Mzt1 is dispensable for the binding of γ-TuSC to receptors but required for the assembly of active γ-TuSC rings. (A) Caγ-TuSC was copurified with CaSpc110-N with (b) and without (a) CaMzt1. The purified complexes were eluted from GST-resin. SDS-PAGE (CBB) and anti-Flag immunoblot (IB; CaMzt1-3Flag) are shown. (B) Coexpression of CaMzt1 and CaSpc110-N promotes oligomerization of Caγ-TuSC. The presence of CaMzt1 further increased oligomerization. Complexes from A were subjected to gel-filtration analysis. SDS-PAGE with CBB. (C) CaSpc110-N has higher affinity to Caγ-TuSC–CaMzt1 than Caγ-TuSC alone. MST was performed as in Fig. 3 C. Kd ± SD; n = 3 independent measurements. (D) CaMzt1 keeps the oligomerization of Caγ-TuSC–CaSpc110-N compact and organized. Caγ-TuSC–CaSpc110-N and CaMzt1–Caγ-TuSC–CaSpc110-N complexes from void (0.9 ml) in B were subjected to negative staining and EM. Bars, 50 nm. (E) The averaged diameter of Caγ-TuSC–CaSpc110-N oligomers was larger than oligomers with CaMzt1. Error bars are SD. n = 15 (number of particles measured in each sample). ***, P < 0.001 with t test. (F) CaMzt1 is required for optimal MT nucleation activity. Left: maximum projection of 20 images for each sample. Bar, 50 µm. Right: statistical analysis of MT nucleation. n = 6–9 independent samples from 3 independent experiments. Error bars are SD. ***, P < 0.001. Fig. S5 D shows DMSO and taxol control. (G) The N-terminal domains of CaSpc98 and CaSpc110 are both required to recruit CaMzt1 to S. cerevisiae SPBs. EGFP-CaMZT1 expression was induced with galactose. Spc42-mCherry is an SPB marker. Bar, 5 µm. Ana, anaphase; M, metaphase.
