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. 2016 Apr 13;5:e15507. doi: 10.7554/eLife.15507

Figure 1. Novel Snf7 point mutations trigger core domain activation.

(A) Domain organization of Vps20(red)-Snf7(blue) chimera (left) and quantitative MVB sorting data (right) for vps20Δ yeast exogenously expressing VPS20, vps201-197-snf7211-240, vps201-172-snf7181-240, vps201-147-snf7150-240, vps201-118-snf7120-240, vps201-105-snf7107-240, vps201-59-snf761-240, vps201-5-snf711-240, and SNF7. Error bars represent standard deviations from 3–5 independent experiments. (B) Screening strategy to identify snf7 suppressors in vps20Δ yeast. (C) Canavanine sensitivity assay for vps20Δ yeast exogenously expressing empty vector, VPS20, SNF7, snf7Q90L, and snf7N100I. (D) Domain organization of Snf7, with the locations of Gln90 and Asn100. WebLogo of protein sequence analysis (Doerks et al., 2002) of Snf7 orthologs from Saccharomyces cerevisiae, Homo sapiens, Mus musculus, Xenopus laevis, Drosophila melanogaster, Caenorhabditis elegans, Schizosaccharomyces pombe. (E) Close-up view of the side chain interactions of Gln90 (left) and Asn100 (right) in a 'closed' Snf7 homology model (Henne et al., 2012). (F) Liposome sedimentation assays of Snf7WT and Snf7Q90L. Liposome-bound (B) proteins and unbound (U) proteins. (G) CD scanning spectra from 200 nm to 260 nm of wild-type Snf7α1–4 (left), Snf7α1–4 Q90L (middle), and Snf7α1–4 N100I proteins with (red) and without (blue) liposomes.

DOI: http://dx.doi.org/10.7554/eLife.15507.002

Figure 1.

Figure 1—figure supplement 1. A full-length Vps20 is required for MVB sorting.

Figure 1—figure supplement 1.

(A–B) Domain organization (left) and quantitative MVB sorting data (right) for (A) snf7Δ yeast exogenously expressing empty vector, SNF7, and VPS20, and for (B) vps20Δ yeast exogenously expressing empty vector, SNF7, VPS20, vps201-200, vps201-172, and vps201-147. Error bars represent standard deviations from 3–5 independent experiments.
DOI: http://dx.doi.org/10.7554/eLife.15507.003
Figure 1—figure supplement 2. Vps20-Snf7 chimera complements Vps20 function.

Figure 1—figure supplement 2.

(A) Canavanine sensitivity assay for vps20Δ yeast exogenously expressing VPS20, empty vector, vps201-147, and vps201-147-snf7150-240. (B) Representative images of vps20Δ SNF7-GFP yeast exogenously expressing VPS20, empty vector, vps201-147, and vps201-147-snf7150-240. GFP images (left) and composite images of GFP and DIC (right).
DOI: http://dx.doi.org/10.7554/eLife.15507.004
Figure 1—figure supplement 3. Snf7Q90L and Snf7N100I trigger core domain activation.

Figure 1—figure supplement 3.

(A) Domain organization of Snf7 closed and open conformations, highlighting the core domain. (B) Ribbon models of Snf7 closed (homology) (Henne et al., 2012) and open conformations (right, PDB: 5FD9), with the locations of Gln90 and Asn100.
DOI: http://dx.doi.org/10.7554/eLife.15507.005
Figure 1—figure supplement 4. Snf7Q90L assembles into protofilaments in vivo and in vitro.

Figure 1—figure supplement 4.

Domain organization and quantitative MVB sorting data (left) for snf7Δ yeast exogenously expressing empty vector, SNF7, snf7Q90L, and snf7N100I. Error bars represent standard deviations from 3–5 independent experiments. Representative TEM images (right) of Snf7Q90L. Scale bars 100 nm.
DOI: http://dx.doi.org/10.7554/eLife.15507.006