Tubulin, hybrid dimers, and tubulin S. Stepwise charge reduction and polymerization

Limited proteolysis of rat brain tubulin (alpha beta) by subtilisin cleaves a 1-2-kDa fragment from the carboxyl-terminal ends of both the alpha and beta subunits with a corresponding loss in negative charge of the proteins. The beta subunit is split much more rapidly (and exclusively at 5 degrees C), yielding a protein with cleaved beta and intact alpha subunit, called alpha beta s, which is of intermediate charge. Further proteolysis cleaves the carboxyl terminus of the alpha subunit leading, irreversibly, to the doubly cleaved product, named tubulin S, with a composition alpha s beta s. Both cleavage products are polymerization-competent and their polymers are resistant to 1 mM Ca2+- and 0.24 M NaCl-induced depolymerization. The two polymers differ in that the alpha beta s polymer is stable to cold, GDP, and podophyllotoxin, whereas tubulin S polymer is disassembled by these agents; moreover, alpha beta s forms ring-shaped polymers, whereas alpha s beta s forms filaments associated into bundles and sheets. Tubulin S co-polymerizes with native tubulin yielding a mixed product of intermediate stability. The presence of low mole fractions of tubulin S leads to a marked reduction in the critical concentration for polymerization of the mixture.