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

Figure 6. Vps24 and Vps2 cooperatively bind Snf7, as over-expressing them suppresses the defects of Snf7 helix-4 mutants.

(A) Mup1-pHluorin endocytosis data after 90 min of methionine addition, showing overexpression of Vps24 or Vps2 rescuing the defect of the helix-4 mutant D131K. 2-fold overexpression (O.E.) corresponds to expressing either Vps24 or Vps2 with a CEN plasmid, while ~16 fold represents experiments performed with these genes on a CMV promoter with a tet-off operator. Error bars represent standard deviation from three to five independent experiments. (B).Overexpression of Vps24 (on a plasmid with a tet-off operator), rescues Mup1-pHluorin degradation defect of the D131K mutant, as shown immunoblotting of pHluorin over 60 min of methionine addition. (C).Model showing the hypothesized recruitment of Vps24/Vps2 in helix-4 mutant under normal (left) or under overexpressed (right) Vps24/Vps2 conditions.

Figure 6—source data 1. Individual data points for the % MVB sorting of Snf7 mutant and suppression through overexpression of Vps24 and Vps2.
elife-46207-fig6-data1.xlsx (8.9KB, xlsx)
DOI: 10.7554/eLife.46207.029

Figure 6.

Figure 6—figure supplement 1. Overexpression of Vps24 or Vps2 do not suppress the defects of longitudinal polymerization mutants of Snf7.

Figure 6—figure supplement 1.

(A) Expression levels of Vps24 at endogenous, overexpressed with CEN plasmid, or overexpressed with the pCM189 plasmid which contains a CMV promoter and a tet-off operator.(B) Cartoon depiction of the locations of the Snf7 mutants used in the subsequent experiments. R25, H29 and K36 fall on helix-1 of Snf7, which make contacts with the Snf7 helix-3 residues E95, E105 and E109 in the longitudinal polymeric arrangement of Snf7. (C) Mup1-pHluorin degradation experiments, blotting for pHluorin over time, with Snf7 mutants, while overexpressing Vps24. (D) Mup1-pHluorin flow-cytometry data under overexpression of Vps24 or Vps2 with Snf7 mutants. Error bars represent standard deviation from three independent experiments.