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The ribosome receptors Mrx15 and Mba1 jointly organize cotranslational insertion and protein biogenesis in mitochondria

The ribosome receptors Mrx15 and Mba1 jointly organize cotranslational insertion and protein... MBoC | ARTICLE The ribosome receptors Mrx15 and Mba1 jointly organize cotranslational insertion and protein biogenesis in mitochondria a a a b Braulio Vargas Möller-Hergt , Andreas Carlström , Katharina Stephan , Axel Imhof , a, and Martin Ott * a b Department of Biochemistry and Biophysics, Stockholm University, SE-10691 Stockholm, Sweden; Protein Analysis Unit, Biomedical Center, Faculty of Medicine, Ludwig Maximilian University of Munich, DE-82152 Planegg-Martinsried, Germany Monitoring Editor ABSTRACT Mitochondrial gene expression in Saccharomyces cerevisiae is responsible for Benjamin S. Glick the production of highly hydrophobic subunits of the oxidative phosphorylation system. University of Chicago Membrane insertion occurs cotranslationally on membrane-bound mitochondrial ribosomes. Here, by employing a systematic mass spectrometry–based approach, we discovered the Received: Apr 13, 2018 previously uncharacterized membrane protein Mrx15 that interacts via a soluble C-terminal Revised: Jul 9, 2018 Accepted: Aug 2, 2018 domain with the large ribosomal subunit. Mrx15 contacts mitochondrial translation products during their synthesis and plays, together with the ribosome receptor Mba1, an overlapping role in cotranslational protein insertion. Taken together, our data reveal how these ribosome receptors organize membrane protein biogenesis in mitochondria. INTRODUCTION The mitochondrial proteome consists of polypeptides from two In addition, mitoribosomes are permanently attached to the in- genetic sources. Most proteins are encoded in the nucleus and ner mitochondrial membrane (Preuss et al., 2001; Ott et al., posttranslationally imported into their respective mitochondrial 2006; Prestele et al., 2009; Pfeffer et al., 2015), in contrast to compartments (Neupert, 2015; Wiedemann and Pfanner, 2017). bacterial ribosomes that are targeted to the membrane by the A small subset of proteins, eight in Saccharomyces cerevisiae, is signal-recognition particle (Bernstein et al., 1989). In yeast mito- encoded in the mitochondrial genome (Foury et al., 1998). Their chondria, cotranslational membrane insertion is mediated in a genes are transcribed and translated by a genetic system resid- concerted manner by Oxa1 and Mba1 (Preuss et al., 2001; Ott ing in the matrix of the organelle. The mitochondrial ribosomes et al., 2006). Oxa1, which belongs to the conserved YidC/Alb3/ (mitoribosomes) synthesize, almost exclusively, membrane pro- Oxa1 family, inserts client proteins into the membrane (He and teins that are subunits of oxidative phosphorylation complexes. Fox, 1997; Hell et al., 2001). The peripheral membrane protein Recent structural data have revealed that mitoribosomes are Mba1 cooperates with the C-terminal domain of Oxa1 in mem- adapted for the production of hydrophobic polypeptides by a brane protein insertion and ribosome–membrane attachment specific makeup of the polypeptide exit tunnel, which is rather (Ott et al., 2006). The human homologue of Mba1 (Mrpl45) is apolar to accommodate the hydrophobic translation products integrated in the large ribosomal subunit and was speculated to (Amunts et al., 2014; Brown et al., 2014; Greber et al., 2014a,b). fulfill a similar role in membrane attachment (Greber et al., 2014b). Additionally, Mba1 shuttles the mitochondrial-encoded cytochrome c oxidase subunit 2 (Cox2) to the assembly factor Cox20 (Lorenzi et al., 2016). This article was published online ahead of print in MBoC in Press (http://www In a previous study, we found that the mitochondrial ribosome is .molbiolcell.org/cgi/doi/10.1091/mbc.E18-04-0227) on August 9, 2018. *Address correspondence to: Martin Ott (martin.ott@dbb.su.se). organized in large assemblies that we termed MIOREX complexes Abbreviations used: BN-PAGE, blue native PAGE; cryo-EM, cryo–electron micros- (Kehrein et al., 2015). These assemblies contain factors responsible copy; LSU, large subunit; PMSF, phenylmethylsulfonyl fluoride; SSU, small subunit. for mRNA maturation and turnover, translation, chaperones, prote- © 2018 Möller-Hergt et al. This article is distributed by The American Society for ases, and a number of previously uncharacterized proteins, some of Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Un- which were later shown to play a role in mitochondrial gene expres- ported Creative Commons License (http://creativecommons.org/licenses/by-nc sion (Moda et al., 2016; Rak et al., 2016). Another study used a SILAC -sa/3.0). (stable isotope labeling of amino acids in cell culture)-based ap- “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology. proach (Woellhaf et al., 2016) to identify several factors comigrating 2386 | B. V. Möller-Hergt et al. Molecular Biology of the Cell FIGURE 1: Proteomic profiling of the small and large mitochondrial ribosomal subunits. (A) Strategy to isolate the MIOREX complex with subsequent separation of mitoribosome subunits. (B) After the procedure described in A, the protein abundance of individual fractions from a continuous sucrose gradient was determined by label-free quantitative mass spectrometry. Sedimentation profiles are depicted for the large subunit (blue) and small subunit (yellow). As a control, the experiment was probed for proteins from the large (Mrpl36) and the small (Mrps5) subunit as well as aconitase (Aco1). (C) Top, summary of known interactors of the large and small subunit. These factors showed the same migration behavior as their described ribosomal-interacting subunit. Bottom, summary of known mitoribosome interactors and the interacting subunit found in this study. (D) Summary of known (star) or newly discovered MIOREX components found in this study that comigrated with either the large or the small ribosomal subunit. LSU, large subunit; P, pellet; qMS, quantitative mass spectrometry; R, resuspended; SN, supernatant; SSU, small subunit; T, total. with the assembled mitoribosome in sucrose gradients, but the ribo- tal Data S1). Proteins from the large subunit (LSU) almost exclusively somal subunits with which they interacted remained unknown. migrated in fractions 2–4, while proteins of the small subunit (SSU) Here, we developed an alternative strategy to further character- were found in fractions 4–6 (Figure 1B). ize the mitoribosomal interactome. We thereby discovered a previ- Next, we cross-referenced our data set with known interactors of ously uncharacterized protein (Mrx15) that interacts with the large the large and small ribosomal subunit. As expected, previously de- ribosomal subunit. Simultaneous absence of Mrx15 and Mba1 pro- scribed mitoribosome interactors like Cbp3, Mhr4, and Mtg2 (Figure vokes a profound respiratory growth defect and decrease in cyto- 1C, top) (Datta et al., 2005; Gruschke et al., 2011; De Silva et al., 2013) chrome c oxidase. We demonstrate that Mba1 and Mrx15 are ribo- comigrated with the LSU. The mitochondrial initiation factor 2 (Ifm1) some receptors with overlapping roles in cotranslational protein (Garofalo et al., 2003) and Cmm1 (Puchta et al., 2010) comigrated insertion, directly interacting with the large ribosomal subunits, na- with the SSU, indicating that we can identify predicted ribosomal in- scent polypeptide chains, and the inner membrane. teraction partners with our methodology. We next checked for pro- teins that have been reported to interact with the mitoribosome, but RESULTS for which the subunit they interact with was unknown (Westermann Identification of mitoribosomal interactors by a systematic et al., 1996; Williams et al., 2007; Bauerschmitt et al., 2008; Paul et al., mass-spectrometric approach 2012; Kehrein et al., 2015). This allowed us to establish that Cbp2, To categorize the extensive interactome of the yeast mitoribosome Pth4, and Mdj1 interact with the LSU, while Rmd9, Gep3, and Guf1 in the MIOREX complexes (Kehrein et al., 2015), we developed a interact with the SSU (Figure 1C, bottom), information likely important strategy to isolate mitoribosome subunits and analyze their respec- to understand their function. For example, the