Post-translational modifications and multiple tubulin isoforms in Nicotiana tabacum L. cells (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.
Mutant Genes of Plant Tubulins As Selective Marker Genes
This review describes different approaches to employment of new marker genes in selection of transformed plant cells, which are based on the use of mutant tubulin genes from natural plant biotypes and, in prospect, induced plant mutants. The results of studies of plant (biotypes, mutants) resistance to herbicides with antimicrotubular mode of action at molecular and cellular levels were summarized. The reports on the transfer and expression of mutant tubulin genes conferring resistance to amiprophosmethyl (phosphorothioamidate herbicide) and trifluralin (dinitroaniline herbicide) from corresponding Nicotiana plumbaginifolia mutants in related and remote plant species by somatic hybridization methods were analyzed. The results of experiments on transformation of monocotyledonous and dicotyledonous plants by mutant α-tubulin gene conferring resistance to dinitroanilines are described to test the possibility of its use as a marker gene and simultaneously obtaining dinitroaniline-resistant plants.
BMC Plant Biology, 2010
Background: The function of the cortical microtubules, composed of αβ-tubulin heterodimers, is linked to their organizational state which is subject to spatial and temporal modulation by environmental cues. The role of tubulin posttranslational modifications in these processes is largely unknown. Although antibodies against small tubulin regions represent useful tool for studying molecular configuration of microtubules, data on the exposure of tubulin epitopes on plant microtubules are still limited. Results: Using homology modeling we have generated an Arabidopsis thaliana microtubule protofilament model that served for the prediction of surface exposure of five β-tubulin epitopes as well as tyrosine residues. Peptide scans newly disclosed the position of epitopes detected by antibodies 18D6 (β1-10), TUB2.1 (β426-435) and . Experimental verification of the results by immunofluorescence microscopy revealed that the exposure of epitopes depended on the mode of fixation. Moreover, homology modeling showed that only tyrosines in the C-terminal region of β-tubulins (behind β425) were exposed on the microtubule external side. Immunofluorescence microscopy revealed tyrosine phosphorylation of microtubules in plant cells, implying that β-tubulins could be one of the targets for tyrosine kinases.
Plant gamma-Tubulin Interacts with alphabeta-Tubulin Dimers and Forms Membrane-Associated Complexes
THE PLANT CELL ONLINE, 2003
␥ -Tubulin is assumed to participate in microtubule nucleation in acentrosomal plant cells, but the underlying molecular mechanisms are still unknown. Here, we show that ␥ -tubulin is present in protein complexes of various sizes and different subcellular locations in Arabidopsis and fava bean. Immunoprecipitation experiments revealed an association of ␥ -tubulin with ␣ -tubulin dimers. ␥ -Tubulin cosedimented with microtubules polymerized in vitro and localized along their whole length. Large ␥ -tubulin complexes resistant to salt treatment were found to be associated with a highspeed microsomal fraction. Blue native electrophoresis of detergent-solubilized microsomes showed that the molecular mass of the complexes was Ͼ 1 MD. Large ␥ -tubulin complexes were active in microtubule nucleation, but nucleation activity was not observed for the smaller complexes. Punctate ␥ -tubulin staining was associated with microtubule arrays, accumulated with short kinetochore microtubules interacting in polar regions with membranes, and localized in the vicinity of nuclei and in the area of cell plate formation. Our results indicate that the association of ␥ -tubulin complexes with dynamic membranes might ensure the flexibility of noncentrosomal microtubule nucleation. Moreover, the presence of other molecular forms of ␥ -tubulin suggests additional roles for this protein species in microtubule organization.
Tubulin evolution: Two major types of α-tubulin
Journal of Molecular Evolution, 1982
Tubulin subunits have been isolated from a variety of protists and marine invertebrates. The sources were: sperm tails of a tunicate (Ciona intestinalisJ, an abalone (Haliotis rufescens) and a sea anemone (Tealia crassicornis), the gall cilia of a clam (Mercenaria mercenaria), the cilia of a ciliate (Tetrahymena pyriformis) and the cytoplasm of a slime mold (Physarum polycephalum). All the/3-tubulins, as characterised by their electropherograms after limited proteolytic cleavage with Staphylococcus aureus protease, were fairly similar.
Two γ -tubulin isoforms are differentially expressed during development in Helianthus annuus
Physiologia Plantarum, 2001
The cytoskeleton is involved in major developmental events in munoblotting, with bands at 52 and 58 kDa. The larger plant cell growth and differentiation. Nucleation events play a key role in the dynamic and organization of the microtubule -tubulin (58 kDa) is present in all the sunflower tissues tested and is associated with the nucleus. The smaller -tubulin (52 (Mt) cytoskeleton. Among many proteins involved in Mt kDa), differing from the former at the carboxy-terminal end, nucleation, -tubulin has been identified as an essential com-