Chemistry And Function Of Microtubules

Wolff, Jan

Abstract

All 20 cysteines are accessible to disulfide reagents in the dimer but only 4 are accessible in taxol-stabilized microtubules. Reaction rates with disulfide reagents are a function of the reagent, are decreased by G nucleotides, and are increased by increasing pH and urea. With transient (stop-flow) kinetics 5,5?-dithio-bis (2-nitrobenzoate) and 2,2?-dithiodipyridine progress curves cannot be fitted by the sum of exponential terms based only on classes of cysteines. The mixed disulfide products react further to form both intra- and inter- monomer disulfide bonds that can be reversed by reducing agents. With methyl methanethiosulfonate or n-octyl-dithio-2-nitrobenzoate (ODNB) virtually no protein/protein disulfide bonds are formed and the ODNB reaction can be resolved into the sum of three exponential terms with pseudo-first order rate constants of 0.206, 0.069, and 0.010 sec-1 at pH 6.9 suggesting three classes of thiol reactivities. Limited cysteine substitution leads to only small changes in tryptophan or CD spectra, whereas complete substitution leads to loss of helix content. Substitution of >2 cysteines with MMTS abolishes polymerization competence. Assembly promoters such as higher protein or buffer concentrations, or taxol facilitate polymerization of tubulin despite substitution of up to 5 cysteines/dimer and despite increases in the critical concentration for polymerization. The substituted tubulin forms protofilament-based structures such as microtubules, open tubules, sheets and/or bundles. We are also investigating the role of tubulin in apopotosis. Most, if not all, antimitotic or antitubulin agents cause apoptosis. Initially it was thought that disruption of microtubule integrity by such drugs would lead to phosphorylation and inactivation of Bcl2. Alternatively, these drugs could interact with non-microtubule tubulin bound to mitochondria (with summer student Jack Shern and in collaboration with Dr. Dan Sackett (NICDH)). We find that: mitochondria from various sources vary tremendously in the tubulin content; tubulin is associated with mitochondrial proteins in a high molecular weight complex; pro- and anti-apototic proteins, such as Bax and Bcl2 etc., interact with tubulin in vitro in a competitive manner. We are now determining whether or not this interaction is specific and plan to study such interactions with intact mitrochondria.