Interaction of microtubule-associated proteins with microtubules: yeast lysyl- and valyl-tRNA synthetases and tau 218-235 synthetic peptide as model systems.
The respective contributions of electrostatic interaction and specific sequence recognition in the binding of microtubule-associated proteins (MAPs) to microtubules have been studied, using as models yeast valyl- and lysyl-tRNA synthetases (VRS, KRS) that carry an exposed basic N-terminal domain, and a synthetic peptide reproducing the sequence 218-235 on tau protein, known to be part of the microtubule-binding site of MAPs. VRS and KRS bind to microtubules with a KD in the 10(-6) M range, and tau 218-235 binds with a KD in the 10(-4) M range. Binding of KRS and tau 218-235 is accompanied by stabilization and bundling of microtubules, without the intervention of an extraneous bundling protein. tau 218-235 binds to microtubules with a stoichiometry of 2 mol/mol of assembled tubulin dimer in agreement with the proposed binding sequences alpha[430-441] and beta[422-434]. Binding stoichiometries of 2/alpha beta S tubulin and 1/alpha S beta S tubulin were observed following partial or complete removal of the tubulin C-terminal regions by subtilisin, which localizes the site of subtilisin cleavage upstream residue alpha-441 and downstream residue beta-434. Quantitative measurements show that binding of MAPs, KRS, VRS, and tau 218-235 is weakened but not abolished following subtilisin digestion of the C-terminus of tubulin, indicating that the binding site of MAPs is not restricted to the extreme C-terminus of tubulin.
Melki
R
R
Laboratoire d'Enzymologie, CNRS, Gif-sur-Yvette, France.
Kerjan
P
P
Waller
J P
JP
Carlier
M F
MF
Pantaloni
D
D
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D001667:Binding, Competitive; D008250:Lysine-tRNA Ligase / Q000737:chemistry*; D008869:Microtubule-Associated Proteins / Q000737:chemistry* / Q000648:ultrastructure; D008870:Microtubules / Q000737:chemistry* / Q000648:ultrastructure; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014404:Tubulin / Q000737:chemistry / Q000648:ultrastructure; D014637:Valine-tRNA Ligase / Q000737:chemistry*
D000596:Amino Acids; D008869:Microtubule-Associated Proteins; D010455:Peptides; D014404:Tubulin; D008250:Lysine-tRNA Ligase; D014637:Valine-tRNA Ligase