Proteolytic analysis of polymerized maize tubulin: regulation of microtubule stability to low temperature and Ca2+ by the carboxyl terminus of β‐tubulin
To obtain information on plant microtubule stability to low temperature and Ca 2+ , the regulatory domain of polymerized tubulin from maize (Zea mays cv. Black Mexican Sweet) was dissected by limited proteolysis with subtilisin. Tubulin in taxol-stabilized microtubules was cleaved in a subtilisin concentration- and time-dependent manner. Immunoblotting of microtubules with antibodies having mapped epitopes on α- and β-tubulins revealed that cleavage initially removed ≤15 residues from the β-tubulin carboxyl terminus to produce αβ s -microtubules. Subsequent cleavage occurred at an extreme site and an internal site within the α-tubulin carboxyl terminus. Electron microscopy revealed that αβ s -microtubules were ultrastructurally indistinguishable from uncleaved control αβ-microtubules. Quantitative polymer sedimentation showed that low temperature treatment (0 °C) caused significant depolymerization of αβ-microtubules, but little depolymerization of αβ s -microtubules. Ca 2+ enhanced the cold-induced depolymerization of both αβ- and αβ s -microtubules. However, αβ s -microtubules were significantly more stable to depolymerization by cold and Ca 2+ than were αβ-micro-tubules. The results showed that maize microtubules containing shortened β-tubulin carboxyl termini are relatively resistant to the combined depolymerizing effects of cold and Ca 2+ . Thus, the extreme carboxyl terminus of β-tubulin is a crucial element of the plant tubulin regulatory domain and may be involved in the modulation of microtubule stability during the chilling response in plants.