An α-Tubulin Mutant Destabilizes the Heterodimer: Phenotypic Consequences and Interactions with Tubulin-binding Proteins

Many effectors of microtubule assembly in vitro enhance the polymerization of subunits. However, several Saccharomyces cerevisiae genes that affect cellular microtubule-dependent processes appear to act at other steps in assembly and to affect polymerization only indirectly. Here we use a mutant α-tubulin to probe cellular regulation of microtubule assembly.tub1-724 mutant cells arrest at low temperature with no assembled microtubules. The results of several assays reported here demonstrate that the heterodimer formed between Tub1-724p and β-tubulin is less stable than wild-type heterodimer. The unstable heterodimer explains several conditional phenotypes conferred by the mutation. These include the lethality of tub1-724haploid cells when the β-tubulin–binding protein Rbl2p is either overexpressed or absent. It also explains why theTUB1/tub1-724 heterozygotes are cold sensitive for growth and why overexpression of Rbl2p rescues that conditional lethality. Both haploid and heterozygous tub1-724 cells are inviable when another microtubule effector, PAC2, is overexpressed. These effects are explained by the ability of Pac2p to bind α-tubulin, a complex we demonstrate directly. The results suggest that tubulin-binding proteins can participate in equilibria between the heterodimer and its components.

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Function of Tubulin Binding Proteins in Vivo

Genetics ◽

10.1093/genetics/156.1.69 ◽

2000 ◽

Vol 156 (1) ◽

pp. 69-80 ◽

Cited By ~ 1

Author(s):

James A Fleming ◽

Leticia R Vega ◽

Frank Solomon

Keyword(s):

Binding Proteins ◽

Binding Protein ◽

Yeast Cells ◽

Gene Products ◽

Salvage Pathway ◽

Tubulin Binding ◽

Mutant Cells ◽

Β Tubulin

Abstract Overexpression of the β-tubulin binding protein Rbl2p/cofactor A is lethal in yeast cells expressing a mutant α-tubulin, tub1-724, that produces unstable heterodimer. Here we use RBL2 overexpression to identify mutations in other genes that affect formation or stability of heterodimer. This approach identifies four genes—CIN1, CIN2, CIN4, and PAC2—as affecting heterodimer formation in vivo. The vertebrate homologues of two of these gene products—Cin1p/cofactor D and Pac2p/cofactor E—can catalyze exchange of tubulin polypeptides into preexisting heterodimer in vitro. Previous work suggests that both Cin2p or Cin4p act in concert with Cin1p in yeast, but no role for vertebrate homologues of either has been reported in the in vitro reaction. Results presented here demonstrate that these proteins can promote heterodimer formation in vivo. RBL2 overexpression in cin1 and pac2 mutant cells causes microtubule disassembly and enhanced formation of Rbl2p-β-tubulin complex, as it does in the α-tubulin mutant that produces weakened heterodimer. Significantly, excess Cin1p/cofactor D suppresses the conditional phenotypes of that mutant α-tubulin. Although none of the four genes is essential for viability under normal conditions, they become essential under conditions where the levels of dissociated tubulin polypeptides increase. Therefore, these proteins may provide a salvage pathway for dissociated tubulin heterodimers and so rescue cells from the deleterious effects of free β-tubulin.

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Protection from Free β-Tubulin by the β-Tubulin Binding Protein Rbl2p

Molecular and Cellular Biology ◽

10.1128/mcb.22.1.138-147.2002 ◽

2002 ◽

Vol 22 (1) ◽

pp. 138-147 ◽

Cited By ~ 24

Author(s):

Katharine C. Abruzzi ◽

Adelle Smith ◽

William Chen ◽

Frank Solomon

Keyword(s):

