Panel A: A line graph comparing size exclusion chromatography elution profiles of M i s 12 c M t w 1 c variants. The x-axis represents volume (m L), and the y-axis represents absorbance at 280 n m. The graphs include wild type M i s 12 c M t w 1 c, M i s 12 c M t w 1 c delta M i s 12 C, M i s 12 c M t w 1 c delta N n f 1 C, and the double deletion delta M i s 12 C-delta N n f 1 C. Panel B: Four Coomassie brilliant blue-stained S D S-PAGE gels show protein composition corresponding to Panel A fractions, with bands for D s n 1, M i s 12, N s l 1, and N n f 1. Panel C: A line graph compares size exclusion chromatography elution profiles of N d c 80 c interaction assays. The x-axis represents volume, and the y-axis represents absorbance at 280 nanometers. The conditions include N d c 80 c alone, N d c 80 c plus M i s 12 c M t w 1 c (wild type), N d c 80 c plus M i s 12 c M t w 1 c delta M i s 12 C, N d c 80 c plus M i s 12 c M t w 1 c delta N n f 1 C, and N d c 80 c plus M i s 12 c M t w 1 c delta M i s 12 C–delta N n f 1 C. Panel D: Four Coomassie brilliant blue-stained S D S-PAGE gels show protein composition of N d c 80 c reconstitution assays, including bands for D s n 1, N d c 80, N u f 2, S p c 25, S p c 24, M i s 12, N s l 1, and N n f 1. Panel E: Yeast growth assays show m t w 1 delta C and n n f 1 delta C mutants in strains with endogenous M T W 1 and N N F 1 tagged with m A I D 3-F L A G 5, comparing growth on Y E P D and Y E P D plus 0.5 m M I A A (indole-3-acetic acid).
Biochemical analysis of Mis12 Mtw1_Cterm-helix and Nnf1 Cterm-helix function. (A) SEC elution chromatograms of Mis12cMtw1c containing either full-length and wild-type proteins, or full-length and wild-type proteins apart from deletion of Mis12Mtw1 residues 272–289 (Mis12Mis12_Δ272-C: Mis12cMtw1c_Mis12ΔC), deletion of Nnf1 residues 180–201 (Nnf1Δ180-C: Mis12cMtw1c_ Nnf1ΔC), or deletion of both Mis12Mtw1 residues 272–289 and Nnf1 residues 180–201 (Mis12cMtw1c_Mis12ΔC-Nnf1ΔC). (B) Coomassie brilliant blue–stained SDS-PAGE gels of all of the experiments presented in A. (C) SEC elution chromatograms of the Mtw1c:Ndc80c interaction reconstitutions. Attempts at reconstituting Mis12cMtw1c, Mis12cMtw1c_Mis12ΔC, Mis12cMtw1c_Nnf1ΔC, and Mis12cMtw1c_Mis12ΔC-Nnf1ΔC with full-length, unmodified Ndc80c are presented alongside the elution profile of Ndc80c alone. (D) Coomassie brilliant blue–stained SDS-PAGE gels of the experiments in C. (E) Analysis of mtw1ΔC and nnf1ΔC mutants in yeast strain backgrounds with endogenous MTW1 and NNF1 gene products translationally fused to mAID3-FLAG5 tag. Cell growth was investigated by plating 1:9 serial dilutions onto either YEPD plates or YEPD plates supplemented with 0.5 mM IAA. - = no rescue allele was expressed. mAID = monomeric auxin-inducible degron. Source data are available for this figure: SourceData FS3.