CaMzt1 directly interacts with the N-terminal domain of CaSpc98. (A) EGFP-CaMzt1 is located near the tail at the base of Caγ-TuSC. Left: CBB-stained gel. Right: boxed negative-stained Caγ-TuSC-EGFP-CaMzt1 particles were classified and averaged. The arrows point to the additional density of EGFP-CaMzt1. Bar, 10 nm. (B) The deletion of N-terminal domain of Spc98 did not affect γ-TuSC assembly. Purified Caγ-TuSC^Spc98ΔN and Scγ-TuSC^Spc98ΔN (left) were subjected to negative staining and EM analysis (right). Bars, 10 nm. (C) The CaSpc98-N determines binding of Caγ-TuSC to CaMzt1. Gel filtration of EGFP-CaMzt1 incubated with purified Scγ-TuSC, Caγ-TuSC^ScSpc98-N, and Scγ-TuSC^CaSpc98-N. Immunoblot of elution fractions with anti-His (for Spc97 and Spc98) and anti-GFP (for CaMzt1). (D) CaMzt1-3Flag was copurified with Scγ-TuSC^CaSpc98-N but not to Caγ-TuSC^ScSpc98-N. γ-TuSCs were purified with Ni-NTA and analyzed with SDS-PAGE. Western blots (WB) are shown. (E) Binding affinity between Caγ-TuSC and Spc110-N by MST, as in Fig. 3 D. Error bars are SD. (F) The ScSpc110-N binds both Scγ-TuSC and Caγ-TuSC but promotes oligomerization of only Scγ-TuSC. As in C. SDS-PAGE and CBB. mAU, milli-absorbance unit.