Binding Protein ◽

In Vitro Assay ◽

In Vitro Studies ◽

Microtubule Assembly ◽

Vitro Assay ◽

Tubulin Binding ◽

And Function ◽

Β Tubulin ◽

In Cells

ABSTRACT Free β-tubulin not in heterodimers with α-tubulin can be toxic, disrupting microtubule assembly and function. We are interested in the mechanisms by which cells protect themselves from free β-tubulin. This study focused specifically on the function of Rbl2p, which, like α-tubulin, can rescue cells from free β-tubulin. In vitro studies of the mammalian homolog of Rbl2p, cofactor A, have suggested that Rbl2p/cofactor A may be involved in tubulin folding. Here we show that Rbl2p becomes essential in cells containing a modest excess of β-tubulin relative to α-tubulin. However, this essential activity of Rbl2p/cofactorA does not depend upon the reactions described by the in vitro assay. Rescue of β-tubulin toxicity requires a minimal but substoichiometric ratio of Rbl2p to β-tubulin. The data suggest that Rbl2p binds transiently to free β-tubulin, which then passes into an aggregated form that is not toxic.

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Microtubule Regulation in Mitosis: Tubulin Phosphorylation by the Cyclin-dependent Kinase Cdk1

Molecular Biology of the Cell ◽

10.1091/mbc.e05-07-0621 ◽

2006 ◽

Vol 17 (3) ◽

pp. 1041-1050 ◽

Cited By ~ 103

Author(s):

Anne Fourest-Lieuvin ◽

Leticia Peris ◽

Vincent Gache ◽

Isabel Garcia-Saez ◽

Céline Juillan-Binard ◽

...

Keyword(s):

Binding Proteins ◽

Microtubule Dynamics ◽

Cyclin Dependent Kinase ◽

Gtp Binding ◽

Tubulin Binding ◽

A Site ◽

Β Tubulin ◽

In Cells

The activation of the cyclin-depdndent kinase Cdk1 at the transition from interphase to mitosis induces important changes in microtubule dynamics. Cdk1 phosphorylates a number of microtubule- or tubulin-binding proteins but, hitherto, tubulin itself has not been detected as a Cdk1 substrate. Here we show that Cdk1 phosphorylates β-tubulin both in vitro and in vivo. Phosphorylation occurs on Ser172 of β-tubulin, a site that is well conserved in evolution. Using a phosphopeptide antibody, we find that a fraction of the cell tubulin is phosphorylated during mitosis, and this tubulin phosphorylation is inhibited by the Cdk1 inhibitor roscovitine. In mitotic cells, phosphorylated tubulin is excluded from microtubules, being present in the soluble tubulin fraction. Consistent with this distribution in cells, the incorporation of Cdk1-phosphorylated tubulin into growing microtubules is impaired in vitro. Additionally, EGFP-β3-tubulinS172D/E mutants that mimic phosphorylated tubulin are unable to incorporate into microtubules when expressed in cells. Modeling shows that the presence of a phosphoserine at position 172 may impair both GTP binding to β-tubulin and interactions between tubulin dimers. These data indicate that phosphorylation of tubulin by Cdk1 could be involved in the regulation of microtubule dynamics during mitosis.

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

Applied Sciences ◽

10.3390/app11156865 ◽

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.

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

Genetics ◽

10.1093/genetics/137.2.381 ◽

1994 ◽

Vol 137 (2) ◽

pp. 381-392 ◽

Cited By ~ 4

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.

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The double-stranded DNA-binding proteins TEBP-1 and TEBP-2 form a telomeric complex with POT-1

10.1101/2020.08.19.256388 ◽

2020 ◽

Author(s):

Sabrina Dietz ◽

Miguel Vasconcelos Almeida ◽

Emily Nischwitz ◽

Jan Schreier ◽

Nikenza Viceconte ◽

...

