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Proteomic Profiling of the Mitochondrial Ribosome Identifies Atp25 As a Composite Mitochondrial Precursor Protein

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Proteomic Profiling of the Mitochondrial Ribosome Identifies Atp25 As a Composite Mitochondrial Precursor Protein M BoC | ARTICLE Proteomic profiling of the mitochondrial ribosome identifies Atp25 as a composite mitochondrial precursor protein Michael W. Woellhafa, Frederik Sommerb, Michael Schrodab, and Johannes M. Herrmanna,* aCell Biology and bMolecular Biotechnology and Systems Biology, University of Kaiserslautern, 67663 Kaiserslautern, Germany ABSTRACT Whereas the structure and function of cytosolic ribosomes are well characterized, Monitoring Editor we only have a limited understanding of the mitochondrial translation apparatus. Using SILAC- Thomas D. Fox based proteomic profiling, we identified 13 proteins that cofractionated with the mitochondrial Cornell University ribosome, most of which play a role in translation or ribosomal biogenesis. One of these pro- Received: Jul 15, 2016 teins is a homologue of the bacterial ribosome-silencing factor (Rsf). This protein is generated Revised: Aug 18, 2016 from the composite precursor protein Atp25 upon internal cleavage by the matrix processing Accepted: Aug 23, 2016 peptidase MPP, and in this respect, it differs from all other characterized mitochondrial pro- teins of baker’s yeast. We observed that cytosolic expression of Rsf, but not of noncleaved Atp25 protein, is toxic. Our results suggest that eukaryotic cells face the challenge of avoiding negative interference from the biogenesis of their two distinct translation machineries. INTRODUCTION In their overall structure and function, mitochondrial ribosomes are both biochemical fractionation studies (Liu and Spremulli, 2000; similar to cytosolic 80S and bacterial 70S ribosomes (Greber and Jia et al., 2003; Szyrach et al., 2003) and in situ visualization by Ban, 2016; Ott et al., 2016). However, mitochondrial ribosomes microscopy (Watson, 1972; Pfeffer et al., 2015). Membrane bind- were remodeled considerably during eukaryotic evolution and show ing is mediated by a protein of the large subunit, called Mba1 in significant differences compared with bacterial ribosomes (Smits yeast and Mrpl45 in mammals, positioned adjacent to the poly- et al., 2007; Beckmann and Herrmann, 2015). A common feature of peptide exit tunnel and directs nascent chains to the Oxa1 inser- all present-day eukaryotes is the highly reduced mitochondrial ge- tion machinery of the inner membrane (Ott et al., 2006; Jia et al., nome, which only encodes a small number of proteins, most of 2009; Greber et al., 2014b). The tight contact to the membrane which are hydrophobic membrane proteins (Gray et al., 1999). Pre- presumably explains the substantial remodeling of the region sumably, this specialization in synthesizing a very small number of around the exit tunnel of the mitochondrial ribosome (Sharma hydrophobic membrane proteins explains the two major differences et al., 2003; Brown et al., 2014; Greber et al., 2014b). between mitochondrial ribosomes and those of bacteria and the 2. The strongly degenerate rRNA core structure is another common eukaryotic cytosol: feature of the mitochondrial ribosome (Amunts et al., 2014; 1. Mitochondrial ribosomes of many organisms show tight physical Brown et al., 2014; Greber et al., 2014a). It is most obvious in connection with the inner membrane, which was reported from animals (and some parasites), in which rRNA content of the ribo- some is reduced close to what might be the minimum required for catalytic activity. Here many structural rRNA regions were lost, This article was published online ahead of print in MBoC in Press (http://www in some cases replaced by proteins (O’Brien, 2002). In fungi, re- .molbiolcell.org/cgi/doi/10.1091/mbc.E16-07-0513) on August 31, 2016. duction of the rRNA is less extreme, but its structure and base *Address correspondence to: Johannes Herrmann (hannes.herrmann@biologie .uni-kl.de). pairing–mediated stability strongly degenerates (van der Sluis Abbreviations used: MPP, mitochondrial processing peptidase; MTS, matrix-tar- et al., 2015). geting sequence; PK, proteinase K; Rsf, ribosome-silencing factor; SILAC, stable isotope labeling by amino acids in cell culture. In the past, the protein composition of high salt–treated, © 2016 Woellhaf et al. This article is distributed by The American Society for Cell gradient-purified ribosomes under stringent conditions was identi- Biology under license from the author(s). Two months after publication it is avail- fied by proteomics (Graack et al., 1999; Koc et al., 2001a,b; Suzuki able to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). et al., 2001). A recent study followed an interesting complemen- “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of