Proteolytic analysis of polymerized maize tubulin: regulation of microtubule stability to low temperature and Ca2+ by the carboxyl terminus of β‐tubulin

ABSTRACT To obtain information on plant microtubule stability to low temperature and Ca2+, the regulatory domain of polymerized tubulin from maize (Zea mays ev. 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 αβs‐microtubules. Subsequent cleavage occurred at an extreme site and an internal site within the α‐tubulin carboxyl terminus. Electron microscopy revealed that αβs‐microtubules were ultra structurally indistinguishable from uncleaved control αβ‐micro‐tubules. Quantitative polymer sedimentation showed that low temperature treatment (0°C) caused significant depolymerization of αβ‐microtubules, but little depolymerization of αβs‐microtubules. Ca2+ enhanced the cold‐induced depolymerization of both αβ‐ and αβs‐microtubules. However, αβs‐microtubules were significantly more stable to depolymerization by cold and Ca2+ 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 Ca2+. 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.