Keyword(s):

Quantitative Proteomics ◽

Binding Proteins ◽

Protein Complexes ◽

Wild Type ◽

C Elegans ◽

Double Stranded Dna ◽

Telomere Sequence ◽

Proteomics Approach

AbstractTelomeres are bound by dedicated protein complexes, like shelterin in mammals, which protect telomeres from DNA damage. In the nematode Caenorhabditis elegans, a comprehensive understanding of the proteins interacting with the telomere sequence is lacking. Here, we harnessed a quantitative proteomics approach to screen for proteins binding to C. elegans telomeres, and identified TEBP-1 and TEBP-2, two paralogs that associate to telomeres in vitro and in vivo. TEBP-1 and TEBP-2 are expressed in the germline and during embryogenesis. tebp-1 and tebp-2 mutants display strikingly distinct phenotypes: tebp-1 mutants have longer telomeres than wild-type animals, while tebp-2 mutants display shorter telomeres and a mortal germline, a phenotype characterized by transgenerational germline deterioration. Notably, tebp-1; tebp-2 double mutant animals have synthetic sterility, with germlines showing signs of severe mitotic and meiotic arrest. TEBP-1 and TEBP-2 form a telomeric complex with the known single-stranded telomere-binding proteins POT-1, POT-2, and MRT-1. Furthermore, we find that POT-1 bridges the double- stranded binders TEBP-1 and TEBP-2, with the single-stranded binders POT-2 and MRT-1. These results describe the first telomere-binding complex in C. elegans, with TEBP-1 and TEBP-2, two double-stranded telomere binders required for fertility and that mediate opposite telomere dynamics.

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Correction of the defect in initiation of DNA replication in a temperature-sensitive mutant hamster cell line by in vitro addition of extracts from normal cells

Molecular and Cellular Biology ◽

10.1128/mcb.5.4.902-905.1985 ◽

1985 ◽

Vol 5 (4) ◽

pp. 902-905

Author(s):

M Narkhammar ◽

R Hand

Keyword(s):

Cell Line ◽

Nuclear Extract ◽

Cell Extract ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Permissive Temperature ◽

Wild Type ◽

Whole Cell ◽

Mutant Cells

ts BN-2 is a temperature-sensitive hamster cell line that is defective in DNA synthesis at the restrictive temperature. The mutant expresses its defect during in vitro replication in whole-cell lysates. Addition of a high-salt-concentration extract from wild-type BHK-21, revertant RBN-2, or CHO cells to mutant cells lysed with 0.01% Brij 58 increased the activity in the mutant three- to fourfold, so that it reached 85% of the control value, and restored replicative synthesis. The presence of extract had an insignificant effect on wild-type and revertant replication and on mutant replication at the permissive temperature. Extract prepared from mutant cells was less effective than the wild-type cell extract was. Also, the stimulatory activity was more heat labile in the mutant than in the wild-type extract. Nuclear extract was as active as whole-cell extract.

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Loss of the F-Actin Binding and Vesicle-Associated Protein Comitin Leads to a Phagocytosis Defect

Eukaryotic Cell ◽

10.1128/ec.1.6.906-914.2002 ◽

2002 ◽

Vol 1 (6) ◽

pp. 906-914 ◽

Cited By ~ 18

Author(s):

Thomas Schreiner ◽

Martina R. Mohrs ◽

Rosemarie Blau-Wasser ◽

Alfred von Krempelhuber ◽

Michael Steinert ◽

...

Keyword(s):

Pathogenic Bacteria ◽

Gene Replacement ◽

Actin Binding ◽

Wild Type ◽

Hyperosmotic Shock ◽

Latex Beads ◽

Knockout Mutants ◽

Mutant Cells

ABSTRACT Comitin is an F-actin binding and membrane-associated protein from Dictyostelium discoideum, which is present on Golgi and vesicle membranes and changes its localization in response to agents affecting the cytoskeleton. To investigate its in vivo functions we have generated knockout mutants by gene replacement. Based on comitin's in vitro functions we examined properties related to vesicular transport and microfilament function. Whereas cell growth, pinocytosis, secretion, chemotaxis, motility, and development were unaltered, comitin-lacking cells were impaired in the early steps of phagocytosis of Saccharomyces cerevisiae particles and of Escherichia coli, whereas uptake of latex beads was unaffected. Furthermore, the lack of comitin positively affected survival of pathogenic bacteria. Mutant cells also showed an altered response to hyperosmotic shock in comparison to the wild type. The redistribution of comitin during hyperosmotic shock in wild-type cells and its presence on early phagosomes suggest a direct involvement of comitin in these processes.

