IDENTIFICATION AND PRELIMINARY CHARACTERIZATION OF A 65-KDA HIGHER-PLANT MICROTUBULE-ASSOCIATED PROTEIN

Microtubules in plant cells, as in animal cells, are dynamic structure s. However, our lack of knowledge about the constituents of microtubul es in plant cells has prevented us from understanding the mechanisms t hat control microtubule dynamics. To characterize some of these consti tuents, a cytoplasmic extract was prepared from evacuolated protoplast s (miniprotoplasts) of tobacco BY-2 cells, and microtubules were assem bled in the presence of taxol and disassembled by cold treatment in th e presence of Ca2+ and a high concentration of NaCl. SDS-PAGE analysis of triple-cycled microtubule protein revealed the presence of 120 kDa , 110 kDa and a group of 60-65 kDa polypeptides in addition to tubulin . Since these polypeptides had copolymerized with tubulin, through the three cycles of assembly and disassembly, and they bundle microtubule s, we tentatively identified the three polypeptides as microtubule-ass ociated proteins (MAPs). To characterize these factors further, triple -cycled microtubule protein was fractionated by Mono-Q anion-exchange chromatography and the microtubule-bundling activity of each fraction was examined. Fractions having microtubule-bundling activity contained only the 65 kDa MAP, an indication that the 65 kDa MAP is responsible for the bundling of microtubules. Purified 65 kDa MAP formed cross-br idge structures between adjacent microtubules in vitro. Polyclonal ant ibodies were raised in mice against the 65 kDa MAP. Immunofluorescence microscopy revealed that the 65 kDa MAP colocalized with microtubules in BY-2 cells throughout the cell cycle. Western blotting analysis of extracts from several species of plants suggested that the 65 kDa MAP and/or related peptides are widely distributed in the plant kingdom.