1p), however, Sto1p does not localize to microtubules but is found throughout the cell. Overexpression of Sto1p has no toxic effects in wild-type cells, suggesting that it is unable to disrupt alpha/beta tubulin dimers in vivo.

scholarly journals The colR4 and colR15 beta-tubulin mutations in Chlamydomonas reinhardtii confer altered sensitivities to microtubule inhibitors and herbicides by enhancing microtubule stability.

1991 ◽  
Vol 113 (3) ◽  
pp. 605-614 ◽  
Author(s):  
M J Schibler ◽  
B Huang
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Missense Mutations ◽  
Wild Type ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Microtubule Inhibitors ◽  
Microtubule Stability ◽  
The Stability ◽  
Tubulin Mutations

The colR4 and colR15 beta 2-tubulin missense mutations for lysine-350 in Chlamydomonas reinhardtii (Lee and Huang, 1990) were originally isolated by selection for resistance to the growth inhibitory effects of colchicine. The colR4 and colR15 mutants have been found to be cross resistant to vinblastine and several classes of antimitotic herbicides, including the dinitroanilines (oryzalin, trifluralin, profluralin, and ethafluralin); the phosphoric amide amiprophos methyl; and the dimethyl propynl benzamide pronamide. Like colchicine and vinblastine, the antimitotic effects of these plant-specific herbicides have been associated with the depolymerization of microtubules. In contrast to their resistance to microtubule-depolymerizing drugs, the mutants have an increased sensitivity to taxol, a drug which enhances the polymerization and stability of microtubules. This pattern of altered sensitivity to different microtubule inhibitors was found to cosegregate and corevert with the beta-tubulin mutations providing the first genetic evidence that the in vivo herbicidal effects of the dinitroanilines, amiprophos methyl, and pronamide are related to microtubule function. Although wild-type like in their growth characteristics, the colR4 and colR15 mutants were found to have an altered pattern of microtubules containing acetylated alpha-tubulin, a posttranslational modification that has been associated with stable subsets of microtubules found in a variety of cells. Microtubules in the interphase cytoplasm and those of the intranuclear spindle of mitotic cells, which in wild-type Chlamydomonas cells do not contain acetylated alpha-tubulin, were found to be acetylated in the mutants. These data taken together suggest that the colR4 and colR15 missense mutations increase the stability of the microtubules into which the mutant beta-tubulins are incorporated and that the altered drug sensitivities of the mutants are a consequence of this enhanced microtubule stability.

scholarly journals Arabidopsis Disulfide Reductase, Trx-h2, Functions as an RNA Chaperone under Cold Stress

2021 ◽  
Vol 11 (15) ◽  
pp. 6865
Author(s):  
Eun Seon Lee ◽  
Joung Hun Park ◽  
Seong Dong Wi ◽  
Ho Byoung Chae ◽  
Seol Ki Paeng ◽  
...  
Keyword(s):  
Cold Stress ◽  
Wild Type ◽  
Rna Chaperone ◽  
E Coli ◽  
Cold Sensitive ◽  
Thioredoxin H ◽  
Functional Rnas

The thioredoxin-h (Trx-h) family of Arabidopsis thaliana comprises cytosolic disulfide reductases. However, the physiological function of Trx-h2, which contains an additional 19 amino acids at its N-terminus, remains unclear. In this study, we investigated the molecular function of Trx-h2 both in vitro and in vivo and found that Arabidopsis Trx-h2 overexpression (Trx-h2OE) lines showed significantly longer roots than wild-type plants under cold stress. Therefore, we further investigated the role of Trx-h2 under cold stress. Our results revealed that Trx-h2 functions as an RNA chaperone by melting misfolded and non-functional RNAs, and by facilitating their correct folding into active forms with native conformation. We showed that Trx-h2 binds to and efficiently melts nucleic acids (ssDNA, dsDNA, and RNA), and facilitates the export of mRNAs from the nucleus to the cytoplasm under cold stress. Moreover, overexpression of Trx-h2 increased the survival rate of the cold-sensitive E. coli BX04 cells under low temperature. Thus, our data show that Trx-h2 performs function as an RNA chaperone under cold stress, thus increasing plant cold tolerance.

scholarly journals Structural analysis of mutations in the Drosophila beta 2-tubulin isoform reveals regions in the beta-tubulin molecular required for general and for tissue-specific microtubule functions.