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Diffusion rather than intraflagellar transport likely provides most of the tubulin required for axonemal assembly in Chlamydomonas

Journal of Cell Science ◽

10.1242/jcs.249805 ◽

2020 ◽

Vol 133 (17) ◽

pp. jcs249805 ◽

Cited By ~ 1

Author(s):

Julie Craft Van De Weghe ◽

J. Aaron Harris ◽

Tomohiro Kubo ◽

George B. Witman ◽

Karl F. Lechtreck

Keyword(s):

Binding Site ◽

Critical Concentration ◽

Intraflagellar Transport ◽

Microtubule Assembly ◽

Strong Reduction ◽

Tubulin Binding ◽

Respective Contribution ◽

Β Tubulin

ABSTRACTTubulin enters the cilium by diffusion and motor-based intraflagellar transport (IFT). However, the respective contribution of each route in providing tubulin for axonemal assembly remains unknown. Using Chlamydomonas, we attenuated IFT-based tubulin transport of GFP–β-tubulin by altering the IFT74N–IFT81N tubulin-binding module and the C-terminal E-hook of tubulin. E-hook-deficient GFP–β-tubulin was incorporated into the axonemal microtubules, but its transport frequency by IFT was reduced by ∼90% in control cells and essentially abolished when the tubulin-binding site of IFT81 was incapacitated. Despite the strong reduction in IFT, the proportion of E-hook-deficient GFP–β-tubulin in the axoneme was only moderately reduced. In vivo imaging showed more GFP–β-tubulin particles entering cilia by diffusion than by IFT. Extrapolated to endogenous tubulin, the data indicate that diffusion provides most of the tubulin required for axonemal assembly. We propose that IFT of tubulin is nevertheless needed for ciliogenesis, because it augments the tubulin pool supplied to the ciliary tip by diffusion, thus ensuring that free tubulin there is maintained at the critical concentration for plus-end microtubule assembly during rapid ciliary growth.

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β-Tubulin C354 Mutations that Severely Decrease Microtubule Dynamics Do Not Prevent Nuclear Migration in Yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e02-01-0003 ◽

2002 ◽

Vol 13 (8) ◽

pp. 2919-2932 ◽

Cited By ~ 51

Author(s):

Mohan L. Gupta ◽

Claudia J. Bode ◽

Douglas A. Thrower ◽

Chad G. Pearson ◽

Kathy A. Suprenant ◽

...

Keyword(s):

Essential Gene ◽

Dynamic Properties ◽

Microtubule Dynamics ◽

Cytoplasmic Microtubule ◽

Bud Emergence ◽

Spindle Elongation ◽

Mutant Cells ◽

Β Tubulin

Microtubule dynamics are influenced by interactions of microtubules with cellular factors and by changes in the primary sequence of the tubulin molecule. Mutations of yeast β-tubulin C354, which is located near the binding site of some antimitotic compounds, reduce microtubule dynamicity greater than 90% in vivo and in vitro. The resulting intrinsically stable microtubules allowed us to determine which, if any, cellular processes are dependent on dynamic microtubules. The average number of cytoplasmic microtubules decreased from 3 in wild-type to 1 in mutant cells. The single microtubule effectively located the bud site before bud emergence. Although spindles were positioned near the bud neck at the onset of anaphase, the mutant cells were deficient in preanaphase spindle alignment along the mother-bud axis. Spindle microtubule dynamics and spindle elongation rates were also severely depressed in the mutants. The pattern and extent of cytoplasmic microtubule dynamics modulation through the cell cycle may reveal the minimum dynamic properties required to support growth. The ability to alter intrinsic microtubule dynamics and determine the in vivo phenotype of cells expressing the mutant tubulin provides a critical advance in assessing the dynamic requirements of an essential gene function.

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