Crystal structure of Ist1NTD. (A) Ribbon representation of Ist1NTD structure shown in two orientations. Secondary structural elements and the N- and C-termini of the structure are labeled. (B) Structure-based sequence alignments of Ist1NTD from various organisms. Secondary structural elements of S. cerevisiae Ist1NTD are shown above the sequence block. Residues involved in forming the conserved Did2-binding groove are highlighted with triangles at the bottom. Residues essential for Did2 binding are also noted with stars. (C) Ist1NTD has an ESCRT-III like fold. Helices present in both structures of Ist1NTD and the ESCRT-III protein CHMP3 are labeled and highlighted.

A conserved surface of Did2 mediates interaction with Ist1NTD. (A) The structure of the Ist1NTD/Did2—MIM1 complex. Side chains of residues at the interface from the two proteins are shown in an enlarged view. The MIM1 helices in B and C are shown in the same general orientation. (B) The structure of the Vps4-MIT/CHMP1A–MIM1 complex (PDB ID: 2JQ9). (C) The structure of the spastin-MIT/CHMP1B–MIM1 complex (PDB ID: 3EAB). (D) MIM1 or MIM1-like sequences in Did2, Vps2, Ist1, and Vps24. Conserved sequence features are highlighted. Residues that have been experimentally demonstrated to be important for protein–protein interaction are shown in red. (E) Conserved residues in Did2 are important for Ist1 binding. GST-tagged wild-type or mutant Did2^176-204 proteins were used to pull down purified Ist1 as indicated. Proteins retained on the beads were analyzed by SDS-PAGE and visualized by Coomassie staining.

Model for the functional cycle of the ESCRT-III proteins. Left panel, the open, active conformation of the ESCRT-III proteins. Helices α1–α4 are adapted from the structure of human CHMP3. Helix α5 and the C-terminal helix α6 are shown as arbitrarily placed cartoons and the linkers as dash lines. Right panel, the speculative closed, inactive conformation of the ESCRT-III proteins. The positions of helices α5–α6 are inspired by the structure of the Ist1NTD/Did2–MIM1 complex.

Analysis of interaction between Ist1 and Did2. (A) Ist1 binds to the C-terminal region of Did2. GST, GST-Did2, or GST-Did2^176-204 was used to pull down N-terminally His-tagged Ist1 as indicated. Proteins retained on the beads were analyzed by SDS-PAGE and visualized by anti-His immunoblotting (top) and Ponceau S staining (bottom). (B) Ist1NTD specifically interacts with Did2-MIM1. His-SUMO-tagged MIM1 fragment from Did2 (Did2^190-204), Vps2 (Vps2^218-232), Vps24 (Vps24^201-224), or Ist1 (Ist1^281-298) was coexpressed with Ist1NTD in E. coli. Proteins retained on the Ni²⁺-NTA beads were analyzed by SDS-PAGE and visualized by Coomassie staining.

Did2-MIM1 binds in a conserved surface groove of Ist1NTD. (A) Fo-Fc difference map (contoured at 3σ) showing the location of Did2-MIM1 helix in the Ist1NTD/Did2-MIM1 structure. Conserved residues of Ist1 at the interface are also shown. (B) Did2-MIM1 binds to Ist1NTD in a highly conserved surface groove. The molecular surface of Ist1NTD is colored based on conservation scores. The most conserved residues are colored in magenta and the least conserved in cyan. (C) Contribution of the conserved residues to the binding of Did2. GST-tagged Did2 fragments were used to pull down wild-type or mutant Ist1 as indicated. Proteins retained on the beads were analyzed by SDS-PAGE and visualized by Coomassie staining. (D) SPR analysis of wild-type and mutant Ist1 proteins binding to Did2^176-204. Data represent the average of three independent measurements for each group. A simple one-to-one binding model was used to fit the data.

Ist1 residues involved in Did2 binding are important for both MVB sorting in yeast and mammalian cytokinesis. (A) The yeast MVB cargo GFP-CPS was visualized by fluorescent and brightfield microscopy in living cells corresponding to wild-type, ist1Δ, vta1Δ, ist1Δvta1Δ, or ist1Δvta1Δ cells expressing wild-type or mutant Ist1 proteins as indicated. The absence of lumenal GFP fluorescence in ist1Δvta1Δ cells expressing HA-Ist1^L168A/Y172A or HA-Ist1^E175R indicated that the Ist1–Did2 interaction contributes to Ist1 function in vivo. (B) In vitro interaction between hIST1 and CHMP1B. Cos-1 cells expressing HA-tagged hIST1 proteins were lysed and subjected to pulldown by GST or GST-CHMP1B. Proteins retained on the beads were analyzed by SDS-PAGE and visualized by anti-HA blotting (top) and Ponceau S staining (bottom). (C) In vivo interaction between hIST1 and CHMP1B. Cos-1 cells expressing FLAG-tagged CHMP1B and HA-tagged hIST1 constructs were lysed and subjected to immunoprecipitation with anti-HA antibody. CHMP1B proteins associated with hIST1 were detected by anti-FLAG blotting. (D) Disruption of hIST1-CHMP1 interaction leads to cytokinesis defects. Hela cells expressing wild-type or mutant HA-hIST1 proteins were fixed and subjected to immunofluorescence with anti-HA (green) and anti-α-tubulin (red). Nuclei were visualized with TORPO 3 (blue). (E) Quantification of the cytokinesis defects shown in C. Multinucleated cells (with two or more nuclei) or cells displaying apparent cytokinesis defect (with clear cytoplasmic bridge between the daughter cells as visualized by α-tubulin staining) were scored. Experiments were repeated four times, with 100 cells counted for each sample.