Genetics ◽  
1995 ◽  
Vol 139 (1) ◽  
pp. 267-286 ◽  
Author(s):  
J D Fackenthal ◽  
J A Hutchens ◽  
F R Turner ◽  
E C Raff
Keyword(s):  
Amino Acid ◽  
Three Dimensional ◽  
Variable Region ◽  
Internal Variable ◽  
Microtubule Assembly ◽  
Dimensional Structure ◽  
Wild Type ◽  
Beta Tubulin ◽  
Assembly Kinetics ◽  
Beta 2

Abstract We have determined the lesions in a number of mutant alleles of beta Tub85D, the gene that encodes the testis-specific beta 2-tubulin isoform in Drosophila melanogaster. Mutations responsible for different classes of functional phenotypes are distributed throughout the beta 2-tubulin molecule. There is a telling correlation between the degree of phylogenetic conservation of the altered residues and the number of different microtubule categories disrupted by the lesions. The majority of lesions occur at positions that are evolutionarily highly conserved in all beta-tubulins; these lesions disrupt general functions common to multiple classes of microtubules. However, a single allele B2t6 contains an amino acid substitution within an internal cluster of variable amino acids that has been identified as an isotype-defining domain in vertebrate beta-tubulins. Correspondingly, B2t6 disrupts only a subset of microtubule functions, resulting in misspecification of the morphology of the doublet microtubules of the sperm tail axoneme. We previously demonstrated that beta 3, a developmentally regulated Drosophila beta-tubulin isoform, confers the same restricted morphological phenotype in a dominant way when it is coexpressed in the testis with wild-type beta 2-tubulin. We show here by complementation analysis that beta 3 and the B2t6 product disrupt a common aspect of microtubule assembly. We therefore conclude that the amino acid sequence of the beta 2-tubulin internal variable region is required for generation of correct axoneme morphology but not for general microtubule functions. As we have previously reported, the beta 2-tubulin carboxy terminal isotype-defining domain is required for suprastructural organization of the axoneme. We demonstrate here that the beta 2 variant lacking the carboxy terminus and the B2t6 variant complement each other for mild-to-moderate meiotic defects but do not complement for proper axonemal morphology. Our results are consistent with the hypothesis drawn from comparisons of vertebrate beta-tubulins that the two isotype-defining domains interact in a three-dimensional structure in wild-type beta-tubulins. We propose that the integrity of this structure in the Drosophila testis beta 2-tubulin isoform is required for proper axoneme assembly but not necessarily for general microtubule functions. On the basis of our observations we present a model for regulation of axoneme microtubule morphology as a function of tubulin assembly kinetics.

Overexpression of yeast homologs of the mammalian checkpoint gene RCC1 suppresses the class of alpha-tubulin mutations that arrest with excess microtubules.

Genetics ◽  
1994 ◽  
Vol 137 (2) ◽  
pp. 381-392 ◽  
Author(s):  
D Kirkpatrick ◽  
F Solomon
Keyword(s):  
Cell Cycle ◽  
Microtubule Assembly ◽  
Functional Requirements ◽  
Cellular Regulation ◽  
Yeast Saccharomyces Cerevisiae ◽  
Alpha Tubulin ◽  
Class 1 ◽  
Cold Sensitive ◽  
Growth Phenotype ◽  
Cytoplasmic Structures

