Abstract
The mitochondrial proteome comprises ~1000 (yeast)–1500 (human) different proteins, which are distributed into four different subcompartments. The sublocalization of these proteins within the organelle in most cases remains poorly defined. Here we describe an integrated approach combining stable isotope labeling, various protein enrichment and extraction strategies and quantitative mass spectrometry to produce a quantitative map of submitochondrial protein distribution in S. cerevisiae. This quantitative landscape enables a proteome-wide classification of 986 proteins into soluble, peripheral, and integral mitochondrial membrane proteins, and the assignment of 818 proteins into the four subcompartments: outer membrane, inner membrane, intermembrane space, or matrix. We also identified 206 proteins that were not previously annotated as localized to mitochondria. Furthermore, the protease Prd1, misannotated as intermembrane space protein, could be re-assigned and characterized as a presequence peptide degrading enzyme in the matrix.
Highlights
The mitochondrial proteome comprises ~1000–1500 different proteins, which are distributed into four different subcompartments
The first part of our experimental approach is based on the dissection and global identification of soluble, peripheral, and integral membrane proteins from mitochondria of the model system yeast
Separation of the three protein classes was achieved by either (i) carbonate treatment at pH 11.5 to separate integral membrane proteins from peripheral membrane and soluble proteins[19] or (ii) sonication of the purified organelles to separate a complete membrane fraction from the soluble proteins (SNson) (Fig. 1b)
Summary
The mitochondrial proteome comprises ~1000 (yeast)–1500 (human) different proteins, which are distributed into four different subcompartments. We describe an integrated approach combining stable isotope labeling, various protein enrichment and extraction strategies and quantitative mass spectrometry to produce a quantitative map of submitochondrial protein distribution in S. cerevisiae This quantitative landscape enables a proteome-wide classification of 986 proteins into soluble, peripheral, and integral mitochondrial membrane proteins, and the assignment of 818 proteins into the four subcompartments: outer membrane, inner membrane, intermembrane space, or matrix. (ii) In 2002, De Hertogh et al.[11] predicted a large number of integral yeast membrane proteins based on phylogenetic classifications and to date all of these proteins are annotated, e.g., in the yeast genome database, as integral membrane proteins[8] This includes many mitochondrial proteins, such as Tim[9], Tim[10], and Tim[13], which are prime examples of soluble proteins of the IMS5, 12, or the peripheral IM subunits of the respiratory complex IV (Cox[4] and Cox12) and the ATP-synthase (Atp[1], Atp[2], or Atp3), all clearly not integral membrane proteins[13]. We generate a high quality map of submitochondrial distribution for native proteins under clearly defined experimental conditions
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