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. 2006 Jun 5;173(5):651–658. doi: 10.1083/jcb.200603092

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Copyright © 2006, The Rockefeller University Press

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Figure 4. — Subcellular localization of Ups1p. (A) Cells expressing Ups1p-myc were grown in YPD, homogenized (H), and separated into a mitochondrial pellet (M) and a post-mitochondrial supernatant (P) by centrifugation. Cell-equivalent amounts of each fraction were analyzed by immunoblotting with the indicated antibodies. (B) ups1Δ cells expressing Ups1p-GFP were stained with 0.1 μM Mitotracker. Bar, 3 μm. (C) WT cells expressing Ups1(1–80)p-GFP or Ups1(1–40)p-GFP were stained with Mitotracker. Bar, 3 μm. (D) Ups1p-myc mitochondria (M) and mitoplasts (MP) were incubated with 0.2 mg/ml trypsin for 20 min on ice and analyzed by immunoblotting. (E) The OM of Ups1p-myc mitochondria was disrupted by osmotic shock. The resulting mitoplasts were separated into supernatant (S) and pellet (P) fractions by centrifugation, and analyzed by immunoblotting. (F) Mitoplasts were pelleted by centrifugation, treated with either 1.5 M NaCl, 0.1 M Na₂CO₃, or buffer, and separated into supernatant (S) and pellet (P) fractions by centrifugation. Each fraction was analyzed by immunoblotting. (G) Ups1p-myc mitochondria were sonicated and membrane vesicles were separated on sucrose gradients. Fractions were analyzed by immunoblotting. (H and I) Mitochondria from the indicated strains grown in YPD were analyzed by blue-native electrophoresis, followed by immunoblotting.