tary approach to identify the proteome of mitochondrial ribo- the Cell®” are registered trademarks of The American Society for Cell Biology. somes of Saccharomyces cerevisiae under very mild conditions Volume 27 October 15, 2016 3031 (10 mM potassium acetate), using an affinity purification strategy mitochondrial extracts and the 73S fractions of the gradients were of an epitope-tagged ribosome (Kehrein et al., 2015). A surpris- mixed, digested with trypsin, and analyzed by mass spectrometry. ingly large number of components copurified with mitochondrial Because each tryptic peptide contains either one lysine or one ar- ribosomes, including many factors involved in mitochondrial gene ginine residue, the ratios of each peptide could be determined expression. accurately in the samples. In this study, we used a third approach, relying on stable iso- Next we compared the peptide ratios of the 73S fractions of tope labeling by amino acids in cell culture (SILAC)–based isotope wild type to rho0 with wild type to Mrp20-depleted cells (Figure 1D). labeling and comparative profiling of mitochondrial fractions con- We identified 466 proteins in both samples (Supplemental Tables S1 taining or lacking ribosomes to reduce the number of false-positive and S2), many of which were enriched in the wild type relative to the interaction partners. We thereby identified 28 (85%) and 37 (84%) mutants. These proteins included ribosomal subunits but also many of the previously described proteins of the small and large subunit, components of the respiratory chain whose stable accumulation in respectively, plus 13 additional proteins, seven of which are essen- mitochondria depends on the function of the mitochondrial ribo- tial for mitochondrial translation. We found that one of these ad- some. However, when we removed all proteins that were not at least ditional proteins, mitochondrial ribosome-silencing factor (Rsf), threefold enriched in the 73S fraction relative to the total, the was generated from a composite precursor protein of Atp25 (Zeng remaining proteins fell into three categories (Figure 1, E–G). 1) Previ- et al., 2008). The Atp25 precursor consisted of a matrix-targeting ously identified subunits of the mitochondrial ribosomes (Supple- sequence (MTS), the Rsf domain, and a C-terminal region (M mental Table S2). We identified 28 (85%) and 37 (84%) of the domain). previously described proteins of the large and small subunit, respec- In bacteria, Rsf was shown to block protein synthesis under star- tively. The few proteins we missed (Mrpl27, Mrpl37, Mrpl44, Mrp10, vation conditions (Häuser et al., 2012), but the function of its mito- Rsm19, Mrps12, and Mrps18) were predominantly very small and chondrial counterpart is largely unknown. Three recent studies on positively charged, which explains why they did not provide enough the mammalian homologue indicate a function in biogenesis or unambiguous peptides. Of interest, the proteins of the small functional maintenance of the large subunit (Rorbach et al., 2012; (Figure 1E, yellow) and large (Figure 1E, blue) subunit were clearly Wanschers et al., 2012; Fung et al., 2013). We observed that the distinct in the segregation pattern due to the specific depletion of expression of Rsf in the yeast cytosol is toxic, whereas the expres- the large-subunit proteins in the Mrp20-depletion sample. 2) We sion of Rsf-M domain fusion, such as present in the Atp25 precursor, identified many proteins of the 80S ribosome that were coisolated was much better tolerated. Thus the unconventional organization with the mitochondria. All of these proteins scattered around the of Atp25 as a tandem protein may serve as a strategy to prevent intersection of the axes and hence were not influenced by the ab- interference of the Rsf precursor with the cytosolic translation sence or presence of mitochondrial ribosomes (Figure 1F). 3) As a machinery. third group, we identified 13 additional proteins that exactly resem- bled in their fractionation profile the subunits of the mitochondrial RESULTS ribosome (Figure 1G). Proteomic analysis of the mitochondrial ribosome To isolate mitochondria devoid of assembled ribosomes, we used Aim23, Atp25, Cbp2, Mhr1, Mss11, Mrx14, and Pth4 are strains that lacked mitochondrial DNA (rho0) and hence mitochon- required for mitochondrial translation drial rRNA. In addition, we generated a strain in which the expression With the exception of Tes1, all of the 13 proteins that cofractionated of a central ribosomal protein (Mrp20, the homologue of bacterial with mitochondrial ribosomes were previously shown to be present L23) was under control of the GAL10 promoter. Mrp20 is essential in mitochondria (Figure 2A). Tes1 is a peroxisomal acyl-CoA thioes- for the assembly of the large ribosomal subunit (Fearon and Mason, terase involved in β-oxidation of oleate.
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