Mechanisms blocking microtubule minus end assembly: evidence for a tubulin dimer-binding protein

We have characterized an activity in sea urchin eggs which prevents microtubule assembly at minus ends. Using Chlamydomonas axoneme fragments to nucleate the assembly of plus and minus end microtubules, we find robust assembly at microtubule plus ends with negligible assembly at minus ends. The minus end assembly inhibitor does not co-pellet with microtubules when assembly is stimulated with DMSO while the resulting pellet of tubulin and microtubule associated proteins readily assembles from both plus and minus ends of axoneme fragments. Addition of increasing concentrations of porcine bran tubulin to the tubulin and MAP-depleted fraction eventually saturates the minus end inhibitory activity. Compared to purified tubulin, cytosolic fractions both increase the minus end critical concentration approximately 3 fold and decrease the plus end critical concentration. The inhibitory activity is removed by heating, trypsin, or by co-immunoprecipitation with tubulin. We hypothesize that a tubulin dimer binding protein is responsible for preventing assembly onto minus ends in our in vitro assays and speculate that this protein functions in vivo to prevent spontaneous nucleation, thus limiting assembly to nucleation sites.