CaMzt1 interacts with CaSpc110-N and CaSpc72-N depending on the CM1 element. (A) CaMzt1 directly interacts with CaSpc110-N. Mixed EGFP-CaMzt1 and GST–CaSpc110-N were subjected to gel filtration. Analysis as in Figs. 3 C and 4 C. The bottom band in the anti-GFP blot is an EGFP-CaMzt1 degradation product. (B) Weblogo of CM1 element of γ-TuCRs. CM1a and CM1b are underlined. Green asterisk marks Y137A. Red asterisks indicate mutated residues in CaSpc110-N^4A. Mutations in CaSpc110-N^9A1 and CaSpc110-N^9A2 are marked blue and orange, respectively. (C) MST measurement of the binding of EGFP-CaMzt1, EGFP-CaMzt1^D22A, or CaMzt1-EGFP to CaSpc110-N^WT (1–222 aa). Error bars are SD. Single representative binding curve out of three independent experiments is shown. (D) MST measurement of the binding of EGFP-CaMzt1 to CaSpc110-N variants. Error bars are SD. Single representative binding curve out of three independent experiments is shown. (E) Summary of MST measurements on the binding of EGFP-CaMzt1 to CaSpc72-N and CaSpc110-N. K_d ± SD; n = 3 independent measurements. mAU, milli-absorbance unit.