CaMzt1 directly interacts with Caγ-TuSC and promotes Caγ-TuSC oligomerization. (A) CaMzt1 forms a stable complex with Caγ-TuSC. Recombinant CaMzt1-3Flag and Caγ-TuSC were copurified. Protein complex was subjected to ion-exchange chromatography (MonoQ; gradient from 50–500 mM NaCl). Caγ-TuSC-CaMzt1 eluted at ∼400 mM NaCl; peak fractions were analyzed by SDS-PAGE. Immunoblot (IB) for 3Flag-tagged CaMzt1. (B) The purified Caγ-TuSC-CaMzt1 was stable and monomeric after ion exchange and gel filtration. SDS-PAGE with CBB; IB for 3Flag-tagged CaMzt1. (C) Caγ-TuSC–CaMzt1 interaction was reconstituted in vitro. Purified EGFP-CaMzt1 was incubated with Caγ-TuSC in HB100, pH 7.4. A₄₉₀ detected EGFP-CaMzt1. The faint band below CaSpc97 in Caγ-TuSC^Spc98ΔN is a degradation product. SDS-PAGE with CBB. (D) Binding affinity between Caγ-TuSC and CaMzt1 was measured with MST. Fluorophore-labeled Caγ-TuSC and Caγ-TuSC^Spc98ΔN (150 nM) were incubated with CaMzt1^WT and CaMzt1^D22A ranging from ∼10 nM to ∼240 µM. Four records from two independent sample preparations were collected, and the mean values were subjected to binding curve fitting with NanoTemper software. Error bars are SD. The binding curve is a single representative out of three independent measurements. (E) Summary of MST measurements of CaMzt1 and CaMzt1^D22A with the indicated γ-TuSCs. K_d ± SD; n = 3 independent measurements. (F) CaMzt1 promoted Caγ-TuSC oligomerization under low-salt buffer. Recombinant CaMzt1 was incubated with Caγ-TuSC and analyzed by gel filtration, SDS-PAGE, and CBB. The control Caγ-TuSC only (150 mM KCl) is the same as shown in Fig. 2 E. (G) Oligomerization of Caγ-TuSC–CaMzt1 complexes under low-salt buffer resulted in large irregular oligomers. Peak fraction of F was subjected to negative staining and EM. Bar, 50 nm. mAU, milli-absorbance unit.