Recycling of cold-stable microtubules: evidence that cold stability is due to substoichiometric polymer blocks.
A substantial subpopulation of mammalian brain crude extract microtubules is resistant to cold-temperature disassembly. We propose here that microtubules are rendered cold stable by rare substoichiometric blocks. Mild shearing of rat brain cold-stable microtubules makes them largely cold labile. In addition, cold-stable microtubules can be destabilized by exposure to low concentrations of calmodulin (5 microM) in the presence of calcium at 0 degree C. Cold-disassembled microtubule protein, obtained from sheared or calmodulin-treated cold-stable preparations, re-forms a cold-stable subpopulation upon reassembly. These observations allow strategies for the recycling purification of cold-stable microtubules. Comparison of purified cold-labile and cold-stable material by gel electrophoresis shows enrichment for a few unique polypeptides, of 135, 70-82, and 56 kilodaltons, in the cold-stable preparation. The 64-kilodalton "switch protein", previously identified as uniquely dephosphorylated in cold-stable microtubules, is equally represented in recycled cold-stable and cold-labile microtubule preparations. Furthermore, when disassembled, cold-stable microtubule proteins are passed through a calmodulin affinity column on which the polypeptides characteristic of cold-stable microtubules are specifically retained, the breakthrough (unbound) material repolymerizes into cold-labile microtubules only. Based on the above data, a model is presented in which microtubules are rendered cold stable by the presence of substoichiometric, calmodulin-sensitive blocks that randomly reshuffle upon reassembly of cold-stable microtubules.
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002147:Calmodulin; D002846:Chromatography, Affinity; D003080:Cold Temperature; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D008869:Microtubule-Associated Proteins; D008870:Microtubules / Q000648:ultrastructure*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats
D002147:Calmodulin; D046911:Macromolecular Substances; D008869:Microtubule-Associated Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins