Autopalmitoylation of tubulin

Purr rat brain tubulin is readily palmitoylated in vitro using [H-3]palmito yl CoA but no added enzymes. A maximum of approximately six palmitic acids are added per dimer in 2-3 h at 36-37 degrees C under native conditions. Bo th alpha and beta tubulin are labeled, and 63-73% of the label was hydroxyl amine-labile, presumed thioesters. Labeling increases with increasing pH an d temperature, and with low concentrations of guanidine HCl or KCl (but not with urea) to a maximum of similar to 13 palmitates/dimer. High SDS and gu anidine HCl concentrations are inhibitory. At no time could all 20 cysteine residues of the dimer be palmitoylated. Polymerization to microtubules, or use of tubulin S, markedly decreases the accessibility of the palmitoylati on sites, Palmitoylation increases the electrophoretic mobility of a portio n of cr tubulin toward the beta band. Palmitoylated tubulin binds a colchic ine analogue normally, but during three warm/cold polymerization/depolymeri zation cycles there is a progressive loss of palmitoylated tubulin, indicat ing decreased polymerization competence. We postulate that local electrosta tic factors are major regulators of reactivity of tubulin cysteine residues toward palmitoyl CoA, and that the negative charges surrounding a number o f the cysteines are sensitive to negative charges on palmitoyl CoA.