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

Abstract

Limited proteolysis of rat brain tubulin (αβ) by subtilisin cleaves a 1-2-kDa fragment from the carboxyl-terminal ends of both the α and β subunits with a corresponding loss in negative charge of the proteins. The β subunit is split much more rapidly (and exclusively at 5 ° C), yielding a protein with cleaved β and intact α subunit, called α β_s, which is of intermediate charge. Further proteolysis cleaves the carboxyl terminus of the α subunit leading, irreversibly, to the doubly cleaved product, named tubulin S, with a composition α _s β _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 αβ_s polymer is stable to cold, GDP, and podophyllotoxin, whereas tubulin S polymer is disassembled by these agents; moreover, αβ_s forms ring-shaped polymers, whereas α_sβ_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.