Binding of Mvb12 to Ub. (A) Recombinant V5 epitope–tagged Mvb12 for S. cerevisiae and S. kluyveri were assayed for Ub binding with Ub-GST. (B) V5 epitope–tagged fusions of the C terminus of either wild-type (WT) Mvb12 or the indicated mutants were expressed in bacteria and assayed for Ub binding with Ub-GST. (C) A sequence of Mvb12 with the Ub-binding region shown in red. Regions that make contact with Vps37 in the ESCRT-I crystal structure (Protein Data Bank accession no. 2P22) are underlined. The asterisk indicates the end of the protein sequence. (D) Cell lysates prepared from mvb12Δ cells transformed with low copy plasmids expressing Mvb12-HA (pPL3713), Mvb12^ΔUb1-HA (pPL3709), or Mvb12^ΔUb2-HA (pPL3711) were immunoblotted with anti-HA and anti-PGK antibodies. (E) Summary of NMR HSQC experiments with ¹⁵N-labeled Ub (50 µM) with the C-terminal fragment of Mvb12 (100 µM). (left) Part of the spectra of ¹⁵N-labeled Ub in the presence (red) and absence (green) of Mvb12 is shown. Chemical shift differences were quantified and plotted on the linear sequence of Ub (middle) or onto the surface of Ub (right). Red indicates (0.2δN² + δH²)^1/2 ≥ 0.02. Black residues were not observed. ppm, parts per million.