Fig. 1.

The CHMP4C A232T substitution reduces ALIX binding. (A) β-Galactosidase activity assays of yeast cotransformed with the indicated full-length CHMP4C constructs fused to VP16 and full-length ALIX or CHMP4C fused to GAL4 (mean ± SD, n = 3). (B) Competitive fluorescence polarization binding assay with an ALIX construct spanning the Bro1 and V domains (residues 1–698) binding to fluorescently labeled CHMP4C^A232 peptide (residues 216–233) competing with the indicated unlabeled CHMP4C peptides. Curves are from a representative experiment. K_i values are expressed as mean ± SD. n ≥ 7. (C, Left) Superposition of ALIX Bro1 domain (gray) complexes with CHMP4C^A232 (green) and CHMP4C^T232 (orange) peptides. (Right) Enlarged view of the CHMP4C peptide superposition highlights the reduction in CHMP4C^T232 helicity. (D) Binding interfaces between ALIX (gray) and CHMP4C^A232 (green) (Left) and CHMP4C^T232 (orange) (Right). Figures show equivalent intermolecular distances (in angstroms) for different atoms of CHMP4C residue W231. See also SI Appendix, Fig. S1 and Table S1.