Taxol has been reported to induce the polymerization of plant tubulin into microtubules, albeit weakly when compared to that of mammalian tubulin [Morejohn, L.C., & Fosket, D.E. (1984) J. Cell Biol. 99, 141-147], suggesting that taxol, a product of plant secondary metabolism, may interact poorly with plant microtubules. To test this idea in detail, we have investigated critical parameters affecting taxol-dependent microtubule polymerization and stability using tubulins from model cell lines of maize [Zea mays cv. Black Mexican Sweet (BMS)] and tobacco [Nicotiana tabacum cv. Bright Yellow 2 (BY-2)]. When plant tubulin dimer is isolated by using a modified version of the original method [Morejohn, L.C., & Fosket, D.E. (1982) Nature 297, 426-428], most of the tubulin polymerizes at 25 degrees C, with critical dimer concentrations (Cc) of 0.06 mg/mL for BMS tubulin and 0.13 mg/mL for BY-2 tubulin. When taxol-induced assembly is initiated with a 0-25 degrees C temperature jump, 42% of polymer is polymorphic, presumably due to aberrant nucleation events. Taxol-induced assembly at 2 degrees C minimizes the formation of polymorphic structures and is much more rapid than that of purified bovine brain tubulin, indicating a functional difference in the polymerization domains of these diverse tubulins. Temperature ramping during taxol-induced polymerization affords > or = 95% assembly of plant tubulin into polymer consisting of 86% microtubules, which may be completely depolymerized by a combined treatment with low temperature and Ca2+. We report for the first time that plant tubulin may be subjected to numerous cycles of efficient taxol-induced polymerization and cold/Ca(2+)-induced depolymerization with little loss of polymerization competence. Gel filtration chromatography at low temperature may be used to separate taxol from soluble plant tubulin dimer, which retains its characteristic polymerization and herbicide-binding properties. Our results demonstrate that despite its origin from plants, taxol is a potent drug for the reversible polymerization of plant microtubules.

The isozyme form of eukaryotic initiation factor 4F [eIF-(iso)4F] from wheat germ is composed of a p28 subunit that binds the 7-methylguanine cap of mRNA and a p86 subunit having unknown function. The p86 subunit was found to have limited sequence similarity to a kinesin-like protein encoded by the katA gene of Arabidopsis thaliana. Native wheat germ eIF-(iso)4F and bacterially expressed p86 subunit and p86-p28 complex bound to taxol-stabilized maize microtubules (MTs) Numerous studies have provided evidence for interactions between the cytoskeleton and the eukaryotic protein synthesis machinery. Polysomal mRNAs, ribosomes, and certain translation factors are associated with the cytoskeleton from detergent-extracted animal cells (1-4). Treatment of HeLa cells with the microfilament-disrupting agent cytochalasin releases mRNA, ribosomes, and initiation factors from the cytoskeletal framework (1, 3, 4). Polyribosomes have been found associated with cytoskeleton preparations from plants as well (5-7). Certain translation factors have been immunolocalized to the cytoskeleton of animal (8-10) and plant (11) cells. The translational machinery appears to have a relatively stable association with the cytoskeleton in animal cells, because detergentextracted cytoskeletons (12) and saponin-permeabilized cells (13) engage in efficient translation without the addition of macromolecular translation components. Thus, these observations indicate that the translational machinery may be bound to and regulated by the cytoskeleton.A different perspective is provided, however, by mounting evidence that protein synthesis factors may influence cytoskeletal function. For example, the existence of homologs of elongation factor la (EF-la) in putative centrosome precursor particles from CHO cells (14) and in microtubule (MT)-organizing centers of sea urchin mitotic spindles (15-17) suggests binding of EF-l1a to the minus ends of MTs and a function in MT nucleation. A similar MT-organizing role of a putative EF-la in tobacco cells was deduced from immuno-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. localization patterns produced by antibodies against a sea urchin centrosomal EF-la homolog (11). However, animal EF-la was recently reported to sever MTs deficient in MTassociated proteins (MAPs) (18), a result we have confirmed with plant EF-la and MTs (L. E. Littlepage and L.C.M., unpublished data). Thus, these observations indicate that, under particular conditions, EF-la may modulate the disposition of MTs. Other than EF-la, however, no other translation factor has been demonstrated to affect MTs.The isozyme form of eukaryotic initiation factor 4F [eIF-(iso)4F] from wheat germ is composed of equimolar amounts of a small subunit (p28) that binds the 7-methylguanine (m7G) cap of mRNAs and a large subunit (p86) that has unknown function during translation and limited sequ...

