Supplemental Material

This article contains the following supporting material:

• Table 1
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• Figure 1 - Mitochondrial localization of proteins identified in the proteome of the outer mitochondrial membrane. (A-C) Erg9, Erg6 and Pho88 are located in the outer mitochondrial membrane. Outer and inner membrane vesicles were generated from highly purified mitochondria containing HA-tagged versions of Erg9, Erg6 or Pho88, respectively. The vesicles were separated on a sucrose gradient and analyzed by SDS-PAGE and Western blotting. Quantification of immunoreactive bands was performed with the NIH Image program. The maximal value was set to 100% for each protein. (D) ³⁵S-labeled precursor proteins of Yor285w and Rho1 were synthesized in rabbit reticulocyte lysate and imported into highly purified mitochondria as described (Meisinger et al., 2000, 2001; Wiedemann et al., 2005). Where indicated, the membrane potential Δ ψ across the inner membrane was dissipated before the import reaction or the mitochondria were treated with proteinase K (Prot. K; 50 μg/ml) after the import reaction. The mitochondria were reisolated and analyzed by SDS-PAGE and digital autoradiography. Prec., 30% of precursor protein added to the import reaction. (E) Highly purified mitochondria were isolated after growth of yeast cells on a fermentable carbon source (YPD). The mitochondria were treated with proteinase K or trypsin as indicated. The reisolated mitochondria were analyzed by SDS-PAGE and Western blotting.

• Figure 2 - Enrichment of proteins/protein fragments with high MLR score in the mitochondrial surface fraction after cell growth on a non-fermentable carbon source. (A) The mitochondrial surface fraction (MSF) was prepared from highly purified mitochondria (400 µg protein) after growth of yeast on non-fermentable (YPG) or fermentable (YPD) media. After SDS-PAGE and silver staining, the indicated bands were excised and analyzed by nano-LC-MS/MS. Numbers in brackets indicate the MLR values of the identified proteins. (B) Western blot analysis of MSF. In the MSF, Sam35 and Tom70 were identified as fragments of 17 kDa and 24 kDa, respectively. Mito, mitochondria were loaded as control.