Fig. 2. A region within the distal half of the NuMA tail domain binds directly to tubulin. (A) Immunoblot for tubulin, showing Xenopus egg extract, and eluates of Niagarose beads after incubation in untreated egg extract (beads only), or egg extract supplemented with hexahistidine-tagged Xenopus NuMA tail I (beads+tail1) or NuMA tail II (beads+tail2). For comparison, phosphocellulose-purified tubulin (tubulin) is shown. (B) Top: Coomassie-stained gel showing purified tubulin (tub.), hexa-histidine tagged SNAP (SNAP), and hexahistidine-tagged fusion proteins of Xenopus NuMA head domain (N. head), a 425-residue fragment of the Xenopus NuMA rod domain (N. rod), the proximal half of the Xenopus NuMA tail (N. tail1), and the distal half of the Xenopus NuMA tail (N. tail2). Middle: immunoblot for tubulin, showing a binding assay of soluble tubulin mixed with hexa-histidine-tagged fusion proteins as shown on the Coomassie-stained gel, and adsorbed to magnetic Niagarose beads. Supernatants (left) and bead eluates (Ni++ beads eluate, right) are shown. `no prot.' indicates a control of soluble tubulin, binding to Ni-beads only; `tub.' indicates purified tubulin only. The relative amounts of tubulin bound in each reaction were measured with a phosphoimager and noted underneath; the NuMA tail2 sample, showing the strongest binding, was set to 100%. Bottom: an identical immunoblot, probed with antibody against the peptide sequence of hexa-His-Gly (anti 6xHis-G). The lack of reactivity against the SNAP fusion protein is due to cloning in a pQE-9 vector, encoding hexa-histidine without glycine. All other fusion proteins were cloned in pRSET, leading to immunoreactive fusion proteins containing hexa-His-Gly. (C) Coomassie stained gel, showing supernatants and pellets of bovine serum albumin (BSA), Xenopus NuMA tail II (NuMA t.2), taxol-stabilized microtubules (tub.), and taxol-stabilized microtubules incubated with bovine serum albumin (BSA+tub.) or Xenopus NuMA tail II (Nu.t2+tub.). (D) Microtubule assembly from phosphocellulose-purified tubulin mixed with rhodamine-labelled tubulin, without additions (left), or with added Xenopus NuMA tail I (middle), or tail II (right). Bar, 20 µm. (E) Scatchard plot, showing Xenopus NuMA tail II binding at increasing concentrations (in mol/l) to taxol-stabilized microtubules. (F) The percentage of bound Xenopus NuMA tail II to taxol-stabilized microtubules, quantifying unphosphorylated protein (open squares), or counts of NuMA tail II phosphorylated with recombinant cdc2 kinase/cyclinB and radioactive ATP (open diamonds).