Abstract Microtubules in eukaryotic cells participate in a variety of nuclear and cytoplasmic structures, reflecting functional requirements and cell cycle position. We are studying the cellular regulation of microtubule assembly and organization in the yeast Saccharomyces cerevisiae. We screened for genes that when overexpressed suppress the growth phenotype of conditional mutants in alpha-tubulin that arrest with excess microtubules at the nonpermissive temperature (class 2 mutations). Here we describe one such suppressing element, called ATS1 (for Alpha Tubulin Suppressor). Overexpression of this gene rescues both the growth and microtubule phenotypes of all class 2 mutations, but not the cold-sensitive mutations that arrest with no microtubules (class 1 mutations). Deletion of ATS1 confers a modest slow growth phenotype which is slightly enhanced in strains containing both a deletion of ATS1 and a class 2 tub 1 mutation. The predicted ATS1 protein contains 333 amino acids and has considerable structural homology to the products of both the mammalian mitotic control gene RCC1 and the S. cerevisiae gene SRM1/PRP20. Overexpression of SRM1/PRP20 also suppresses class 2 mutants. The results suggest that this family of genes may participate in regulatory interactions between microtubules and the cell cycle.

Phenotypic consequences of tubulin overproduction in Saccharomyces cerevisiae: differences between alpha-tubulin and beta-tubulin

1990 ◽  
Vol 10 (10) ◽  
pp. 5295-5304
Author(s):  
B Weinstein ◽  
F Solomon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Division ◽  
Microtubule Assembly ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Tubulin Genes ◽  
Data Support ◽  
Cell Biol

Overexpression of alpha- and beta-tubulin genes in Saccharomyces cerevisiae, separately or together, leads to accumulation of large excesses of each of the polypeptides and arrest of cell division. However, other consequences of overexpression of these genes differ in several ways. As shown previously (D. Burke, P. Gasdaska, and L. Hartwell, Mol. Cell. Biol. 9:1049-1059, 1989), overexpression of beta-tubulin leads, at early times, to loss of microtubule structures and loss of viability. Eventually, the excess beta-tubulin forms abnormal structures. We show here that, in contrast, overexpression of alpha-tubulin led to none of these phenotypes and in fact could suppress each of the phenotypes associated with beta-tubulin accumulation. Truncated forms of beta-tubulin that were not competent to carry out microtubule functions also failed to elicit the beta-tubulin-specific phenotypes when overexpressed. The data support the hypothesis that beta-tubulin in excess over alpha-tubulin is uniquely toxic, perhaps because it interferes with normal microtubule assembly.

scholarly journals An amino-terminal tetrapeptide specifies cotranslational degradation of beta-tubulin but not alpha-tubulin mRNAs.

1994 ◽  
Vol 14 (6) ◽  
pp. 4076-4086 ◽  
Author(s):  
C J Bachurski ◽  
N G Theodorakis ◽  
R M Coulson ◽  
D W Cleveland
Keyword(s):  
Degradation Mechanism ◽  
Full Range ◽  
Mrna Degradation ◽  
Mrna Levels ◽  
Wild Type ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Amino Terminal ◽  
Tubulin Subunit ◽  
Tubulin Mrna

The steady-state level of alpha- and beta-tubulin synthesis is autoregulated by a posttranscriptional mechanism that selectively alters alpha- and beta-tubulin mRNA levels in response to changes in the unassembled tubulin subunit concentration. For beta-tubulin mRNAs, previous efforts have shown that this is the result of a selective mRNA degradation mechanism which involves cotranslational recognition of the nascent amino-terminal beta-tubulin tetrapeptide as it emerges from the ribosome. Site-directed mutagenesis is now used to determine that the minimal sequence requirement for conferring the full range of beta-tubulin autoregulation is the amino-terminal tetrapeptide MR(E/D)I. Although tubulin-dependent changes in alpha-tubulin mRNA levels are shown to result from changes in cytoplasmic mRNA stability, transfection of wild-type and mutated alpha-tubulin genes reveals that alpha- and beta-tubulin mRNA degradation is not mediated through a common pathway. Not only does the amino-terminal alpha-tubulin tetrapeptide MREC fail to confer regulated mRNA degradation, neither wild-type alpha-tubulin transgenes nor an alpha-tubulin gene mutated to encode an amino-terminal MREI yields mRNAs that are autoregulated. Further, although slowing ribosome transit accelerates the autoregulated degradation of endogenous alpha- and beta-tubulin mRNAs, degradation of alpha-tubulin transgene mRNAs is not enhanced, and in one case, the mRNA is actually stabilized. We conclude that, despite similarities, alpha- and beta-tubulin mRNA destabilization pathways utilize divergent determinants to link RNA instability to tubulin subunit concentrations.

