Abstract
The intermembrane space (IMS) represents the smallest subcompartment of mitochondria. Nevertheless, it plays important roles in the transport and modification of proteins, lipids, and metal ions and in the regulation and assembly of the respiratory chain complexes. Moreover, it is involved in many redox processes and coordinates key steps in programmed cell death. A comprehensive profiling of IMS proteins has not been performed so far. We have established a method that uses the proapoptotic protein Bax to release IMS proteins from isolated mitochondria, and we profiled the protein composition of this compartment. Using stable isotope-labeled mitochondria from Saccharomyces cerevisiae, we were able to measure specific Bax-dependent protein release and distinguish between quantitatively released IMS proteins and the background efflux of matrix proteins. From the known 31 soluble IMS proteins, 29 proteins were reproducibly identified, corresponding to a coverage of >90%. In addition, we found 20 novel intermembrane space proteins, out of which 10 had not been localized to mitochondria before. Many of these novel IMS proteins have unknown functions or have been reported to play a role in redox regulation. We confirmed IMS localization for 15 proteins using in organello import, protease accessibility upon osmotic swelling, and Bax-release assays. Moreover, we identified two novel mitochondrial proteins, Ymr244c-a (Coa6) and Ybl107c (Mic23), as substrates of the MIA import pathway that have unusual cysteine motifs and found the protein phosphatase Ptc5 to be a novel substrate of the inner membrane protease (IMP). For Coa6 we discovered a role as a novel assembly factor of the cytochrome c oxidase complex. We present here the first and comprehensive proteome of IMS proteins of yeast mitochondria with 51 proteins in total. The IMS proteome will serve as a valuable source for further studies on the role of the IMS in cell life and death.
Highlights
Mitochondria are double-membrane-bound organelles that fulfill a multitude of important cellular functions
We found that cytochrome c and other soluble intermembrane space (IMS) proteins are released from Bax-treated yeast mitochondria, whereas soluble matrix proteins largely remain within the organelle [30]
We identified novel proteins that fall into several classes: (i) those that are involved in maintaining protein redox homeostasis, (ii) those that undergo proteolytic processing by inner membrane protease (IMP) (Ptc5), (iii) those that utilize the MIA pathway for their import (Mic23 and Coa6), and (iv) those that play a role in the assembly of cytochrome c oxidase (Coa6)
Summary
Yeast Strains and Isolation of Mitochondria—The following Saccharomyces cerevisiae strains were used: YPH499 [31], BY4741 (Euroscarf, Frankfurt, Germany), BY4741 rho0 [32], tom5⌬ [33], tom22⌬ [34], and mia40 –3 [35]. imp1⌬, imp2⌬, and coa6⌬ strains were from Euroscarf. For discrete candidate proteins, SILAC ratios could not be determined for all biological replicates and all different IMS samples To compensate for those missing SILAC ratios, additional data interpretation was conducted using PeptideShaker version 0.12.0 (http://code.google.com/p/peptide-shaker/) in order to identify missing candidate peptides based on multi-search engine database searches, including Mascot, OMSSA [44], and X!Tandem [45], combined with a thorough FDR assessment [46]. One missing candidate peptide (supplemental Table S3; supplemental Fig. S1) could be detected within the final 1% FDR (protein level) results list, and m/z value and retention time were used to manually determine the SILAC ratios based on monoisotopic MS signal intensities in the raw data using Xcalibur Qual Browser 2.1 (supplemental Table S3). A comprehensive list of all reproducibly identified proteins, including their SNBax/SNCon and SNBax/Tot ratios, is available in supplemental Table S5
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