Unique Functional Characteristics of the Polymerization and MAP Binding Regulatory Domains of Plant Tubulin

Abstract

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.

Function of the p86 subunit of eukaryotic initiation factor (iso)4F as a microtubule-associated protein in plant cells.

“…1 shows that NaCl inhibited eIF-(iso)4F binding to MTs. Because MAP2 binding to plant and animal MTs is little affected by 75 mM KCl (24,25), eIF-(iso)4F binding to MTs must be of relatively low affinity.…”

“…Polymer sedimentation analysis and EM were as described (25). Protein determinations were done by using dye binding (26) as described (27).…”

“…Proteins were analyzed by SDS/PAGE (8 or 10% polyacrylamide) (29). Gels were stained with Coomassie blue and analyzed by quantitative densitometry as described (25). In cases where bands were distorted, gel images were produced with a Color OneScanner (Apple, Cupertino, CA), saved as 8-bit TIFF files, and analyzed with NIH IMAGE program (version 1.52) (30).…”

Identification of a Rice RNA- and Microtubule-binding Protein as the Multifunctional Protein, a Peroxisomal Enzyme Involved in the β-Oxidation of Fatty Acids

“…Due to the similarity of the MAP fraction profiles from each column, bovine tubulin columns were used for all subsequent large scale isolations of MAP fractions because of the ease of purifying high quantities of tubulin from bovine brain and the increased stability of the tubulin columns compared with microtubule columns. The interaction of plant MAPs with animal tubulin has been well documented (35,43,44). Two-dimensional NEPHGE demonstrated that the rice MAP fraction that bound to the bovine tubulin column contained ϳ40 -50 abundant proteins and about 100 less abundant proteins (Fig.…”

A calmodulin-sensitive interaction between microtubules and a higher plant homolog of elongation factor-1 alpha.

“…Tubulin prepared from brain has been used because it can be more highly purified in greater yields, and studies on heterologous versus homologous MT-MAP interactions using brain or plant proteins show that either type of interaction produces similar results except for some quantitative variation (Hugdahl et al, 1993;Schellenbaum et al, 1993). Nevertheless, we investigated the interaction of ppSO with taxol-stabilized MTs of pure carrot tubulin.…”