Exchange of tubulin dimer into rings in microtubule assembly--disassembly

Abstract

We have prepared native radioactive tubulin dimer from two species: [35S]tubulin dimer, by in vivo labeling of rat brain, and porcine [3H]ethyltubulin, as previously described [Zeeberg, B., Cheek, J., & Caplow, M. (1980) Anal. Biochem. 104, 321--327]. After microtubule assembly with radioactive tubulin dimer and nonradioactive dimer and rings, the tubulin in the rings and the dimer obtained upon disassembly have approximately equal specific activities. Therefore, during the reaction sequence dimer + rings leads to 37 degrees C microtubules leads to 0 degrees C dimer + rings the tubulin initially in rings becomes indistinguishable from tubulin initially in dimer. Under nonpolymerizing conditions (0 degrees C) radioactive tubulin dimer and radioactive guanine nucleotide are incorporated into rings at approximately equal rates. This indicates that there is a pathway for nucleotide incorporation into rings under nonpolymerizing conditions which involves the incorporation of dimer-bound nucleotide. We also report results on the lack of the mirror image equilibrium during the disassembly process, using porcine [3H]-ethyltubulin dimer, rat [35S]tubulin dimer, and a [3H]-GDP.porcine tubulin dimer complex. In all three cases there is no significant disassembly-dependent incorporatioin of radioactivity into rings when microtubules are disassembled in the presence of radioactive dimer. These results demonstrate that, for rat and porcine tubulin, rings are formed during microtubule disassembly by direct cleavage of intact rings, without a tubulin dimer intermediate.