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 intbrination on plant microtubuic stability to low temperature and Ca^*, the regulatory domain of polymerized tubulin from maize {Zea mays cv. Black Mexican Sweet) was dissected by limited proteolysis with snbtilisin. Tubulin in taxol-stabilized microtubules was cleaved in a subtilisin concentration-and time-dependent manner. Immunoblotting of microtubules with antibodies having mapped epitopes on a-and /^tubnlins revealed that eleavage initially removed <15 residues IVom the /3-tubulin carboxyl terminus to prodnec a/i^-microtubules. Subsequent cleavage occurred at an extreme site and an internal site within the a-tubniin carboxyl terminus. Electron microscopy revealed that a/3s-microtnbules were ultrastructurally indistinguishable from uncleaved control a^microtubules. Quantitative polymer sedimentation showed that low temperature treatment (0 °C) caused significant depolymcrization of a/J-microtnbules, but little depolymerization of a/Jj-microtubules. Ca^* enhanced the cold-induced depolymerization of both aft-and a/Js-microtubnles. However, a/iJ^-microtubules were significantly more stable to depolymerization by cold and Ca^* than were a/iJ-niicrotubules. The results showed that maize microtubules containing shortened /^tubiilin carboxyl termini are relatively resistant to the combined depolymerizing effects of cold and Ca^*. Thus, the extreme carboxyl terminus of /3-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.

In vitro assembled plant microtubules exhibit a high state of dynamic instability

“…Among vertebrates, the majority of ␤-tubulins have 10 acidic residues, or less, in this region [Ludueña, 1993]. This region of ␤-tubulin has been implicated in affecting microtubule dynamics specifically by virtue of its cationic charge, which correlates well with increasing instability [Burns and Surridge, 1990;Bokros et al, 1996]. The results reported herein using carrot tubulin are consistent with this hypothesis.…”

Tubulin isoform usage in maize microtubules

“…It is known also that changes in the extreme carboxyl terminus of maize tubulin affect the sensitivity of MTs to depolymerization in vitro by low temperature and Ca 2+ (Bokros et al 1996). In view of this information, we believe it is reasonable to ask whether the many c~-tubulin and ~-tubulin genes of higher plants are entirely redundant, or whether at least some of the different tubulin gene products have unique functions during plant growth and development and during exposure to varying environmental conditions.…”

A dual effect on protein synthesis and degradation modulates the tubulin level in rice cells treated with oryzalin

“…In addition, oryzalin has been shown to activate membrane-bound Ca 2+ channels with a corresponding increase in the concentration of cytosolic Ca 2+ . Also, Ca 2+ promotes the disassembly of maize microtubules reconstituted in vitro (Bokros et al 1996).…”

“…Ca 2+ is known to destabilize MTs, possibly by binding to the C-terminal end of tubulin (Bokros et al 1996). Ca 2+ may act directly or through the involvement of Ca 2+ -modulated protein kinase activities.…”