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. 2019 Jun 27;8:e46207. doi: 10.7554/eLife.46207

Figure 2. Helix-4 region of Snf7 is involved in recruiting Vps24.

(A) Helix-4 mutants fail to recruit Vps24-GFP. Vps24-GFP is present in endosomal dots in cells expressing SNF7 WT on a plasmid in a snf7Δ;Vps24-GFP strain, while with the mutation on Snf7, Vps24-GFP is more diffuse in the cytoplasm. (B) Coimmunoprecipitation experiments, immunoprecipitating Vps24 and blotting for either wild-type Snf7 or the mutant D131K. (C) Electron microscopy assay on lipid monolayers, depicting co-assembly of Snf7 R52E with Vps24 and Vps2 into helices. Snf7 R52E D131K fails to form similar structures. Experiments were done with 1 μM of each protein incubated for 1 hr on lipid monolayers. (D) ‘In vivo’ glycerol-gradient experiments using a gradient of 10% to 40%, using various mutant strains annotated on the far-right. Western-blots were performed against Snf7 (left) or Vps24 (right).

Figure 2.

Figure 2—figure supplement 1. Helix-4 contacts a trans Snf7 polymer in the crystal lattice.

Figure 2—figure supplement 1.

(A) Depiction of the contact between helix-4 of Snf7 with residues of helix-1, a trans polymer in the lattice. Zoomed-in box shows image of the D131 interacting with K21 and R25 of helix-1. (B) MVB-sorting (Mup-pHluorin degradation) of helix-1 mutants of Snf7. Error bars represent standard deviation from three independent experiments. (C) Canavanine sensitivity assay of helix-1 mutants of Snf7.
Figure 2—figure supplement 2. Helix-4 region of Snf7 is involved in recruiting Vps24.

Figure 2—figure supplement 2.

(A) Vps24-GFP recruitment experiment in a snf7Δ;Vps24-GFP strain expressing different mutants of Snf7 on a plasmid. (B) Membrane-fractionation experiments with wild-type or mutant Snf7 D131K showing relative amounts of Vps24 pelleted to endosomal (P13) or cytosolic (S13) fractions.
Figure 2—figure supplement 3. CHMP1B helix-4 contacts the helix-1 of IST1.

Figure 2—figure supplement 3.

(A).The cryo-EM structure of CHMP1B-IST1 copolymer (PDB 3JC1, McCullough et al., 2015). CHMP1B is colored in orange-red and IST1 is depicted in cyan. (B).Zoomed-in image of the contact between the helix-1 residues of IST1 with helix-4 residues of CHMP1B. (C).Sequence alignment of a part of helix-4 of human CHMP1B and yeast Snf7. (D).Overlay of the IST1 structure (PDB 3FRR, Bajorek et al., 2009b) with the homology model of yeast Vps24 (yVps24). The crystal structure of CHMP3 (PDB 3FRT, Bajorek et al., 2009b) was used to create the homology model.