An understanding of the regulation of microtubule polymerization and dynamics in plant cells requires biochemical information on the structures, functions, and molecular interactions of plant tubulin and microtubule-associated proteins (MAPs) that regulate microtubule function. We have probed the regulatory domain and polymerization domain of purified maize tubulin using MAP2, an extensively characterized mammalian neumnal MAP. MAP2 bound to the surface of preformed, taxol-stabilized maize microtubules, with binding saturation occurring with one MAP2 molecule per five to six tubulin dimers, as it does with mammalian microtubules. MAP2 binding and dissociation analyses revealed two affinity classes of binding sites on maize microtubules: a high-affinity site 12 dimers apart that may be homologous to the cognate mammalian MAP2 binding site and an additional low-affinity site also 12 dimers apart that may be homologous to the mammalian tau binding site. MAPS corrected in vitro folding errors in taxol-stabilized maize microtubules and reduced the critical concentration of maize tubulin polymerization eightfold, from 8.3 to 1.0 pM. However, MAPS dissociated much more readily from maize microtubules than from mammalian microtubules and induced the assembly of maize tubulin into aberrant helical ribbon polymers that remained stable for prolonged periods. Our results indicated that MAPS binds to maize tubulin via a partially specific, low-fidelity interaction that reflects unique structural and functional properties of the polymerization and regulatory domains of plant tubulin and possibly of the tubulin binding domains of undocumented MAPs that regulate microtubule function in plant cells.

An understanding of the regulation of microtubule polymerization and dynamics in plant cells requires biochemical information on the structures, functions, and molecular interactions of plant tubulin and microtubule-associated proteins (MAPs) that regulate microtubule function. We have probed the regulatory domain and polymerization domain of purified maize tubulin using MAP2, an extensively characterized mammalian neumnal MAP. MAP2 bound to the surface of preformed, taxol-stabilized maize microtubules, with binding saturation occurring with one MAP2 molecule per five to six tubulin dimers, as it does with mammalian microtubules. MAP2 binding and dissociation analyses revealed two affinity classes of binding sites on maize microtubules: a high-affinity site 12 dimers apart that may be homologous to the cognate mammalian MAP2 binding site and an additional low-affinity site also 12 dimers apart that may be homologous to the mammalian tau binding site. MAPS corrected in vitro folding errors in taxol-stabilized maize microtubules and reduced the critical concentration of maize tubulin polymerization eightfold, from 8.3 to 1.0 pM. However, MAPS dissociated much more readily from maize microtubules than from mammalian microtubules and induced the assembly of maize tubulin into aberrant helical ribbon polymers that remained stable for prolonged periods. Our results indicated that MAPS binds to maize tubulin via a partially specific, low-fidelity interaction that reflects unique structural and functional properties of the polymerization and regulatory domains of plant tubulin and possibly of the tubulin binding domains of undocumented MAPs that regulate microtubule function in plant cells.

To obtain intbrination on plant microtubuic stability to low temperature and Ca^*, the regulatory domain of polymerized tubulin from maize {Zea mays cv. Black Mexican Sweet) was dissected by limited proteolysis with snbtilisin. Tubulin in taxol-stabilized microtubules was cleaved in a subtilisin concentration-and time-dependent manner. Immunoblotting of microtubules with antibodies having mapped epitopes on a-and /^tubnlins revealed that eleavage initially removed <15 residues IVom the /3-tubulin carboxyl terminus to prodnec a/i^-microtubules. Subsequent cleavage occurred at an extreme site and an internal site within the a-tubniin carboxyl terminus. Electron microscopy revealed that a/3s-microtnbules were ultrastructurally indistinguishable from uncleaved control a^microtubules. Quantitative polymer sedimentation showed that low temperature treatment (0 °C) caused significant depolymcrization of a/J-microtnbules, but little depolymerization of a/Jj-microtubules. Ca^* enhanced the cold-induced depolymerization of both aft-and a/Js-microtubnles. However, a/iJ^-microtubules were significantly more stable to depolymerization by cold and Ca^* than were a/iJ-niicrotubules. The results showed that maize microtubules containing shortened /^tubiilin carboxyl termini are relatively resistant to the combined depolymerizing effects of cold and Ca^*. Thus, the extreme carboxyl terminus of /3-tubulin is a crucial element of the plant tubulin regulatory domain and may be involved in the modulation of microtubule stability during the chilling response in plants.