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Proteolytic analysis of polymerized maize tubulin: regulation of microtubule stability to low temperature and Ca 2+ by the carboxyl terminus of β‐tubulin
Author(s) -
BOKROS C. L.,
HUGDAHL J. D.,
BLUMENTHAL S. S. D.,
MOREJOHN L. C.
Publication year - 1996
Publication title -
plant, cell and environment
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.646
H-Index - 200
eISSN - 1365-3040
pISSN - 0140-7791
DOI - 10.1111/j.1365-3040.1996.tb00387.x
Subject(s) - microtubule , tubulin , depolymerization , subtilisin , cleavage (geology) , biophysics , biochemistry , chemistry , microtubule associated protein , cytoskeleton , proteolysis , biology , microbiology and biotechnology , enzyme , cell , paleontology , organic chemistry , fracture (geology)
To obtain information on plant microtubule stability to low temperature and Ca 2+ , 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 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 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 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.