FIG 2.

Proteasome defects increase Mia40. (A) Spores of FZO1/fzo1Δ cells harboring the Mia40 or Erv1 expression plasmid were dissected on YPAD prior to growth at 26°C for 4 days. Triangles and pentagons indicate fzo1Δ cells with or without the Mia40 or Erv1 expression plasmid, respectively. (B) Lysates of cells expressing chromosomally encoded Mia40-Flag were subjected to Western blotting, and proteins were detected using the indicated antibodies. Pgk1 and porin were used as cytosolic and mitochondrial loading controls, respectively. (C) Total RNA of the indicated strains was extracted. The amount of MIA40 mRNA was measured and standardized to TOM20 or ACT1 mRNA. TOM20 and ACT1 were used as mitochondrial and nonmitochondrial standards, respectively. (D) The indicated yeast cells were treated with Zymolyase 100T to digest the cell wall. Spheroplasts were homogenized, and the resultant homogenates were fractionated by differential centrifugation. Total, total homogenate; PMS, postmitochondrial supernatant; Mito, rough mitochondria. The total and rough mitochondrial fractions were diluted by 5-fold and 8-fold, respectively, so that Mia40-Flag in the PMS fraction could be detected. Pgk1 was used as a cytosolic loading control, and Om45-GFP was used as a mitochondrial loading control.