Inhibition of plant cell proteolytic activities that degrade tubulin (original) (raw)
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Monoclonal antibodies specific to plant tubulin
Protoplasma, 1985
Tubulin was isolated from mung bean seedling by a combination of affinity (ethyl N-phenylcarbamate-Sepharose 4 B) and ion exchange (DEAE-Sephacel) chromatography. Using SDS-PAGE together with blotting with subunit-specific antitubulins, mung bean tubulin has been shown to consist of two a-tubulin subunits, MBT 2 and MBT3, of which MBT 3 is a minor component, and one J3-tubulin, MBT 1 . Monoclonal antibodies were produced by fusing mouse myeloma cells and spleen cells from a Balb/c mouse immunized with mung bean tubulin. Antibody producing cell lines were identified by an ELISA assay and immunofluorescence microscopy and subsequently cloned by limiting dilution. The properties of monoclonal antibody (K4ETG3) were examined by Western blot analysis and indirect immunofluoreseence studies. K4ETG 3 reacts with MBT 2 and MBT 3 u-tubulin subunits of mung bean tubulin, but not with MBT 1 13-tubulin nor with the c~-and [3subunits of sheep brain tubulin. Peptide fragments transferred onto nitrocellulose papers were treated with K4E7G 3 and with other monoclonal antibodies that are known to be specific to the c~-subunit of yeast tubulin and ct-or 13-subunit of mammalian brain tubulin. MBT 2 and MBT 3 are shown to be similar but not identical and are quite different from MBT 1 and the 13-subunit of sheep brain tubulin. K4E7G 3 reacts with peptide fragments in MBT 2 and MBT 3 that are not found in digests of brain tubulin, and that are either not reactive or only weakly reactive to the antibodies to yeast and brain ct-tubulin. It is concluded that K4E7G 3 and another monoclonal antibody, K2D7B 8, which has similar properties, are relatively specific for plant u-tubulin. In indirect immunofhiorescence studies on a wide range of plant cells, the epitopes recognised by these monoclonal antibodies are shown to be present in all types of microtubule array that were investigated.
Changes in the accumulation of α- and β-tubulin during bud development in Vitis vinifera L
Planta, 2010
Microtubules play important roles during growth and morphogenesis of plant cells. Multiple isoforms of -and -tubulin accumulate in higher plant cells and originate either by transcription of diVerent genes or by post-translational modiWcations. The use of diVerent tubulin isoforms involves the binding of microtubules to diVerent associated proteins and therefore generates microtubules with diVerent organizations and functions. Tubulin isoforms are diVerentially expressed in vegetative and reproductive structures according to the developmental program of plants. In grapevine (Vitis vinifera L.), vegetative and reproductive structures appear on the same stem, making this plant species an excellent model to study the accumulation of tubulin isoforms. Proteins were extracted from grapevine samples (buds, leaves, Xowers and tendrils) using an optimized extraction protocol, separated by twodimensional electrophoresis and analyzed by immunoblot with anti-tubulin antibodies. We identiWed eight -tubulin and seven -tubulin isoforms with pI around 4.8-5 that group into separate clusters. More acidic -tubulin isoforms were detected in buds, while more basic -isoforms were prevalently found in tendrils and Xowers. Similarly, more acidic -tubulin isoforms were used in the bud stage while a basic -tubulin isoform was essentially used in leaves and two central -tubulin isoforms were characteristically used in tendrils and Xowers. Acetylated -tubulin was not detected in any sample while tyrosinated -tubulin was essentially found in large latent buds and in bursting buds in association with a distinct subset of tubulin isoforms.
Biochemistry, 1988
W e synthesized five peptides homologous to the potentially antigenic positions 4 2 14-226), a(430-443), 4 4 1 5-443), @(241-256), and p(412-431) of the porcine brain tubulin sequences. These peptides were successfully employed to raise tubulin-cross-reactive antibodies. The antibodies are specific of the regions of tubulin spanned by the peptides. They react specifically with the tubulin bands in immunoblots and with microtubules in immunofluorescence assays of cytoskeletons. The peptides of the C-terminal regions have also been employed to localize determinants recognized by two available monoclonal antibodies to tubulin in the positions a(415-430) and @(412-431), respectively. In a first application of the anti-peptide antibodies, we have mapped the fragments of limited proteolysis of purified calf brain tubulin by trypsin, chymotrypsin, papain, thermolysin, subtilisin, and protease V8 from Staphylococcus aureus. Thirty-seven peptides have been identified, of which 32 have been unequivocally aligned into the tubulin sequences on the basis of their antigenic reactivity. There are three major, well-defined zones of preferential cleavage by the proteases: the C-termini and two internal zones in each chain. C-Terminal cleavages of both chains by subtilisin do not remove the antigenic reactivity of the zones a(415-430) and p(412-431). C-Terminal cleavages by protease V8 are preferential of P-tubulin. All six proteases tested cleave a-and/or &tubulin at one or both of the internal zones. These zones are located roughly a t one-third and two-thirds of the chain length in both subunits. Therefore, a model of the tubulin monomers is proposed which consists of three major, proteolytically defined, compact regions (N-terminal, middle, and C-terminal thirds) and the cleavable zones. This model is discussed with the tubulin structural information presently available.
