Chemistry And Function Of Microtubules

Wolff, Jan

Abstract

Membrane Tubulin Many years ago we discovered that plasma membranes contain a significant fraction of the cellular tubulin. This was surprising since that protein is very polar and exhibits a very high charge density. We have now returned to this puzzling phenomemon. In our effort to understand the association of microtubules to the plasma membrane we have used two approaches: 1. Direct attachment of tubulin to the membrane via palmitoylation of some of its many thiol groups (1,2). This occurs with a preferential insertion of the palmitoylated tubulin into the plasma membrane of PC12 cells(3). As a model we have also used autopalmitoylation of tubulin (2). We are currently examining which isoelectric variant serves as the preferred substrate for thioester formation(4). After considerable difficulties in identifying the substituted cysteines (because of the extreme hydrophobicity of the tryptic peptides, we are identifying the most reactive SH groups by first using less hydrophobic reagents, then move to less hydrophobic thioesters and thence, hopefully, to palmitic acid.Alternatively, we will displaced thioesters already formed with a more p olar reagent after the previously unreacted thiols have been blocked. This has led us to investigate the electrostatic factors that lower pKa to form the thiolate ion such as helix dipole, neighboring basic or acidic amino acids etc.. These neighborhoods are identified with the help of the electron diffraction structure and the peptides with the most reactive thiol. 2. It is also possible that linker proteins attach microtubules to the plasma membrane. To this effect we have identified one such protein. 2’,3’ Cyclic nucleotide-3’-phosphodiesterase (CNP) is firmly associated with tubulin from brain tissue and from FRTL-5 thyroid cells as demonstrated by a) co-polymerization through several warm/cold cycles, b)the presence of diesterase activity in partially purified tubulin, c) and identical behavior during various extraction procedures. CNP acts like a microtubule-associated protein in promoting microtubule assembly at low mole ratios. This activity resides in the C-terminus of CNP, and a C-terminal tridecapeptide also promotes assembly. Phosphorylation of CNP and loss of the C-terminus abolish assembly promoting activity; the latter also leads to abnormal microtubule distribution in COS cells. Finally, we have demonstrated co-localization of the two proteins in the plasma membrane (5).