RESUMEN
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) in vitro. Binding saturation occurred at 1 mol of p86 per 5-6 mol of polymerized tubulin dimer, demonstrating a substoichiometric interaction of p86 with MTs. No evidence was found for a direct interaction of the p28 subunit with MTs. Unlike kinesin, cosedimentation of eIF-(iso)4F with MTs was neither reduced by MgATP nor enhanced by adenosine 5'-[gamma-imido]triphosphate. Both p86 subunit and p86-p28 complex induced the bundling of MTs in vitro. The p86 subunit was immunolocalized to the cytosol in root maize cells and existed in three forms: fine particles, coarse particles, and linear patches. Many coarse particles and linear patches were colocalized or closely associated with cortical MT bundles in interphase cells. The results indicate that the p86 subunit of eIF-(iso)4F is a MT-associated protein that may simultaneously link the translational machinery to the cytoskeleton and regulate MT disposition in plant cells.
Asunto(s)
Proteínas de Arabidopsis , Isoenzimas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Zea mays/metabolismo , Factor 4F Eucariótico de Iniciación , Técnica del Anticuerpo Fluorescente , Immunoblotting , Isoenzimas/genética , Isoenzimas/aislamiento & purificación , Isoenzimas/ultraestructura , Cinesinas/genética , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/aislamiento & purificación , Proteínas Asociadas a Microtúbulos/ultraestructura , Microtúbulos/ultraestructura , Factores de Iniciación de Péptidos/genética , Factores de Iniciación de Péptidos/aislamiento & purificación , Factores de Iniciación de Péptidos/ultraestructura , Proteínas de Plantas/genética , Raíces de Plantas/ultraestructura , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/metabolismo , Zea mays/química , Zea mays/genéticaRESUMEN
Amiprophos-methyl (APM), a phosphoric amide herbicide, was previously reported to inhibit the in vitro polymerization of isolated plant tubulin (L.C. Morejohn, D.E. Fosket [1984] Science 224: 874-876), yet little other biochemical information exists concerning this compound. To characterize further the mechanism of action of APM, its interactions with tubulin and microtubules purified from cultured cells of tobacco (Nicotiana tabacum cv Bright Yellow-2) were investigated. Low micromolar concentrations of APM depolymerized preformed, taxol-stabilized tobacco microtubules. Remarkably, at the lowest APM concentration examined, many short microtubules were redistributed into fewer but 2.7-fold longer microtubules without a substantial decrease in total polymer mass, a result consistent with an end-to-end annealing of microtubules with enhanced kinetic properties. Quasi-equilibrium binding measurements showed that tobacco tubulin binds [14C]oryzalin with high affinity to produce a tubulin-oryzalin complex having a dissociation constant (Kd) = 117 nM (pH 6.9; 23[deg]C). Also, an estimated maximum molar binding stoichiometry of 0.32 indicates pharamacological heterogeneity of tobacco dimers and may be related to structural heterogeneity of tobacco tubulin subunits. APM inhibits competitively the binding of [14C]oryzalin to tubulin with an inhibition constant (Ki) = 5 [mu]M, indicating the formation of a moderate affinity tubulin-APM complex that may interact with the ends of microtubules. APM concentrations inhibiting tobacco cell growth were within the threshold range of APM concentrations that depolymerized cellular microtubules, indicating that growth inhibition is caused by microtubules depolymerization. APM had no apparent effect on microtubules in mouse 3T3 fibroblasts. Because cellular microtubules were depolymerized at APM and oryzalin concentrations below their respective Ki and Kd values, both herbicides are proposed to depolymerize microtubules by a substoichiometric endwise mechanism.
RESUMEN
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 neuronal 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 mammalian MAP2 binding site and an additional low-affinity site also 12 dimers apart that may be homologous to the mammalian tau binding site. MAP2 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 microM. However, MAP2 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 MAP2 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.
Asunto(s)
Proteínas Asociadas a Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Zea mays/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Biopolímeros/metabolismo , Encéfalo/metabolismo , Bovinos , Técnicas In Vitro , Microscopía Electrónica , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Datos de Secuencia Molecular , Paclitaxel/farmacología , Unión Proteica/genética , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Tubulina (Proteína)/genética , Tubulina (Proteína)/ultraestructura , Zea mays/genética , Zea mays/ultraestructuraRESUMEN
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.