Identification of multiple tubulins in taxol microtubules purified from carrot suspension cells

Abstract

Tubulin has been purified from carrot suspension cells by ion-exchange chromatography and assembled into microtubules in the presence of 20 microM taxol. One-dimensional SDS-PAGE suggested that the alpha band migrated faster than the beta band (as has been established for some lower eukaryotic tubulins) and this heterology with brain tubulins was confirmed by peptide mapping. When subjected to two-dimensional gel electrophoresis, the plant tubulins could be separated into multiple alpha and beta isotypes. Immunoblotting, using monoclonal anti-tubulins, confirmed that the tubulin isotypes identified in taxol microtubules represent all of the tubulins present in homogenates of unsynchronised log-phase carrot suspension cells. All identified tubulins are therefore assembly-competent under these conditions. Plant cells can contain four different microtubule arrays, but cells arrested in G0/G1 contain only cortical microtubule arrays; such cells, however, exhibit the same tubulin profile as non-synchronised cells, thereby showing no restriction in the number of subunits during this phase of the cell cycle.