Figure 1.

mia40-4pHA is an unstable mitochondrial protein. (A) Ten-fold serial dilutions of mia40Δ cells expressing mia40-4-HA or MIA40-HA from CEN plasmids at permissive (25°) or nonpermissive (37°) temperatures were spotted to minimal media lacking leucine. (B) WT yeast were treated with CHX at 25 or 37° for the indicated times to assess the degradation of CEN plasmid-expressed mia40-4pHA or Mia40pHA. Proteins were detected by immunoblotting with HA antibody. Phosphoglycerate kinase (PGK) served as a protein loading control. (C) Lysates from cells expressing Mia40pHA (top) or mia40-4pHA (bottom) from CEN plasmids at 25 or 37° were fractionated into mitochondrial pellets (P) and postmitochondrial supernatants (S). Fractions were subject to immunoblotting with antibodies to HA, Porin, or PGK. (D) Differential interference contrast (DIC) and fluorescence microscopy at 25 and 37° of cells coexpressing mia40-4pGFP or Mia40pGFP and a mitochondrial matrix-targeted RFP variant, mtERFP. Bar, 10 μm and “merge” is an overlay of GFP and RFP channels. (E) Mitochondria isolated as in (C) were treated with proteinase K (PrK) alone, or in combination with Triton X-100 (Tx-100), and subject to immunoblotting with antibodies specific to the OM protein Sam35p, the IM protein Cox1p, or the matrix protein Hsp60p.