Higher plant tubulin identified by self-assembly into microtubules in vitro

Abstract

Microtubules are filamentous, subcellular structures present in virtually all eukaryotes. In higher plants, microtubules form the mitotic spindle¹, determine the plane of cell division^2,3 and orient cellulose microfibril deposition in growing cells^4,5, and thus are significant determinants of morphogenesis^6,7. The main structural component of microtubules is tubulin, a conserved, heterodimeric protein having a molecular weight of 110,000 and composed of α- and β-subunits (≃55,000 each)⁸. The resistance of plant microtubules to several antimitotic drugs has suggested that plant tubulins differ from animal tubulins (see refs 5, 9, 10 for review), but further characterization has awaited the development of a method to isolate plant tubulin. As tubulin has no characteristic enzymatic activity, the positive identification of this protein depends on its ability to self-assemble in vitro to form structures which have the morphological features of microtubules. Recent studies^11–17 have used indirect methods to isolate tubulin-like proteins from higher plant cell extracts, but none has conclusively identified tubulin by demonstrating its self-assembly into microtubules. We report here the isolation of tubulin from cultured cells of a higher plant and its unequivocal identification by self-assembly into microtubules in vitro. Furthermore, we report peptide mapping data which indicate that although the β-summits of mammalian and higher plant tubulins have been conserved, the α-subunits have diverged during evolution.