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. 2018 Jun 4;217(6):2087–2102. doi: 10.1083/jcb.201706044

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© 2018 Mast et al.

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Figure 7. — ESCRT-III is required for PPV budding from the ER. (A) PYCs containing microsomes and expressing Pex3-pA were incubated with the S100 fraction of cytosol isolated from WT (lanes 2–4), vps25Δ (lane 5), vps20Δ (lane 6), snf7Δ (lane 7), did4Δ (lane 8), vps24Δ (lane 9), or ist1Δ (lane 10) for 90 min at room temperature in the presence of an ATP-regenerating system. Controls included incubating the PYCs alone (lane 1), with cytosol but no ATP (lane 2), or with cytosol and ATP but at 4°C (lane 3). (B) As in A but with the S100 fraction of cytosol isolated from W303 WT and deletion strains. (C and D) Deficiencies in PPV budding are complemented by mixing the S100 fractions of defective cytosols. PYCs were incubated with WT (lanes 2–4), vps20Δ (lanes 5, 8, and 9), snf7Δ (lanes 6, 8, and 10), and pex19Δ (lanes 7, 9, and 10) S100 fractions of cytosol for 90 min at room temperature in the presence of an ATP-regenerating system. Controls included incubating the PYCs alone (lane 1), with cytosol but no ATP (lane 2), or with cytosol and ATP but at 4°C (lane 3). (E and F) VPS20 and SNF7 complement deficiencies in PPV budding in their respective deletion mutants. PYCs were incubated with S100 fractions of cytosol from WT carrying pCM189 (lane 2), vps20Δ carrying pCM189 (lane 3) or pCM189-VPS20 (lane 4), or snf7Δ carrying pCM189 (lane 5) or pCM189-SNF7 (lane 6). (G) ATP is not required for PPV release but rather to recycle scission components. PYCs expressing Pex3-GFP and the S100 fraction of cytosol isolated from WT were pretreated with apyrase before starting the reaction (lanes 3 and 5). The reaction was also sampled at the indicated times (lanes 6–10). For each panel, bar graphs represent the relative percentage of PPV release in AU with error bars representing the SEM of three biological replicates.