Experimental strategy and purity of mitochondrial fractions from S. cerevisiae. (A) Flow chart showing purification of mitochondrial outer membranes and mitochondrial surface fraction followed by MS analysis. (B) Isolation of highly purified mitochondria. Crude mitochondrial fractions (P₁₂) were purified twice via sucrose gradients, yielding purified mitochondria essentially devoid of marker proteins from other cell compartments. Equal-volume aliquots from mitochondria and total yeast extracts were separated by SDS-PAGE, followed by Western blotting and immunodecoration. Immunoreactive bands were quantified and the value for total yeast extract of each protein was set to 100%. Porin, mitochondrial outer membrane protein; PGK, phosphoglycerate kinase (cytosol); Sss1, subunit of Sec61 translocation complex (endoplasmic reticulum); Nsp1, subunit of nuclear pore complex; ALP, vacuolar ALP; Pex13, peroxin13. (C) Purification of outer membrane vesicles. Purified mitochondria and outer membrane vesicles were separated by SDS-PAGE and then either stained with Coomassie brilliant blue (lanes 1 and 2) or transferred onto PVDF membranes followed by immunodecoration (lanes 3 and 4). Tom40, Tom70, subunits of the preprotein translocase of the outer membrane; Sam35, subunit of the sorting and assembly machinery of the outer membrane; CCHL, cytochrome c heme lyase of the intermembrane space; Tim23, subunit of the presequence translocase of the inner membrane; Mge1, soluble matrix protein. (D) Generation of the MSF. Highly purified mitochondria were incubated with or without trypsin (20 μg/ml) in SEM buffer for 15 min on ice. After pelleting, the mitochondria were separated by SDS-PAGE and analyzed by Western blotting and immunodecoration, whereas the supernatant (MSF) was subjected to nano-LC-MS/MS analysis (Supplemental Table 4).

The homologue of human fatty aldehyde dehydrogenase is mainly located in the mitochondrial outer membrane. (A) Comparison of the predicted primary structures of S. cerevisiae Hfd1, encoded by the open reading frame YMR110c (S.c.), and the human fatty aldehyde dehydrogenase FALDH (H.s.). Black, identical residues; gray, similar residues; bars, predicted hydrophobic transmembrane segments found by the HMMTOP program (Tusnady and Simon, 2001). (B) Subcellular fractionation of yeast strains, containing HA-tagged versions of Hfd1 or Eht1 (Open Biosystems, Brussels, Belgium), according to Meisinger et al. (2000), followed by SDS-PAGE and Western blot analysis. Equal amounts of proteins were loaded. The mitochondrial fraction was purified by sucrose gradient purification. P₁₀₀, S₁₀₀, pellet, and supernatant of 100,000 × g centrifugation. Sss1, subunit of the Sec61 protein translocase (endoplasmic reticulum); Sse1, cytosolic heat-shock protein 70. (C) Hfd1_HA is located in the outer membrane. Separation of outer and inner membrane vesicles from Hfd1_HA mitochondria on a sucrose gradient was performed as described in Materials and Methods. Fractions were analyzed by SDS-PAGE and immunodecoration using antisera against HA, the outer membrane protein Tom40, and the inner membrane protein Tim23. (D) Mitochondrial Hfd1 is accessible to proteases. Highly purified mitochondria (50 μg of protein) isolated from the Hfd1_HA strain were resuspended in 100 μl of SEM buffer containing either proteinase K or trypsin and incubated for 15 min on ice. Proteinase K was inhibited by adding 2 mM PMSF, and trypsin was blocked by a 30-fold excess of soybean pancreatic trypsin inhibitor. After washing with SEM, mitochondria were separated on SDS-PAGE and analyzed by Western blotting and immunodecoration.

Accumulation of the precursor form of a cleavable mitochondrial protein in the mitochondrial outer membrane. (A) Two presequence peptides of cytochrome b₂ identified from the 2D-BAC/SDS-PAGE of purified outer membranes vesicles (Figure 2). Numbers indicate amino acid residues. The processing of cytochrome b₂ occurs in two steps, after residue 31 and after residue 80. (B) The precursor form of F₁β migrates in the outer membrane fraction. Outer and inner membrane vesicles from wild-type yeast mitochondria were separated on a sucrose gradient, and fractions were analyzed by SDS-PAGE and Western blotting. Immunoreactive bands were quantified and the maximal value was set to 100% for each protein. p-F₁β, precursor form; m-F₁β, mature protein.

2D-gel analysis of purified mitochondrial outer membranes. Purified outer membranes (∼30 μg of protein) of S. cerevisiae mitochondria were separated by BAC-PAGE in the first dimension followed by SDS-PAGE in the second dimension. Coomassie-stained spots were excised and analyzed either by nano-LC-MS/MS or MALDI-MS/MS as described in Materials and Methods. Thirty-six different proteins were identified and listed in Supplemental Table 1.

Comparison of the mitochondrial outer membrane proteome to a genome-wide analysis of mRNAs targeted to mitochondria. MLR values of 70 or higher (dark columns) indicate a high probability of mRNA localization to the mitochondrial environment (values are accessible at www.biologie.ens.fr/lgmgml/publication/mitoloc/) (Marc et al., 2002). Shown are the MLR fractions (MLR values grouped in blocks of 10: ≥90, ≥80, ≥70, and so on) calculated for all proteins of the classes below for which MLR values are available. (A) Proteins predicted from the S. cerevisiae genome (n = 3190). (B) Mitochondrial proteome (n = 422; Sickmann et al., 2003). (C) Known inner membrane (IM) and matrix proteins found in the outer membrane proteome (n = 21; Table 1). (D) Proteome of outer membrane vesicles (OMV) (n = 79; Table 1). (E) MSF (n = 47; Supplemental Table 4).