Chemistry and Function of Microtubules

Wolff, Jan

Abstract

In continuation of studies on the palmitoylation of tubulin (Biochem. Biophys. Res. Commun. 239: 650-654, 1997), rat pheochromocytoma PC12 cells metabolically labeled with [3H]palmitate were fractionated to obtain various particulate fractions. The bulk of the label was found in plasma membranes (identified by ouabain-sensitive Na/K ATPase). Tubulin from these membranes was detergent extracted, immunoprecipitated, and shown to contain [3H]palmitate in both alpha and beta monomers with alpha greater than beta. The palmitic acid is present both as thioesters and other linkages which we are currently trying to identify.In order to obtain a better understanding of the mechanism of acylation, we have studied autopalmitoylation in vitro. Pure rat brain tubulin is readily palmitoylated in vitro using [3H]palmitoyl CoA but no added enzymes. A maximum of ~6 palmitic acids are added per dimer in 2-3 h at 36-7C under native conditions. Both alpha and beta tubulin are labeled. Palmitoylation increases the electrophoretic mobility of a portion of alpha tubulin toward the beta band, apparently as a function of the degree of palmitoylation. Labeling increases with increasing pH and temperature, and with low concentrations of guanidine HCl or KCl (but not urea) to a maximum of 13 palmitates/dimer. High SDS and guanidine HCl concentrations are inhibitory. At no time could all twenty cysteine residues of the dimer be palmitoylated. Polymerization to microtubules, or use of tubulin S,(in which the C-termini of both alpha and beta tubulin are cleaved and for which we developed a new method (Cell Motility and Cytoskeleton 43: 63?71, 1999) markedly decreases the accessibility of the palmitoylation sites. Palmitoylated tubulin binds a colchicine analogue normally, but during 3 warm/cold polymerization/depolymerization cycles there is a progressive loss of palmitoylated tubulin, indicating decreased polymerization competence. We postulate that local electrostatic factors are major regulators of reactivity of tubulin cysteine residues toward palmitoyl CoA, and that the negative charges surrounding a number of the cysteines are sensitive to negative charges on palmitoyl CoA, thus hindering palmitoylation (submitted to J. Biol. Chem.).Current plans are to assess the hydrophobicity of the modified tubulin as a function of the degree of substitution using a modified Triton-X114 method with rhodium-catalyzed hydrogenated detergent. We are also comparing the in vivo and auto- acylated sites of palmitoylation and hope to identify hydroxylamine-resistant linkages. - hydrophobicity, electrostatics, tubulin S, polymerization