Isolation of cortical MTs from tobacco BY-2 cells

We isolated the cortical microtubules (CMTs) from tobacco BY-2 cells to ide ntify their components. By centrifugation of protoplasts homogenized in the presence of taxol, a MT-stabilizing reagent, in a density gradient of Perc oll, we obtained membranous vesicles to which MTs forming a sheet-like bund le were attached. Rhodamine-conjugated Ricinus communis agglutinin I (RCA-I ), a lectin that bound to the surface of protoplasts, stained these vesicle s, indicating that they were plasma membrane (PM) vesicles that retained CM Ts, CMTs were released by solubilization of PM vesicles with Triton X-100, A sheet-like array of CMTs was retained even after solubilization of PM ves icles. Immunoblot analysis of the isolated CMTs demonstrated the presence o f tubulin, actin, the 65 kDa microtubule-associated protein (MAP) and a 130 kDa RCA-I binding protein. Purification of the isolated CMTs by the temper ature dependent disassembly-reassembly cycling method revealed four polypep tides, 190, 120, 85 and 65 kDa, co-assembling with CMTs.