proposed role of Rmd9 tive interactomes (Figure 1A). After isolation of MIOREX complexes, in delivering mRNAs to the ribosome (Nouet et al., 2007; Williams ribosomes were split into their subunits by magnesium depletion et al., 2007) is supported by our finding that it interacts with the SSU. and separated on a continuous sucrose gradient. Western blotting To identify novel ribosomal interaction partners, we screened the confirmed that both subunits migrated at distinct peaks (Figure 1B). data set for uncharacterized proteins with a predicted or confirmed Next, we repeated the experiment and determined proteins in all mitochondrial localization that comigrate with the LSU or SSU (Figure fractions by label-free quantitative mass spectrometry (Supplemen- 1D). We thereby identified proteins found in the original MIOREX Volume 29 October 1, 2018 Mrx15 acts as mitoribosome receptor | 2387 data set (Kehrein et al., 2015), which were termed Mrx proteins flanked by N- and C-terminal domains. A chromosomally protein A (Figure 1D, star). Consequently, new and uncharacterized ribosomal (PA)-tagged variant (Mrx15-PA) quantitatively comigrated with the interaction partners found in this study were named according to LSU (Figure 2A) and was present in mitochondria in similar quantities this convention (Mrx15–17), as they apparently play a role in expres- as the mitoribosomal subunits (Figure 2B). To investigate the submi- sion of mitochondrially encoded proteins. We concluded that this tochondrial localization of Mrx15, we performed carbonate extrac- approach allowed us to identify new ribosomal interaction partners tion and protease protection assays. Mrx15 behaved during carbon- and to determine the subunit with which they are interacting. ate extraction like the integral membrane protein Cbp4 (Figure 2C) (Crivellone, 1994). This is in line with a high-throughput proteomic Mrx15 is a mitochondrial inner membrane protein that study that also identified Mrx15 as an integral membrane protein of interacts with the LSU the inner mitochondrial membrane (Morgenstern et al., 2017). Fur- Mrx15 is encoded by the open reading frame YNR040W and shows thermore, Mrx15 was resistant toward externally added proteinase K the same sedimentation profile as the LSU (Figure 1D). Mrx15 is a when mitochondria were intact, but was partly degraded to a trun- 29-kDa protein with two predicted transmembrane segments cated form upon removal of the outer membrane (dMrx15-PA; FIGURE 2: Mrx15 is an inner membrane mitochondrial protein that interacts with the large ribosomal subunit. (A) Western blot of mitochondrial extracts containing a chromosomally PA-tagged variant of Mrx15. Mitochondria were lysed in 10 mM EDTA for Mg depletion and separated afterward on a continuous sucrose gradient. Individual fractions were probed for aconitase (Aco1), proteins from the LSU (Mrpl36) and SSU (Mrps5), and PA for Mrx15 detection. (B) Abundancy of PA-tagged variants of the SSU (Mrp1PA), LSU (Mhr1PA), and Mrx15 were compared by Western blotting. Aconitase (Aco1) and Mrpl4 were used as loading controls. (C) Carbonate extraction of mitochondrial extracts. After incubation with either 0.2 M Na CO or 0.2 M NaCl, the membrane and soluble fractions of mitochondrial extracts 2 3 were separated by high-velocity centrifugation. Supernatant and pellet fractions were probed for membrane (Cbp4) and soluble (Aco1) proteins and PA for Mrx15 detection. (D) Protease protection assay of mitochondria (Mitos), mitoplasts, and lysed mitochondria. Mitochondria, mitoplasts created by hypotonic swelling of mitochondria, and detergent-lysed mitochondria were treated for 20 min with proteinase K (PK). Afterward, a Western blot of each sample was probed for proteins of the outer membrane (Tom70), inner membrane (Cbp4), and the matrix (Mrpl36). The PA antibody was used for detection of the PA-tagged variant of Mrx15. (E) Schematic representation of full-length Mrx15 tagged with PA and a C-terminal truncation variant. Cell extracts of strains grown to log phase containing either variant were probed for PA to determine abundance of each construct. (F) Western blot of mitochondrial extracts containing C-terminally truncated variant of Mrx15. Interaction with the mitoribosome was tested by Mg depletion and separation of ribosomal subunits on a continuous sucrose gradient. Individual fractions were probed for aconitase (Aco1), proteins from the LSU (Mrpl36) and SSU (Mrp1), and PA for Mrx15 detection. (G) Mrx15 inner membrane topology. According to our results, the Mrx15 protein is a component of the inner mitochondrial membrane. The N- and C-termini of the protein face the matrix. The C-terminal domain is necessary for interaction with the large ribosomal subunit. IMS, intermembrane space; Mitopl., mitoplasts; P, pellet; SN, supernatant; T, total; TM, transmembrane segment; WT, wild type. 2388 | B. V. Möller-Hergt et al. Molecular Biology of the Cell Figure 2D). Because Mrx15 was detected via its C-terminal PA tag, this shows that the N- and C-termini are facing the mitochondrial matrix. In silico analysis of Mrx15 revealed that it belongs to a family of uncharacterized proteins that are conserved within fungi (Supple- mental Figure 1). All members contain two predicted transmem- brane segments and a charged C-terminal domain. Next, we tested whether the positively charged, soluble C-termi- nal domain of Mrx15 is important for the interaction with the mitori- bosome. We constructed a mutant in which the C-terminal 126 amino acids were deleted (mrx15ΔC; Figure 2E). This deletion leads to decreased levels of Mrx15ΔC-PA (Figure 2E). Importantly, Mrx15ΔC-PA did not comigrate with the LSU (Figure 2F). Accord- ingly, the positively charged C-terminal domain is necessary for ri- bosomal interaction. We concluded that Mrx15 is an integral protein of the inner mitochondrial membrane that interacts with the LSU of the mitoribosome via a C-terminal domain (Figure 2G). Synthetic genetic interaction between MRX15 and MBA1 suggests a common function To investigate the function of Mrx15, we created a chromosomal deletion strain and tested the growth of this mutant. The mrx15Δ mutant did not show a growth defect on fermentable and nonfer- mentable media at 30 or 37°C (Figure 3A). Mba1 is a peripheral inner membrane protein involved in membrane insertion of mito- chondrial translation products (Preuss et al., 2001; Ott et al., 2006). It directly interacts with the LSU of the mitoribosome and aligns the tunnel exit for membrane protein insertion (Gruschke et al., 2010; Pfeffer et al., 2015). Because Mba1 and Mrx15 both bind to the membrane and the ribosome, but are not necessary for respiratory growth, we asked whether the combined absence of Mrx15 and Mba1 would result in a synthetic growth phenotype suggesting a common function. As reported previously, the mba1Δ mutant showed only a mild growth defect on a nonfermentable carbon source (Figure 3A) (Ott et al., 2006). Strikingly, the mrx15Δmba1Δ mutant was respiratory deficient at 30 and 37°C, while fermentative growth was not affected. We concluded that loss of Mrx15 is toler- ated under the tested conditions but required for respiratory growth in the absence of Mba1, suggesting that both proteins have over- lapping functions in the biogenesis of the respiratory chain. Next, we tested whether deletion of the C-terminus of Mrx15 is sufficient to provoke the combined respiratory growth phenotype and found that, while the single mutant did not show a growth phenotype un- der the tested conditions, cells became respiratory deficient in the mrx15ΔCmba1Δ strain (Figure 2A), thereby demonstrating that the C-terminal domain of Mrx15 is required for ribosome interaction and respiratory growth upon deletion of MBA1. Mba1 cooperates with Oxa1 in the insertion of mitochondrial translation products into the inner membrane (Preuss et al., 2001). deleted alone or together with MRX15. Cells were grown to logarithmic phase, spotted in 10-fold dilutions, and incubated at 30 and 37°C. (B) Ribosome pelletation at different ionic strengths. The ribosome and attached factors were separated after lysis in different salt conditions by a sucrose cushion and high-velocity centrifugation. The different fractions were probed for a soluble protein (Aco1), a ribosomal marker (Mrpl36), Mba1, and PA for Mrx15 detection. FIGURE 3: Deletion of MRX15 together with MBA1 leads to (C) Flotation gradient of mitochondrial extracts from wild-type or respiratory deficiency and altered membrane attachment. (A) Serial- mrx15Δmba1Δ strains. Soluble and membrane fractions were dilution growth test on full medium fermentable (glucose) and separated after freeze–thaw cycles by a sucrose step gradient and nonfermentable (glycerol) carbon sources of indicated strains. In the high-velocity centrifugation. Fractions were probed for the LSU mrx15ΔC mutant, the terminal 126 amino acids were replaced by a PA (Mrpl36, Mrpl4, and Mrpl40), membrane (Cbp4), and soluble proteins tag. In the C-terminal Oxa1 mutant the 71 C-terminal residues were (Aco1). P, pellet; SN, supernatant; T, total; WT, wild type. Volume 29 October 1, 2018 Mrx15 acts as mitoribosome receptor | 2389 Simultaneous deletion of Mba1 and of 71 C-terminal residues of complexes (Figure 4B). Importantly, upon simultaneous deletion of Oxa1 leads to a severe respiratory growth inhibition combined with MRX15 and MBA1, complex IV levels were further reduced, while an insertion defect of mitochondrially encoded proteins (Ott et al., complex III did not change. ATP synthase (complex V), which also 2006). We tested whether deletion of the C-terminal tail of Oxa1 in contains mitochondrially encoded subunits, was unchanged in the absence of Mrx15 would impair respiratory growth. However, these strains, pointing to a specific defect in accumulating complex unlike Mba1, Mrx15 did not show a synthetic genetic interaction IV in the mrx15Δmba1Δ cells. with the C-terminal domain of Oxa1 (Figure 3A). This suggests that To confirm this conclusion, we analyzed respiratory chain super - Mrx15 and the C-terminal ribosome-binding domain of Oxa1 do not complex formation and respiratory chain activity. Consistent with have overlapping roles for membrane protein insertion, in contrast the steady-state analyses, blue native PAGE (BN-PAGE) revealed to Mrx15 and Mba1, which apparently share a common function. that complex IV abundance in respiratory supercomplexes was Next, we investigated the salt sensitivity of the Mrx15-LSU and lower in the mba1Δ mutant and severely reduced in the double- Mba1-LSU interactions and pelleted ribosomes through a sucrose deletion mutant, where only the smaller isoform of the supercom- cushion at different ionic strengths (Figure 3B). At low ionic strength plexes (III IV) was detected (Figure 4C, middle). Complex III, the conditions, Mrx15 interacted with the LSU, while Mba1 did not co- partner of complex IV in respiratory supercomplexes, as revealed by migrate with the LSU under any of the tested conditions. Consis- the late-assembling subunit Rip1 (Ndi et al., 2018), was mainly pres- tently, Mba1 comigration with any of the mitoribosomal subunits ent in the dimeric form, indicating severe reduction of complex IV. was not observed in the mass spectrometry data set (Figure 1C). ATP synthase was present as a monomer and dimer in all strains, Instead, interaction of Mba1 with the LSU was found through chemi- and the mrx15Δmba1Δ cells had slightly reduced levels of the di- cal cross-linking or ribosome comigration upon magnesium deple- mer. The specific decrease in complex IV levels upon simultaneous tion only, pointing to a rather transient interaction of Mba1 with the deletion of Mrx15 and Mba1 was also reflected in a significant de - mitoribosome (Ott et al., 2006; Gruschke et al., 2010). crease in complex IV activity, confirming that both Mba1 and Mrx15 Mitoribosomes are permanently associated with the inner mito- are specifically important for the biogenesis of complex IV. In con - chondrial membrane (Ott et al., 2006; Prestele et al., 2009; Pfeffer trast, complex III activity was lower upon MBA1 deletion (Bauer- et al., 2015). Based on the high-resolution cryo–electron microscopy schmitt et al., 2010), but not further decreased in the mrx15Δmba1Δ (cryo-EM) structure of the LSU and cryo-EM tomography, a second mutant. point of membrane attachment was proposed, consisting of a mem- brane-facing protuberance buildup by mitoribosome-specific pro - Mrx15 and Mba1 interact with nascent polypeptide chains teins and rRNA expansion segments (Amunts et al., 2014; Pfeffer Mba1 interacts with nascent polypeptide chains as they emerge et al., 2015). This is supported by data showing that the mitoribo- from the mitoribosome (Preuss et al., 2001; Ott et al., 2006; some stays membrane bound upon deletion of MBA1, either alone Gruschke et al., 2010). Owing to the synthetic genetic interaction or in combination with the C-terminal tail of Oxa1 (Ott et al., 2006). and the cofractionation of Mrx15 and Mba1 with the LSU, we Because we found the Mba1-LSU interaction to be rather weak, we hypothesized that Mrx15 could be in contact with nascent chains. To tested whether Mba1 and Mrx15 showed overlapping functions in test this, we performed in organello labeling of mitochondrial trans- ribosome–membrane attachment. We therefore followed ribo- lation products followed by chemical cross-linking and purification some–membrane interaction upon simultaneous deletion of MRX15 of Mrx15. We chose a cross-linker (DFDNB) with a very short linker and MBA1 in a flotation gradient (Figure 3C). In wild-type mitochon - length (0.3 nm) that can only covalently connect molecules that are dria, a fraction of the mitoribosomes cofractionated with the mem- in very close proximity. Autoradiography of the elution fraction brane marker Cbp4, indicating membrane interaction. In contrast, showed multiple species with electrophoretic mobilities above only a minor fraction of the mitoribosome in the mrx15Δmba1Δ mu- 25 kDa (Figure 5A). In a control experiment with mitochondria from tant floated with the membranes. Therefore, we concluded that the wild type, these species were not detected. This indicated that interaction of mitoribosomes with the membrane is decreased upon Mrx15 was cross-linked to nascent chains. Mba1 also cross-linked to simultaneous deletion of Mba1 and Mrx15. nascent chains, which is in line with previous results (Preuss et al., 2001; Gruschke et al., 2010). We concluded that Mrx15, like Mba1, Absence of Mba1 and Mrx15 leads to complex IV deficiency is not only in contact with the LSU but directly interacts with nascent To further characterize the growth defect in the mutants lacking chains emerging from the tunnel exit. Mrx15, Mba1, or both, we determined steady-state levels of mito- Mba1 forms a stable complex with Mdm38 that can be purified chondrial proteins in the strains. Amounts of proteins implicated in (Bauerschmitt et al., 2010). We hypothesized that Mrx15 could also mitochondrial protein biogenesis did not change in the strains (Figure physically interact with Mba1 or Mdm38. Therefore, we purified 4A). Levels of the cytochrome c oxidase (complex IV) subunit Cox2 FLAG-tagged variants of Mrx15 from mitochondrial extracts with were not changed upon loss of Mrx15, but were reduced in the ab- two different detergents, but no stable complex between these pro- sence of Mba1, with a concomitant accumulation of the precursor teins was detected (Figure 5B). Likewise, we did not observe a com- form of Cox2 (pCox2), in line with previous data (Preuss et al., 2001; bined growth defect in the mrx15Δmdm38Δ cells (Figure 5C). Unlike Bauerschmitt et al., 2010). After synthesis and membrane insertion, Mdm38 and Mba1, Mrx15 and Mba1 apparently carry out their the Cox2 precursor is processed into a mature from (mCox2) by pro- overlapping functions without forming a stable complex. We con- teolytic removal of an N-terminal domain by Imp1 and Cox20, thereby cluded that Mrx15, like Mba1, binds to nascent polypeptide chains generating mature, assembly-competent Cox2 (Nunnari et al., 1993; but does not form a stable complex with Mba1 or Mdm38. Jan et al., 2000). Interestingly, simultaneous loss of Mba1 and Mrx15 leads to a further increase in pCox2 levels relative to mCox2. Mrx15 and Mba1 jointly mediate biogenesis Employing Western blot followed by densitometry analyses, we of the Cox2 precursor found that cytochrome bc complex (complex III) and complex IV The direct interaction of Mrx15 with nascent polypeptide chains subunits were accumulating normally in the