Chemistry and Function of Microtubules

Wolff, Jan

Abstract

PC12 pheochromocytoma cells incorporate [3H]palmitate into alpha and beta tubulin both in cytoplasmic and membrane tubulin. The bulk of the label goes to the plasma membrane tubulin. NGF enhances palmitoylation of alpha and EGF enhances beta tubulin labeling, whereas cAMP inhibits palmitoylation. The presence of even one palmitate per dimer prevents microtubule assembly which may favor localizatiion to the plasma membrane (Zambito and Wolff, submitted). Current attempts to use Triton X-114 or reduced Triton X-114 phase separation to assess the distribution of palmitoylated tubulin on the basis of hydrophobicity are complicated by the fact that the distribution is the sum of extensive hydrophilic forces and the introduced hydrophobic forces; we are planning to use phospholipid micelles where this will be less of a problem. Autopalmitoylation of tubulin by palmitoylCoA at physiological pH yields 1-2 palmitates/dimer; at higher pH 6-7 palmitates are added, but at no time are all 20 SH reacted, even after complete denaturation. Low concentrations of guanidine HCl (but not urea)markedly promote tubulin palmitoylation to as many as 12-13 of the 20 SH groups per tubulin dimer, presumably by charge shielding since KCl does the same. Similar effects are seen in microtubule assembly (J. Wolff, Biochemistry 38: 10722-10729, 1998). This suggests that electrostatics play a major role in regulating SH reactivity toward palmitoylCoA. The electron diffraction structure of tubulin confirms the nearby unfavorable electrostatic environment of r a number of cysteine residues (Wolff, Zambito, Britto & Knipling - Protein Science 9: 1357-1364, 2000). In line with this, we have just started to collaborate in a study to identify the H-bonding environment of the SH groups by Raman spectroscopy. We are also expanding the study to acquire a better understanding of the role of the 20 tubulin SH groups which are apparently not directly catalytic. It has been very difficult to assign the palmitic acids to specific SH groups because of the extroadinary hydrophobicity of the substituted peptides and we are developing a subtraction method for sequencing purposes as well as analysis by Mass Spec. Other publication: Wolff, EC, Wolff, J, & Park, MH. Deoxyhypusine synthase generates and uses bound NADH in a transient hydride transfer mechanism. J. Biol. Chem, 275: 9170-9177, 2000.