scholarly journals Quality Control of a Transcriptional Regulator by SUMO-Targeted Degradation

2009 ◽  
Vol 29 (7) ◽  
pp. 1694-1706 ◽  
Author(s):  
Zheng Wang ◽  
Gregory Prelich
Keyword(s):  
Quality Control ◽  
Protein Quality ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Physiological Importance ◽  
Sensitive Allele ◽  
Quality Control Mechanism ◽  
Mutant Phenotypes

ABSTRACT Slx5 and Slx8 are heterodimeric RING domain-containing proteins that possess SUMO-targeted ubiquitin ligase (STUbL) activity in vitro. Slx5-Slx8 and its orthologs are proposed to target SUMO conjugates for ubiquitin-mediated proteolysis, but the only in vivo substrate identified to date is mammalian PML, and the physiological importance of SUMO-targeted ubiquitylation remains largely unknown. We previously identified mutations in SLX5 and SLX8 by selecting for suppressors of a temperature-sensitive allele of MOT1, which encodes a regulator of TATA-binding protein. Here, we demonstrate that Mot1 is SUMOylated in vivo and that disrupting the Slx5-Slx8 pathway by mutation of the target lysines in Mot1, by deletion of SLX5 or the ubiquitin E2 UBC4, or by inhibition of the proteosome suppresses mot1-301 mutant phenotypes and increases the stability of the Mot1-301 protein. The Mot1-301 mutant protein is targeted for proteolysis by SUMOylation to a much greater extent than wild-type Mot1, suggesting a quality control mechanism. In support of this idea, growth of Saccharomyces cerevisiae in the presence of the arginine analog canavanine results in increased SUMOylation and Slx5-Slx8-mediated degradation of wild-type Mot1. These results therefore demonstrate that Mot1 is an in vivo STUbL target in yeast and suggest a role for SUMO-targeted degradation in protein quality control.

scholarly journals Tubulin-tyrosine ligase has a binding site on beta-tubulin: a two-domain structure of the enzyme

1987 ◽  
Vol 104 (4) ◽  
pp. 1059-1067 ◽  
Author(s):  
J Wehland ◽  
K Weber
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Enzymatic Activity ◽  
Binding Site ◽  
Gradient Centrifugation ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Alpha Beta ◽  
Tubulin Tyrosine Ligase ◽  
Carboxy Terminal

Tubulin-tyrosine ligase and alpha beta-tubulin form a tight complex which is conveniently monitored by glycerol gradient centrifugation. Using two distinct ligase monoclonal antibodies, several subunit-specific tubulin monoclonal antibodies, and chemical cross-linking, a ligase-binding site was identified on beta-tubulin. This site is retained when the carboxy-terminal domains of both tubulin subunits are removed by subtilisin treatment. The ligase-tubulin complex is also formed when ligase is added to alpha beta-tubulin carrying the monoclonal antibody YL 1/2 which binds only to the carboxyl end of tyrosinated alpha-tubulin. The beta-tubulin-binding site described here explains the extreme substrate specificity of ligase, which does not act on other cellular proteins or carboxy-terminal peptides derived from detyrosinated alpha-tubulin. Differential accessibility of this site in tubulin and in microtubules seems to explain why ligase acts preferentially on unpolymerized tubulin. Ligase exposed to V8-protease is converted to a nicked derivative. This is devoid of enzymatic activity but still forms the complex with tubulin. Gel electrophoresis documents both 30- and a 14-kD domains, each which is immunologically and biochemically distinct and seems to cover the entire molecule. The two domains interact tightly under physiological conditions. The 30-kD domain carries the binding sites for beta-tubulin and ATP. The 14-kD domain can possibly form an additional part of the catalytic site as it harbors the epitope for the monoclonal antibody ID3 which inhibits enzymatic activity but not the formation of the ligase-tubulin complex.

scholarly journals Tubulin is phosphorylated at tyrosine by pp60c-src in nerve growth cone membranes.