absence of Mrx15, while prompted us to investigate whether the protein together with mitochondria from mba1Δ mutants had diminished amounts of both Mba1 plays a role in protein insertion. Therefore, we radiolabeled 2390 | B. V. Möller-Hergt et al. Molecular Biology of the Cell FIGURE 4: Mrx15 and Mba1 are required for complex IV accumulation. (A) Steady-state levels of mitochondrial proteins in all indicated strains. Cells were grown in galactose to logarithmic phase, and Western blots probed for proteins necessary for mitochondrial protein biogenesis and complex IV accumulation. (B) Mitochondrial extracts of indicated strains were probed for subunits of complex III (CytB, Qcr7), complex IV (Cox1, Cox13), and complex V (Atp4, F1β). Signal intensities of three independent experiments were quantified and normalized. Significance of observed changes was assessed by a Student’s t test. (C) BN-PAGE of digitonin-solubilized mitochondria from indicated strains. Separated protein complexes were analyzed by Western blotting against subunits of complex III (Rip1), complex IV (Cox1), and complex V (Atp4) (D) Complex III and IV activity measurement. Activity of complex III was followed by measuring cytochrome c reduction, complex IV activity by measuring cytochrome c oxidation at 550 nm. CIII, complex III; CIV, complex IV; CV, complex V; n.s., p > 0.05; **, p ≤ 0.01. mitochondrial translation products in vivo in a pulse–chase experi- enriched, only a smear of nascent chains and some mitoribosomes ment (Figure 6A). Mitochondrial protein synthesis and stability were were copurified (Figure 6C). This indicates that Mrx15, in contrast to not affected in the mrx15Δ mutant compared with the wild type. As Mba1, which interacts specifically with Cox2 (Lorenzi et al., 2016), reported previously (Preuss et al., 2001), the MBA1 deletion mutants does not bind posttranslationally to mitochondrially encoded pro- showed an accumulation of pCox2, and this was enhanced in the teins. Taken together, these results suggest that the observed com- mrx15Δmba1Δ mutant. To quantify this effect, we determined the plex IV deficiency and the respiratory growth defect in the pCox2/mCox2 ratio in both mutants after a 15-min pulse and 90-min mrx15Δmba1Δ mutant are caused by hampered Cox2 biogenesis chase from five independent experiments (Figure 6B). The pCox2/ due to disturbances of ribosome–membrane interactions required mCox2 ratio after 15 min of protein synthesis and a 90-min chase for efficient cotranslational insertion combined with a specific defect was significantly higher in the mrx15Δmba1Δ mutant, revealing an in Cox2 maturation that is carried out by Mba1 (Lorenzi et al., 2016). increased accumulation of the pCox2 variant when both Mrx15 and Mba1 were absent. To check whether Mrx15 interacts with specific DISCUSSION mitochondrial translation products, we purified Mrx15-containing In this study, we identified Mrx15, which helps to tether the mitori - complexes after in organello labeling of mitochondrial translation bosome to the inner membrane for efficient cotranslational inser - products from digitonin lysates. While Mrx15-FLAG could be clearly tion. On the basis of results presented in this study, we propose the Volume 29 October 1, 2018 Mrx15 acts as mitoribosome receptor | 2391 study of the human mitoribosome estab- lished that mL45 is the single contact site to the inner membrane, while the general architecture of the membrane–mitoribo- some contact is conserved between yeast and human (Englmeier et al., 2017). Because homologous proteins of Mrx15 are found only in fungi, it is tempting to speculate that Mrx15 substitutes Mba1 as a permanently bound mitoribosome receptor. Therefore, Mrx15 could occupy a similar binding site on the ribosome as Mba1, ensuring correct mi- toribosome–membrane attachment while Mba1 is escorting Cox2 toward respiratory chain assembly. Permanent membrane attachment of ri- bosomes appears to be specifically impor - FIGURE 5: Mrx15 interacts with nascent chains but does not form a stable complex with Mba1. tant for mitochondrial gene expression. A (A) Cross-linking to nascent mitochondrial polypeptide chains. Mitochondrial translation recent study of mitoribosome assembly in products were radiolabeled in wild-type mitochondria or mitochondria containing a FLAG- yeast found that the mitoribosomal LSU is tagged variant of Mrx15. After 15 min of labeling, the chemical cross-linker DFDNB was added already attached to the membrane during to covalently link molecules in direct physical contact. The different reactions were stopped, and assembly (Zeng et al., 2018). This study Mrx15 cross-linking products were purified with a FLAG resin. Mba1 and cross-linked molecules found mitoribosome–membrane attach- were iummunoprecipitated employing a PA resin and an Mba1 antibody. As a control, the ment even upon simultaneous deletion of reactions were performed without a FLAG tag or an Mba1 antibody. (B) Purification of FLAG- MBA1, MDM38, and OXA1. Therefore mito- tagged Mrx15 variants. Mitochondria were lysed in two detergents (Triton X-100, digitonin) and Mrx15 purified by employing a FLAG resin. Western blots of the purification were probed ribosome–membrane attachment likely for FLAG, Mba1, Mdm38, and Aco1. (C) Serial-dilution growth test on fermentable (YPD) mainly depends on 21S rRNA incorporation and nonfermentable (YPG) carbon sources of indicated strains. E, elution; NB, not bound; into the LSU. This notion is supported by T, total; WT, wild type. data showing that mutations in 21S rRNA can alter mitoribosome–membrane attach- following model (Figure 6D): Mba1 and Mrx15 bind to the LSU of ment (Spithill et al., 1978). According to data presented here, mito- the mitoribosome and interact with nascent chains emerging from ribosomes from the double-deletion mutant of MBA1 and MRX15 the exit tunnel. Both proteins tether the LSU to the inner mitochon- were still partly membrane attached, suggesting that the 21S rRNA drial membrane to ensure proper alignment of the LSU with the in- of LSU could connect to the inner mitochondrial membrane via the sertion site. Mba1 and Mrx15 are in proximity to nascent chains to expansion segment E29 (Pfeffer et al., 2015). ensure efficient membrane insertion by the insertase Oxa1. Upon Combined absence of Mba1 and Mrx15 provokes a strong respi- completion of Cox2 synthesis, Mba1 dissociates from the mitoribo- ratory growth defect, which at least partly reflects the strong com - some to escort the newly synthesized protein toward downstream plex IV deficiency that exhibits a specific accumulation of the Cox2 assembly processes, while Mrx15 remains ribosome bound. precursor. Cox2 precursor accumulation could be caused by a gen- In the simultaneous absence of Mrx15 and Mba1, a large part of eral defect of membrane insertion or the disruption of shuttling the mitoribosomes is liberated from the inner membrane, pointing newly synthesized Cox2 to the maturation complex consisting of to a critical function of both proteins as ribosome–membrane teth- Cox20 and Imp1/2 (Lorenzi et al., 2016). A general insertion defect ers. However, Mrx15 and Mba1 greatly differ in their mitoribosome- in the mrx15Δmba1Δ cells that exacerbates the insertion defect of binding efficiencies. While the integral inner membrane protein mba1Δ cells would be revealed as a pleiotropic defect in the assem- Mrx15 stayed ribosome bound in the presence of magnesium and at bly of all OXPHOS complexes, as has been observed in mutants moderate ionic strength, we did not detect a permanent interaction affected in common processes for synthesis and insertion of the between the peripheral membrane protein Mba1 and the LSU of the mitochondrially encoded membrane proteins (Altamura et al., 1996; mitoribosome under these conditions. This suggests that Mrx15 is a Prestele et al., 2009; Bauerschmitt et al., 2010; Kuzmenko et al., constant ribosome tether, while Mba1 dynamically interacts with the 2016; Ostojic et al., 2016; Suhm et al., 2018). This, however, is not ribosome. This is in line with a recent study demonstrating a post- the case in the mrx15Δmba1Δ cells. Furthermore, our data, which translational function of Mba1 for complex IV biogenesis (Lorenzi indicate that MRX15 does not show a genetic interaction with the et