Figure 7. Biochemical complementation of... | Rockefeller University Press

Figure 7.

$Figure 7. Biochemical complementation of the AP closure defect of ESCRT mutants by purified recombinant proteins. Membrane fractions from ESCRT mutants snf7Δ and vps4Δ were incubated with Snf7 or Vps4 purified from bacteria. Samples were then subjected to the protease-protection assay combined with immunoblot analysis (see Materials and methods). (A and B) Partial protection of the autophagy cargos GFP-Atg8 (A) and prApe1 (B) from protease (PK) in membrane fractions of snf7Δ mutant cells by Snf7 protein. (C and D) Partial protection of the autophagy cargos GFP-Atg8 (C) and prApe1 (D) from PK in vps4Δ mutant fractions by Vps4 WT, but not Vps4K179A mutant protein. Vps4 or the ATPase-defective Vps4K179A mutant protein was added in the presence of ATP regeneration system (ATP-RS). Columns represent mean, error bars represent SD. Results represent three independent experiments. **P < 0.01; ***P < 0.001. Recom, recombinant.$

Biochemical complementation of the AP closure defect of ESCRT mutants by purified recombinant proteins. Membrane fractions from ESCRT mutants snf7Δ and vps4Δ were incubated with Snf7 or Vps4 purified from bacteria. Samples were then subjected to the protease-protection assay combined with immunoblot analysis (see Materials and methods). (A and B) Partial protection of the autophagy cargos GFP-Atg8 (A) and prApe1 (B) from protease (PK) in membrane fractions of snf7Δ mutant cells by Snf7 protein. (C and D) Partial protection of the autophagy cargos GFP-Atg8 (C) and prApe1 (D) from PK in vps4Δ mutant fractions by Vps4 WT, but not Vps4^K179A mutant protein. Vps4 or the ATPase-defective Vps4^K179A mutant protein was added in the presence of ATP regeneration system (ATP-RS). Columns represent mean, error bars represent SD. Results represent three independent experiments. **P < 0.01; ***P < 0.001. Recom, recombinant.