ISOLATION OF A 90-KD MICROTUBULE-ASSOCIATED PROTEIN FROM TOBACCO MEMBRANES

The organization and function of microtubules in plant cells are impor tant in key developmental events, including the regulation of directio nal cellulose deposition. Bridges connecting microtubules to each othe r and to membranes and other organelles have been documented by electr on microscopy; however, the biochemical and molecular nature of these linkages is not known. We have partitioned proteins from a suspension culture of tobacco into cytosolic and membrane fractions, solubilized the membrane fraction with a zwitterionic detergent, and then used aff inity chromatography and salt elution to isolate tubulin binding prote ins. Dark-field microscopy of in vitro-assembled microtubules showed t hat the eluted proteins from both fractions induce microtubule bundlin g and, in the presence of purified tubulin, promote microtubule elonga tion. Gel electrophoresis of the eluted proteins revealed two distinct sets of polypeptides. Those in the membrane eluate included unique ba nds with apparent molecular masses of 98, 90, and 75 kD in addition to bands present in both eluates. The cytosolic eluate, in contrast, typ ically included relatively smaller proteins. The eluted proteins also bound to taxol-stabilized microtubules. Initial immunological characte rization using monoclonal antibodies raised against the 90-kD polypept ide showed that it is colocalized in situ with cortical microtubules i n tobacco protoplast ghosts.