al., 2016), whereby Mba1 shuttles Cox2 from synthesis to its as- C-terminal domain of Oxa1, the insertase, or Mdm38, are in line sembly factor Cox20 for maturation and respiratory chain assembly. with a context-specific function Mrx15 exerts together with Mba1 Cryo-EM tomography has revealed the organization of mitoribo- during Cox2 biogenesis. A special aspect of Cox2 biogenesis is that somes in isolated organelles, where Mba1 occupies a position con- the protein contains by far the largest and most charged soluble necting the tunnel exit with the insertion site. In the absence of Mba1, domains of all mitochondrially encoded proteins, and these do- a specific density was absent, but the general arrangement of the ri - mains need to be translocated over the inner membrane (Herrmann bosome relative to the inner membrane was unchanged (Pfeffer et al., 1995). A disturbed ribosome–membrane contact could et al., 2015), suggesting the presence of other ribosome tethers like specifically impact the translocation of the highly hydrophilic Cox2 the Mrx15 protein identified here. In contrast to yeast Mba1, its mam - domains, particularly when connected with the absence of the shut- malian homologue mL45 is an integral and firmly bound subunit of tling factor Mba1 (Lorenzi et al., 2016) that presumably removes the mitoribosomal LSU (Greber et al., 2014b). A recent tomographic pCox2 from the insertion site. 2392 | B. V. Möller-Hergt et al. Molecular Biology of the Cell In addition to the clear energetic advantages of tethering ribo- somes to membranes to facilitate cotranslational protein insertion, the firm interaction of the mitoribosome and the inner mitochondrial membrane allows further specialization in how translation is orga- nized. For example, translational activators regulate translation of specific client mRNA in yeast mitochondria (Fox, 2012). This class of proteins is firmly membrane bound and essential for translation in yeast mitochondria. Because of these characteristics, it was sug- gested that a main function of translational activators is to localize the synthesis of the hydrophobic translation products to the inner membrane (McMullin and Fox, 1993; Fox, 1996). Moreover, by gath- ering several translational activators controlling synthesis of the three cytochrome oxidase subunits (Naithani et al., 2003), localized assembly sites are established that likely facilitate joining of the sub- units. These sites of translation and subsequent biogenesis are not evenly distributed in the inner membrane, as assembly of each OX- PHOS complex preferentially occurs at different subcompartments within the inner membrane (Stoldt et al., 2018), highlighting the in- tricate organization of mitochondrial gene expression. We have previously found mitoribosomes to interact with many proteins to organize biogenesis of the organellar-encoded proteins. These interactions give rise to large assemblies called MIOREX complexes (Kehrein et al., 2015) that unite many steps of mitochon- drial gene expression, including mRNA maturation, translation, as- sembly, and turnover. Here, employing an alternative strategy to identify and classify components interacting with the mitoribosome provided support for this general concept. Specifically, at least parts of mRNA maturation occur on the LSU, as revealed by its interaction with the COB mRNA splicing factor Cbp2. It remains an exciting task to develop strategies to further categorize the interactome of the yeast mitoribosome and to establish how the different processes are organized on the mitoribosome. MATERIALS AND METHODS Yeast strains and growth media All yeast strains used in this study (Supplemental Table 1) were iso- genic to either the wild-type strains W303a or CW04. MRX15 was disrupted with a kanamycin resistance cassette. The MBA1 coding sequence was replaced with an HIS3 selection cassette. FLAG- or PA-tagged variants of Mrx15 were created by replacing the endog- enous stop codon with a FLAG sequence or a PA sequence employ- ing a TRP1 or HIS3 selection cassette. Cells were grown at 30°C in YP medium (2% peptone, 1% yeast extract) in the presence of either 2% dextrose, 2% galactose, or 2% glycerol. Isolation of mitochondria Mitochondria were isolated as described previously (Gruschke et al., 2011). For experiments analyzed by mass spectrometry, mi- tochondria were isolated by a sucrose step gradient. The mito- chondrial pellet was resuspended in SHE buffer (20 mM HEPES/ KOH, pH 7.4, 250 mM sucrose, 1 mM EDTA) and loaded on a step polypeptides with Mrx15. After in organello labeling of mitochondrial translation products, a FLAG-tagged variant of Mrx15 was purified, and eluates were analyzed by Western blotting and autoradiography. FIGURE 6: Mrx15 is involved in accumulation of premature Cox2. (D) Model of Mrx15 and Mba1 function in Cox2 membrane insertion. (A) In vivo radiolabeling of all indicated strains. Mitochondrial protein In wild-type cells, Mrx15 and Mba1 bind to the LSU and nascent synthesis of all mutants was followed for 15 min The stability of the chains to ensure ribosome–membrane attachment and proper synthesized polypeptides was chased for 90 min. (B) Quantification of membrane insertion. Upon deletion of both factors, membrane the pCox2 to mCox2 ratio after 15 min of synthesis and a 90-min insertion is hampered, and pCox2 accumulates. Consequently, this chase. Bands of five independent experiments were quantified, and leads to decreased Cox2 maturation, complex IV assembly, and significance between displayed values was assessed by a Student’s respiratory deficiency. E, elution; IMS, intermembrane space; NB, not t test. (C) Copurification of newly synthesized mitochondrial bound; T, total; WT, wild type; n.s., p > 0.05; **, p ≤ 0.01. Volume 29 October 1, 2018 Mrx15 acts as mitoribosome receptor | 2393 gradient. The layers consisted of 1.75, 0.95, 0.68, and 0.45 M su- (Thermo Fisher). When the dye front had migrated one-third of the crose in 20 mM HEPES/KOH (pH 7.4) and 1 mM EDTA. Mitochon- way through the gel, the dark blue cathode buffer was replaced with dria were spun down (134,000 × g, 1 h, 4°C), recovered from the a light blue cathode buffer. Afterward, separated protein complexes 1.74–0.95 interface, and resuspended in SH buffer (20 mM HEPES, were blotted onto polyvinylidene difluoride membranes. pH 7.4, 0.6 M sorbitol). At a concentration of 10 mg/ml, mitochon- dria were snap-frozen in liquid nitrogen. Complex III and IV activity assay For respiratory chain activity measurements, 0.5 mg of mitochondria Sucrose gradients were lysed (50 mM Tris/HCl, pH 7.4, 100 mM KCl, 2.5 MgCl , Isolated mitochondria were lysed (10 mM Tris/HCl, pH 7.4, 10 mM 0.1 mM EDTA, 1X complete, 1 mM PMSF, 2% digitonin) for 10 min KOAc, 0.5 mM Mg(OAc) , 10 mM EDTA, 5 mM β-mercaptoethanol, on ice. Afterward, the lysate was cleared by centrifugation (16,000 × 1% dodecylmaltoside, 1 mM phenylmethylsulfonyl fluoride [PMSF], g, 10 min, 4°C) and diluted 50-fold in 50 mM Tris/HCl (pH 7.4). 1X complete, 0.1 mM spermidine, 5% glycerol), and sucrose gradi- For complex III activity measurements, KCN (1 mM) and cyto- ent experiments were performed as described previously (Kehrein chrome c (0.5 mg/ml) were added to the lysate. The reduction of et al., 2015). Collected samples were precipitated by addition of cytochrome c was monitored by measuring the change in absor- 12% trichloroacetic acid. The resuspended samples were analyzed bance at 550 nm upon addition of quinol (0.08 mg/ml). Activity was by SDS–PAGE and Western blotting or mass spectrometry accord- calculated by determining the slope of the reaction. ing to published procedures (Chiurillo et al., 2016). For complex IV activity measurements, antimycin A (0.2 mg/ml) was added to the lysate. The oxidation of cytochrome c was moni- Carbonate extraction and protease protection tored by measuring the change in absorbance at 550 nm upon ad- Isolated mitochondria (100 µg) were resuspended in 0.1 M Na CO or dition of reduced cytochrome c (0.33 mg/ml). Activity was calcu- 2 3 NaCl and incubated 30 min on ice. Membrane and soluble fractions lated as for complex III. were separated by centrifugation (100,000 × g, 30 min, 4°C), TCA Purification of FLAG-tagged Mrx15 precipitated, and analyzed by SDS–PAGE and Western blotting. Mitochondria