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

Figure 2. Auto-activated Snf7 functionally bypasses Vps20.

(A) Domain organization of Snf7 mutants (left) and quantitative MVB sorting data (right) for vps20Δ yeast exogenously expressing empty vector, VPS20, SNF7, snf7Q90L, snf7N100I, snf7R52E, snf7R52E Q90L, snf7R52E Q90L N100I, myr-snf7, myr-snf7R52E Q90L, and myr-snf7R52E Q90L N100I. Error bars represent standard deviations from 3–5 independent experiments. The data of myr-snf7 (vps201-5-snf711-240) and SNF7 were re-plotted from Figure 1A for comparsion. Mutants myr-snf7R52E Q90L and myr-snf7R52E Q90L N100I are referred to snf7** and snf7***, respectively. (B) Representative images of vps20Δ yeast exogenously expressing GFP-CPS1 with VPS20, snf7**, and snf7***. GFP images (left) and composite images of GFP and DIC (right). (C) Canavanine sensitivity assay for vps20Δ yeast exogenously expressing empty vector, VPS20, SNF7, snf7**, and snf7***. (D) Representative TEM images of ILV-containing MVBs from vps20Δ vam7Δ yeast exogenously expressing vam7tsf, with VPS20, snf7**, and snf7***. Scale bars 100 nm. (E–F) Quantitation of ILV (N=150 ILV summed per sample) outer diameter from (D) in frequency distributions (E), and averaged measurements (F). Error bars represent standard deviations.

DOI: http://dx.doi.org/10.7554/eLife.15507.007

Figure 2.

Figure 2—figure supplement 1. Activation mutants complement snf7Δ in vivo.

Figure 2—figure supplement 1.

Domain organization of Snf7 mutants (left) and quantitative MVB sorting data (right) for snf7Δ yeast exogenously expressing empty vector, VPS20, SNF7, snf7Q90L, snf7N100I, snf7R52E, snf7R52E Q90L, myr-snf7, and myr-snf7R52E Q90L. Error bars represent standard deviations from 3–5 independent experiments. The data from snf7Q90L and snf7N100I were partially re-plotted from Figure 1—figure supplement 4 for comparison.
DOI: http://dx.doi.org/10.7554/eLife.15507.008
Figure 2—figure supplement 2. Activation mutants complement vps20Δ snf7Δ in vivo.

Figure 2—figure supplement 2.

(A) Domain organization of Snf7 mutants (left) and quantitative MVB sorting data (right) for vps20Δ snf7Δ yeast exogenously expressing empty vector and empty vector, VPS20 and SNF7, VPS20 and empty vector, empty vector and SNF7, empty vector and myr-snf7R52E Q90L, and empty vector and myr-snf7R52E Q90L N100I. Error bars represent standard deviations from 3–5 independent experiments. (B) Western blotting analyses of vps20Δ snf7Δ yeast expressing SNF7, snf7Q90L, snf7N100I, snf7R52E, myr-snf7, snf7**, and snf7***. G6PDH used as loading controls.
DOI: http://dx.doi.org/10.7554/eLife.15507.009