Post-translational modifications and multiple tubulin isoforms in Nicotiana tabacum L. cells
Planta, 1997
Distribution of post-translationally modified tubulins in cells of Nicotiana tabacum L. was analysed using a panel of specific antibodies. Polyglutamylated, tyrosinated, nontyrosinated, acetylated and Δ2-tubulin variants were detected on -tubulin subunits; polyglutamylation was also found on -tubulin subunits. Modified tubulins were detected by immunofluorescence microscopy in interphase microtubules, preprophase bands, mitotic spindles as well as in phragmoplasts. They were, however, located differently in the various microtubule structures. The antibodies against tyrosinated, acetylated and polyglutamylated tubulins gave uniform staining along all microtubules, while antibodies against nontyrosinated and Δ2-tubulin provided dotlike staining of interphase microtubules. Additionally, immunoreactivity of antibodies against acetylated and Δ2-tubulins was strong in the pole regions of mitotic spindles. High-resolution isoelectric focusing revealed 22 tubulin charge variants in N. tabacum suspension cells. Immunoblotting with antibodies TU-01 and TU-06 against conserved antigenic determinants of -andtubulin molecules, respectively, revealed that 11 isoforms belonged to the -subunit and 11 isoforms to thesubunit. Whereas antibodies against polyglutamylated, tyrosinated and acetylated tubulins reacted with several -tubulin isoforms, antibodies against nontyrosinated and Δ2-tubulin reacted with only one. The combined data demonstrate that plant tubulin is extensively posttranslationally modified and that these modifications participate in the generation of plant tubulin polymorphism.
Proceedings of the National Academy of Sciences, 1984
We have initiated immunological and drug-binding studies on the tubulins from different higher plant species. Antibodies were raised against electrophoretically separated rose ( Rosa sp.) tubulin α- and β-subunits and characterized by immunoblot autoradiographic assays. Each IgG preparation bound to its antigen and cross-reacted differentially with the respective tubulin subunits from an alga, sea urchin, rabbit, and cow. Antigenic determinants were shared more among the β-subunits than among the α-subunits from these organisms. Tubulins were isolated from cultured cells of carrot ( Daucus carota ) and hibiscus ( Hibiscus rosa-senensis ). Immunoautoradiography and quantitation of cross-reactivity on blots showed nonidentity among homologous subunits from rose, carrot, hibiscus, and alga tubulins, with more antigenic differences among α-subunits than among β-subunits. Comparative colchicine-binding assays showed that rose and hibiscus tubulins bound 33% and 65%, respectively, of the ...
Detection of β-tubulin in tomato seeds: Optimization of extraction and immunodetection
Phytochemistry, 1998
SDS-PAGE profiles of SDS-denaturated total protein (MODIL) extracts of imbibing tomato seeds showed no large qualitative differences from those of water-soluble (HEPES) proteins. However, the protein concentrations of the HEPES samples were significantly lower than the MODIL ones. The presence of SDS and DTT in MODIL buffer at an alkaline pH were essential for high yields of fl-tubulin. The absence of DTT in HEPES extraction buffer may have allowed oxidation and subsequent loss of fl-tubulin extracted from root tips of germinated seeds. Optimization of the fl-tubulin extraction procedure and improvements of the immunodetection system enabled us to demonstrate the fl-tubulin accumulation pattern in whole tomato seeds and in the different tissues, fl-Tubulin accumulation in seeds was shown to be tissue-dependent, the highest concentration being found in embryo root tip tissue
PLANT PHYSIOLOGY, 1997
The cortical microtubules determine how cellulose microfibrils are deposited in the plant cell wall and are thus important for the control of cell expansion. To understand how microtubules can control microfibril deposition, the components that link the microtubules to the plasma membrane (PM) of plant cells must be isolated. To obtain information on the properties of the tubulin-membrane associations, cauliflower (Brassica oleracea) PM was subjected to Triton X-114 fractionation, and the distribution of α-- and β-tubulin was analyzed using immunoblotting. Approximately one-half of the PM-associated tubulin was solubilized by Triton X-114 and 10 to 15% of both α-- and β-tubulin was recovered in the detergent phase (indicative of hydrophobic properties) and 30 to 40% was recovered in the aqueous phase. The hydrophobic tubulin could be released from the membrane by high pH extraction with preserved hydrophobicity. A large part of the PM-associated tubulin was found in the Triton-insol...
Tyrosine phosphorylation of plant tubulin
Planta, 2008
Phosphorylation of -tubulins dimers by protein tyrosine kinases plays an important role in the regulation of cellular growth and diVerentiation in animal cells. In plants, however, the role of tubulin tyrosine phosphorylation is unknown and data on this tubulin modiWcation are limited. In this study, we used an immunochemical approach to demonstrate that tubulin isolated by both immunoprecipitation and DEAE-chromatography is phosphorylated on tyrosine residues in cultured cells of Nicotiana tabacum. This opens up the possibility that tyrosine phosphorylation of tubulin could be involved in modulating the properties of plant microtubules.