from yeast strains expressing FLAG-tagged variants Mitochondria (100 µg) were incubated in SH buffer or 20 mM of Mrx15 were lysed for 10 min on ice (10 mM Tris/HCl, pH 7.4, HEPES/KOH or lysis buffer (SH buffer with 0.2% Triton X-100) for 150 mM NaCl, 1 mM EDTA, 1% digitonin or Triton X-100, 1 mM 30 min at 4°C. Afterward, 0.1 mg/ml proteinase K was added, and PMSF, 1X complete). Subsequently, samples were diluted 1:1 in the reaction was incubated for 20 min at 4°C. Intact mitochondria or dilution buffer (10 mM, Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, mitoplasts were spun down (25,000 × g, 10 min, 4°C). The superna- 0.1% digitonin or Triton X-100, 1 mM PMSF, 1x complete), and the tant and pellet were analyzed by SDS–PAGE and Western blotting. lysate was cleared by centrifugation (16,000 × g, 10 min, 4°C). The cleared lysate was incubated with anti-FLAG M2 affinity gel for 2 h Ribosome sedimentation at 4°C. The affinity gel was washed three times with dilution buffer. Isolated mitochondria were lysed in different buffers (10 mM Tris/HCl, Purified proteins were eluted by incubation for 20 min with dilu - pH 7.4, 10/25/100/250 mM KOAc, 5/5/20/50 mM Mg(OAc) , 0.1 mM tion buffer supplemented with 0.15 µg/µl 3× FLAG peptide. Sam- spermidine, 5% glycerol, 1% DDM, 1 mM PMSF, 1X complete) for ples of all relevant fractions were precipitated by addition of 12% 10 min on ice. The lysate was diluted in one volume of the respective TCA. The resuspended samples were analyzed by SDS–PAGE and lysis buffer without DDM and cleared by centrifugation (16,000 × g, Western blotting. 10 min, 4°C). The cleared lysate was underlaid with half a volume of For testing copurification of newly synthesized mitochondrial sucrose cushion (10 mM Tris/HCl, pH 7.4, 10/25/100/250 mM KOAc, translation products with Mrx15, a FLAG-tagged variant was puri- 5/5/20/50 mM Mg(OAc) , 0.1 mM spermidine, 5% glycerol, 1% fied after in organello labeling (Gruschke et al., 2010). Mitochondria DDM, 1 mM PMSF, 1X complete, 1.2 M sucrose) and centrifuged were lysed after the labeling reaction (10 mM Tris/HCl, pH 7.4, 100 (189,062 × g, 105 min, 4°C). Afterward, the supernatant and pellet mM NaCl, 1 mM EDTA, 2% digitonin, 1 mM PMSF, 1X complete), fractions were separated and analyzed by Western blotting. and the purification was carried out as described earlier. Flotation gradients Miscellaneous methods Isolated mitochondria were resuspended in buffer A (20 mM Tris/ Polyclonal antisera against Mrp1 and Mrx15 were generated by in- HCl, pH 7.4, 25 mM KOAc, 5 mM Mg(OAc) , 1 mM PMSF) and jecting rabbits with recombinantly expressed and purified antigens. treated with three consecutive freeze–thaw cycles. Mitochondria The antibodies against PA and FLAG were obtained from Sigma. were frozen in liquid nitrogen and thawed at 37°C. Afterward, the Labeling of mitochondrial translation products and chemical cross- reaction was resuspended in 10 volumes of 2.5 M sucrose in linking of nascent chains were carried out as previously described 20 mM Tris/HCl (pH 7.4). The suspension was overlaid with one (Gruschke et al., 2010). volume of 2.2, 1.5, and 1 M sucrose and centrifuged (40,000 × g, 16 h, 4°C). The gradient was fractionated, and individual fractions were analyzed by Western blotting. ACKNOWLEDGMENTS We thank members of our laboratories for stimulating discussions BN-PAGE and Western blotting and Marc Wirth and Ignasi Forne (Ludwig Maximilian University of Isolated mitochondria (150 µg) were resuspended in BN-PAGE buffer Munich, Germany) for help with mass spectrometric analyses. This (50 mM Bis-Tris/HCl, pH 7.2, 150 mM NaCl, 2 mM aminohexanoic work was supported by grants from the Swedish Research Coun- acid, 1 mM EDTA, 1X complete, 1 mM PMSF, 1% digitonin or 1% cil, the Carl Tryggers Foundation, and the Knut and Alice Wallen- DDM, 12% glycerol) and lysed 10 min on ice. The lysate was cleared berg Foundation. 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The ribosome receptors Mrx15 and Mba1 jointly organize cotranslational insertion and protein biogenesis in mitochondria

Möller-Hergt, Braulio Vargas; Carlström, Andreas; Stephan, Katharina; Imhof, Axel; Ott, Martin
Molecular Biology of the Cell , Volume 29 (20) – Oct 1, 2018
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Abstract

MBoC | ARTICLE The ribosome receptors Mrx15 and Mba1 jointly organize cotranslational insertion and protein biogenesis in mitochondria a a a b Braulio Vargas Möller-Hergt , Andreas Carlström , Katharina Stephan , Axel Imhof , a, and Martin Ott * a b Department of Biochemistry and Biophysics, Stockholm University, SE-10691 Stockholm, Sweden; Protein Analysis Unit, Biomedical Center, Faculty of Medicine, Ludwig Maximilian University of Munich, DE-82152 Planegg-Martinsried, Germany Monitoring Editor ABSTRACT Mitochondrial gene expression in Saccharomyces cerevisiae is responsible for Benjamin S. Glick the production of highly hydrophobic subunits of the oxidative phosphorylation system. University of Chicago Membrane insertion occurs cotranslationally on membrane-bound mitochondrial ribosomes. Here, by employing a systematic mass spectrometry–based approach, we discovered the Received: Apr 13, 2018 previously uncharacterized membrane protein Mrx15 that interacts via a soluble C-terminal Revised: Jul 9, 2018 Accepted: Aug 2, 2018 domain with the large ribosomal subunit. Mrx15 contacts mitochondrial translation products during their synthesis and plays, together with the ribosome receptor Mba1, an overlapping role in cotranslational protein insertion. Taken together, our data reveal how these ribosome receptors organize membrane protein biogenesis in mitochondria. INTRODUCTION The mitochondrial proteome consists of polypeptides from two In addition, mitoribosomes are permanently attached to the in- genetic sources. Most proteins are encoded in the nucleus and ner mitochondrial membrane (Preuss et al., 2001; Ott et al., posttranslationally imported into their respective mitochondrial 2006; Prestele et al., 2009; Pfeffer et al., 2015), in contrast to compartments (Neupert, 2015; Wiedemann and Pfanner, 2017). bacterial ribosomes that are targeted to the membrane by the A small subset of proteins, eight in Saccharomyces cerevisiae, is signal-recognition particle (Bernstein et al., 1989). In yeast mito- encoded in the mitochondrial genome (Foury et al., 1998). Their chondria, cotranslational membrane insertion is mediated in a genes are transcribed and translated by a genetic system resid- concerted manner by Oxa1 and Mba1 (Preuss et al., 2001; Ott ing in the matrix of the organelle. The mitochondrial ribosomes et al., 2006). Oxa1, which belongs to the conserved YidC/Alb3/ (mitoribosomes) synthesize, almost exclusively, membrane pro- Oxa1 family, inserts client proteins into the membrane (He and teins that are subunits of oxidative phosphorylation complexes. Fox, 1997; Hell et al., 2001). The peripheral membrane protein Recent structural data have revealed that mitoribosomes are Mba1 cooperates with the C-terminal domain of Oxa1 in mem- adapted for the production of hydrophobic polypeptides by a brane protein insertion and ribosome–membrane attachment specific makeup of the polypeptide exit tunnel, which is rather (Ott et al., 2006). The human homologue of Mba1 (Mrpl45) is apolar to accommodate the hydrophobic translation products integrated in the large ribosomal subunit and was speculated to (Amunts et al., 2014; Brown et al., 2014; Greber et al., 2014a,b). fulfill a similar role in membrane attachment (Greber et al., 2014b). Additionally, Mba1 shuttles the mitochondrial-encoded cytochrome c oxidase subunit 2 (Cox2) to the assembly factor Cox20 (Lorenzi et al., 2016). This article was published online ahead of print in MBoC in Press (http://www In a previous study, we found that the mitochondrial ribosome is .molbiolcell.org/cgi/doi/10.1091/mbc.E18-04-0227) on August 9, 2018. *Address correspondence to: Martin Ott (martin.ott@dbb.su.se). organized in large assemblies that we termed MIOREX complexes Abbreviations used: BN-PAGE, blue native PAGE; cryo-EM, cryo–electron micros- (Kehrein et al., 2015). These assemblies contain factors responsible copy; LSU, large subunit; PMSF, phenylmethylsulfonyl fluoride; SSU, small subunit. for mRNA maturation and