Isolation of cortical MTs from tobacco BY-2 cells.

We isolated the cortical microtubules (CMTs) from tobacco BY-2 cells to identify their components. By centrifugation of protoplasts homogenized in the presence of taxol, a MT-stabilizing reagent, in a density gradient of Percoll, we obtained membranous vesicles to which MTs forming a sheet-like bundle were attached. Rhodamine-conjugated Ricinus communis agglutinin I (RCA-I), a lectin that bound to the surface of protoplasts, stained these vesicles, indicating that they were plasma membrane (PM) vesicles that retained CMTs. 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 vesicles. Immunoblot analysis of the isolated CMTs demonstrated the presence of tubulin, actin, the 65 kDa microtubule-associated protein (MAP) and a 130 kDa RCA-I binding protein. Purification of the isolated CMTs by the temperature dependent disassembly-reassembly cycling method revealed four polypeptides, 190, 120, 85 and 65 kDa, co-assembling with CMTs.