1990 ◽  
Vol 111 (5) ◽  
pp. 1959-1970 ◽  
Author(s):  
W T Matten ◽  
M Aubry ◽  
J West ◽  
P F Maness
Keyword(s):  
Growth Cone ◽  
Cortical Neurons ◽  
Specific Protein ◽  
Beta Tubulin ◽  
Nerve Growth ◽  
Alpha Tubulin ◽  
Nerve Growth Cone ◽  
Phosphopeptide Mapping

We show here that tubulin is the major in vivo substrate of the tyrosine-specific protein kinase pp60c-src in nerve growth cone membranes. Phosphotyrosine antibodies were used to demonstrate phosphotyrosyl residues in a subpopulation of alpha- and beta-tubulin that was highly enriched in a subcellular fraction of growth cone membranes from fetal rat brain. The presence of phosphotyrosine-modified isoforms of alpha- and beta-tubulin in vivo was confirmed by 32p labeling of rat cortical neurons in culture. Tubulin in growth cone membranes was phosphorylated at tyrosine in endogenous membrane phosphorylation reactions (0.068 mol phosphotyrosine/mol alpha-tubulin and 0.045 mol phosphotyrosine/mol beta-tubulin), and phosphorylation was specifically inhibited by antibodies directed against pp60c-src, which is localized in the growth cone membranes. pp60c-src was capable of directly phosphorylating tubulin as shown in immune complex kinase assays with purified brain tubulin. Phosphopeptide mapping revealed a limited number of sites of tyrosine phosphorylation in alpha- and beta-tubulin, with similar phosphopeptides observed in vivo and in vitro. These results reveal a novel posttranslational modification of tubulin that could regulate microtubule dynamics at the growth cone.

Suppression of a conditional mutation in alpha-tubulin by overexpression of two checkpoint genes

1995 ◽  
Vol 108 (3) ◽  
pp. 1195-1204 ◽  
Author(s):  
S. Guenette ◽  
M. Magendantz ◽  
F. Solomon
Keyword(s):  
Microtubule Assembly ◽  
Cold Sensitivity ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Wild Type ◽  
Checkpoint Gene ◽  
Genomic Libraries ◽  
Alpha Tubulin ◽  
Tubulin Genes ◽  
Checkpoint Pathway

To identify proteins that regulate microtubule assembly in Saccharomyces cerevisiae, we screened for multicopy suppressors of a conditional mutation in alpha-tubulin. Cells expressing the recessive allele tub1-729 as their sole alpha-tubulin gene grow normally at permissive temperature. However, at 15 degrees C the cells lose viability and arrest primarily with large buds and quantitatively diminished microtubule structures. Transformation of mutant cells with genomic libraries repeatedly identified three different suppressors: the two wild-type alpha-tubulin genes, TUB1 and TUB3; and BUB3. BUB3 is a checkpoint gene that permits entry into mitosis depending upon the assembly state of microtubules. Excess BUB3 rescues both the loss of viability and microtubule defects but not the benomyl supersensitivity associated with tub1-729. The suppression is specific for the mutation ALA422VAL in TUB1, and does not affect several other mutations in TUB1 that produce the ‘no microtubule’ phenotype. Overexpression of BUB1, which interacts genetically with BUB3 and which is involved in the same checkpoint pathway, also rescues the cold sensitivity of tub1-729, but another checkpoint gene, MAD2, does not. Overexpression of BUB3 in wild-type cells has no detectable growth or microtubule defect, but disruption of the BUB3 gene produces slow growth and benomyl supersensitivity. Our results suggest that BUB1 and BUB3 overexpression modulate an event required for mitotic spindle function which is rate limiting for tub1-729 cells at the restrictive temperature.

Sign in / Sign up

Export Citation Format

Share Document