turnover, translation, chaperones, prote- © 2018 Möller-Hergt et al. This article is distributed by The American Society for ases, and a number of previously uncharacterized proteins, some of Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Un- which were later shown to play a role in mitochondrial gene expres- ported Creative Commons License (http://creativecommons.org/licenses/by-nc sion (Moda et al., 2016; Rak et al., 2016). Another study used a SILAC -sa/3.0). (stable isotope labeling of amino acids in cell culture)-based ap- “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology. proach (Woellhaf et al., 2016) to identify several factors comigrating 2386 | B. V. Möller-Hergt et al. Molecular Biology of the Cell FIGURE 1: Proteomic profiling of the small and large mitochondrial ribosomal subunits. (A) Strategy to isolate the MIOREX complex with subsequent separation of mitoribosome subunits. (B) After the procedure described in A, the protein abundance of individual fractions from a continuous sucrose gradient was determined by label-free quantitative mass spectrometry. Sedimentation profiles are depicted for the large subunit (blue) and small subunit (yellow). As a control, the experiment was probed for proteins from the large (Mrpl36) and the small (Mrps5) subunit as well as aconitase (Aco1). (C) Top, summary of known interactors of the large and small subunit. These factors showed the same migration behavior as their described ribosomal-interacting subunit. Bottom, summary of known mitoribosome interactors and the interacting subunit found in this study. (D) Summary of known (star) or newly discovered MIOREX components found in this study that comigrated with either the large or the small ribosomal subunit. LSU, large subunit; P, pellet; qMS, quantitative mass spectrometry; R, resuspended; SN, supernatant; SSU, small subunit; T, total. with the assembled mitoribosome in sucrose gradients, but the ribo- tal Data S1). Proteins from the large subunit (LSU) almost exclusively somal subunits with which they interacted remained unknown. migrated in fractions 2–4, while proteins of the small subunit (SSU) Here, we developed an alternative strategy to further character- were found in fractions 4–6 (Figure 1B). ize the mitoribosomal interactome. We thereby discovered a previ- Next, we cross-referenced our data set with known interactors of ously uncharacterized protein (Mrx15) that interacts with the large the large and small ribosomal subunit. As expected, previously de- ribosomal subunit. Simultaneous absence of Mrx15 and Mba1 pro- scribed mitoribosome interactors like Cbp3, Mhr4, and Mtg2 (Figure vokes a profound respiratory growth defect and decrease in cyto- 1C, top) (Datta et al., 2005; Gruschke et al., 2011; De Silva et al., 2013) chrome c oxidase. We demonstrate that Mba1 and Mrx15 are ribo- comigrated with the LSU. The mitochondrial initiation factor 2 (Ifm1) some receptors with overlapping roles in cotranslational protein (Garofalo et al., 2003) and Cmm1 (Puchta et al., 2010) comigrated insertion, directly interacting with the large ribosomal subunits, na- with the SSU, indicating that we can identify predicted ribosomal in- scent polypeptide chains, and the inner membrane. teraction partners with our methodology. We next checked for pro- teins that have been reported to interact with the mitoribosome, but RESULTS for which the subunit they interact with was unknown (Westermann Identification of mitoribosomal interactors by a systematic et al., 1996; Williams et al., 2007; Bauerschmitt et al., 2008; Paul et al., mass-spectrometric approach 2012; Kehrein et al., 2015). This allowed us to establish that Cbp2, To categorize the extensive interactome of the yeast mitoribosome Pth4, and Mdj1 interact with the LSU, while Rmd9, Gep3, and Guf1 in the MIOREX complexes (Kehrein et al., 2015), we developed a interact with the SSU (Figure 1C, bottom), information likely important strategy to isolate mitoribosome subunits and analyze their respec- to understand their function. For example, the proposed role of Rmd9 tive interactomes (Figure 1A). After isolation of MIOREX complexes, in delivering mRNAs to the ribosome (Nouet et al., 2007; Williams ribosomes were split into their subunits by magnesium depletion et al., 2007) is supported by our finding that it interacts with the SSU. and separated on a continuous sucrose gradient. Western blotting To identify novel ribosomal interaction partners, we screened the confirmed that both subunits migrated at distinct peaks (Figure 1B). data set for uncharacterized proteins with a predicted or confirmed Next, we repeated the experiment and determined proteins in all mitochondrial localization that comigrate with the LSU or SSU (Figure fractions by label-free quantitative mass spectrometry (Supplemen- 1D). We thereby identified proteins found in the original MIOREX Volume 29 October 1, 2018 Mrx15 acts as mitoribosome receptor | 2387 data set (Kehrein et al., 2015), which were termed Mrx proteins flanked by N- and C-terminal domains. A chromosomally protein A (Figure 1D, star). Consequently, new and uncharacterized ribosomal (PA)-tagged variant (Mrx15-PA) quantitatively comigrated with the interaction partners found in this study were named according to LSU (Figure 2A) and was present in mitochondria in similar quantities this convention (Mrx15–17), as they apparently play a role in expres- as the mitoribosomal subunits (Figure 2B). To investigate the submi- sion of mitochondrially encoded proteins. We concluded that this tochondrial localization of Mrx15, we performed carbonate extrac- approach allowed us to identify new ribosomal interaction partners tion and protease protection assays. Mrx15 behaved during carbon- and to determine the subunit with which they are interacting. ate extraction like the integral membrane protein Cbp4 (Figure 2C) (Crivellone, 1994). This is in line with a high-throughput proteomic Mrx15 is a mitochondrial inner membrane protein that study that also identified Mrx15 as an integral membrane protein of interacts with the LSU the inner mitochondrial membrane (Morgenstern et al., 2017). Fur- Mrx15 is encoded by the open reading frame YNR040W and shows thermore, Mrx15 was resistant toward externally added proteinase K the same sedimentation profile as the LSU (Figure 1D). Mrx15 is a when mitochondria were intact, but was partly degraded to a trun- 29-kDa protein with two predicted transmembrane segments cated form upon removal of the outer membrane (dMrx15-PA; FIGURE 2: Mrx15 is an inner membrane mitochondrial protein that interacts with the large ribosomal subunit. (A) Western blot of mitochondrial extracts containing a chromosomally PA-tagged variant of Mrx15. Mitochondria were lysed in 10 mM EDTA for Mg depletion and separated afterward on a continuous sucrose gradient. Individual fractions were probed for aconitase (Aco1), proteins from the LSU (Mrpl36) and SSU (Mrps5), and PA for Mrx15 detection. (B) Abundancy of PA-tagged variants of the SSU (Mrp1PA), LSU (Mhr1PA), and Mrx15 were compared by Western blotting. Aconitase (Aco1) and Mrpl4 were used as loading controls. (C) Carbonate extraction of mitochondrial extracts. After incubation with either 0.2 M Na CO or 0.2 M NaCl, the membrane and soluble fractions of mitochondrial extracts 2 3 were separated by high-velocity centrifugation. Supernatant and pellet fractions were probed for membrane (Cbp4) and soluble (Aco1) proteins and PA for Mrx15 detection. (D) Protease protection assay of mitochondria (Mitos), mitoplasts, and lysed mitochondria. Mitochondria, mitoplasts created by hypotonic swelling of mitochondria, and detergent-lysed mitochondria were treated for 20 min with proteinase K (PK). Afterward, a Western blot of each sample was probed for proteins of the outer membrane (Tom70), inner membrane (Cbp4), and the matrix (Mrpl36). The PA antibody was used for detection of the PA-tagged variant of Mrx15. (E) Schematic representation of full-length Mrx15 tagged with PA and a C-terminal truncation variant. Cell extracts of strains grown to log phase containing either variant were probed for PA to determine abundance of each construct. (F) Western blot of mitochondrial extracts containing C-terminally truncated variant of Mrx15. Interaction with the mitoribosome was tested by Mg depletion and separation of ribosomal subunits on a continuous sucrose gradient. Individual fractions were probed for aconitase (Aco1), proteins from the LSU (Mrpl36) and SSU (Mrp1), and PA for Mrx15 detection. (G) Mrx15 inner membrane topology. According to our results, the Mrx15 protein is a component of the inner mitochondrial membrane. The N- and C-termini of the protein face the matrix. The C-terminal domain is necessary for interaction with the large ribosomal subunit. IMS, intermembrane space; Mitopl., mitoplasts; P, pellet; SN, supernatant; T, total; TM, transmembrane segment; WT, wild type. 2388 | B. V. Möller-Hergt et al. Molecular Biology of the Cell Figure 2D). Because Mrx15 was detected via its C-terminal PA tag, this shows that the N- and C-termini are facing the mitochondrial matrix. In silico analysis of Mrx15 revealed that it belongs to a family of uncharacterized proteins that are conserved within fungi (Supple- mental Figure 1). All members contain two predicted transmem- brane segments and a charged C-terminal domain. Next, we tested whether the positively charged, soluble C-termi- nal domain of Mrx15 is important for the interaction with the mitori- bosome. We constructed a mutant in which the C-terminal 126 amino acids were deleted (mrx15ΔC; Figure 2E). This deletion leads to decreased levels of Mrx15ΔC-PA (Figure 2E). Importantly, Mrx15ΔC-PA did not comigrate with the LSU (Figure 2F). Accord- ingly, the positively charged C-terminal domain is necessary for ri- bosomal interaction. We concluded that Mrx15 is an integral protein of the inner mitochondrial membrane that interacts with the LSU of the mitoribosome via a C-terminal domain (Figure 2G). Synthetic genetic interaction between MRX15 and MBA1 suggests a common function To investigate the function of Mrx15, we created a chromosomal deletion strain and tested the growth of this mutant. The mrx15Δ mutant did not show a growth defect on fermentable and nonfer- mentable media at 30 or 37°C (Figure 3A). Mba1 is a peripheral inner membrane protein involved in membrane insertion of mito- chondrial translation products (Preuss et al., 2001; Ott et al., 2006). It directly interacts with the LSU of the mitoribosome and aligns the tunnel exit for membrane protein insertion (Gruschke et al., 2010; Pfeffer et al., 2015). Because Mba1 and Mrx15 both bind to the membrane and the ribosome, but are not necessary for respiratory growth, we asked whether the combined absence of Mrx15 and Mba1 would result in a synthetic growth phenotype suggesting a common function. As reported previously, the mba1Δ mutant showed only a mild growth defect on a nonfermentable carbon source (Figure 3A) (Ott et al., 2006). Strikingly, the mrx15Δmba1Δ mutant was respiratory deficient at 30 and 37°C, while fermentative growth was not affected. We concluded that loss of Mrx15 is toler- ated under the tested conditions but required for respiratory growth in the absence of Mba1, suggesting that both proteins have over- lapping functions in the biogenesis of the respiratory chain. Next, we tested whether deletion of the C-terminus of Mrx15 is sufficient to provoke the combined respiratory growth phenotype and found that, while the single mutant did not show a growth phenotype un- der the tested conditions, cells became respiratory deficient in the mrx15ΔCmba1Δ strain (Figure 2A), thereby demonstrating that the C-terminal domain of Mrx15 is required for ribosome interaction and respiratory growth upon deletion of MBA1. Mba1 cooperates with Oxa1 in the insertion of mitochondrial translation products into the inner membrane (Preuss et al., 2001). deleted alone or together with MRX15. Cells were grown to logarithmic phase, spotted in 10-fold dilutions, and incubated at 30 and 37°C. (B) Ribosome pelletation at different ionic strengths. The ribosome and attached factors were separated after lysis in different salt conditions by a sucrose cushion and high-velocity centrifugation. The different fractions were probed for a soluble protein (Aco1), a ribosomal marker (Mrpl36), Mba1, and PA for Mrx15 detection. FIGURE 3: Deletion of MRX15 together with MBA1 leads to (C) Flotation gradient of mitochondrial extracts from wild-type or respiratory deficiency and altered membrane attachment. (A) Serial- mrx15Δmba1Δ strains. Soluble and membrane fractions were dilution growth test on full medium fermentable (glucose) and separated after freeze–thaw cycles by a sucrose step gradient and nonfermentable (glycerol) carbon sources of indicated strains. In the high-velocity centrifugation. Fractions were probed for the LSU mrx15ΔC mutant, the terminal 126 amino acids were replaced by a PA (Mrpl36, Mrpl4, and Mrpl40), membrane (Cbp4), and soluble proteins tag. In the C-terminal Oxa1 mutant the 71 C-terminal residues were (Aco1). P, pellet; SN, supernatant; T, total; WT, wild type. Volume 29 October 1, 2018 Mrx15 acts as mitoribosome receptor | 2389 Simultaneous deletion of Mba1 and of 71 C-terminal residues of complexes (Figure 4B). Importantly, upon simultaneous deletion of Oxa1 leads to a severe respiratory growth inhibition combined with MRX15 and MBA1, complex IV levels were further reduced, while an insertion defect of mitochondrially encoded proteins (Ott et al., complex III did not change. ATP synthase (complex V), which also 2006). We tested whether deletion of the C-terminal tail of Oxa1 in contains mitochondrially encoded subunits, was unchanged in the absence of Mrx15 would impair respiratory growth. However, these strains, pointing to a specific defect in accumulating complex unlike Mba1, Mrx15 did not show a synthetic genetic interaction IV in the mrx15Δmba1Δ cells. with the C-terminal domain of Oxa1 (Figure 3A). This suggests that To confirm this conclusion, we analyzed respiratory chain super - Mrx15 and the C-terminal ribosome-binding domain of Oxa1 do not complex formation and respiratory chain activity. Consistent with have overlapping roles for membrane protein insertion, in contrast the steady-state analyses, blue native PAGE (BN-PAGE) revealed to Mrx15 and Mba1, which apparently share a common function. that complex IV abundance in respiratory supercomplexes was Next, we investigated the salt sensitivity of the Mrx15-LSU and lower in the mba1Δ mutant and severely reduced in the double- Mba1-LSU interactions and pelleted ribosomes through a sucrose deletion mutant, where only the smaller isoform of the supercom- cushion at different ionic strengths (Figure 3B). At low ionic strength plexes (III IV) was detected (Figure 4C, middle). Complex III, the conditions, Mrx15 interacted with the LSU, while Mba1 did not co- partner of complex IV in respiratory supercomplexes, as revealed by migrate with the LSU under any of the tested conditions. Consis- the late-assembling subunit Rip1 (Ndi et al., 2018), was mainly pres- tently, Mba1 comigration with any of the mitoribosomal subunits ent in the dimeric form, indicating severe reduction of complex IV. was not observed in the mass spectrometry data set (Figure 1C). ATP synthase was present as a monomer and dimer in all strains, Instead, interaction of Mba1 with the LSU was found through chemi- and the mrx15Δmba1Δ cells had slightly reduced levels of the di- cal cross-linking or ribosome comigration upon magnesium deple- mer. The specific decrease in complex IV levels upon simultaneous tion only, pointing to a rather transient interaction of Mba1 with the deletion of Mrx15 and Mba1 was also reflected in a significant de - mitoribosome (Ott et al., 2006; Gruschke et al., 2010). crease in complex IV activity, confirming that both Mba1 and Mrx15 Mitoribosomes are permanently associated with the inner mito- are specifically important for the biogenesis of complex IV. In con - chondrial membrane (Ott et al., 2006; Prestele et al., 2009; Pfeffer trast, complex III activity was lower upon MBA1 deletion (Bauer- et al., 2015). Based on the high-resolution cryo–electron microscopy schmitt et al., 2010), but not further decreased in the mrx15Δmba1Δ (cryo-EM) structure of the LSU and cryo-EM tomography, a second mutant. point of membrane attachment was proposed, consisting of a mem- brane-facing protuberance buildup by mitoribosome-specific pro - Mrx15 and Mba1 interact with nascent polypeptide chains teins and rRNA expansion segments (Amunts et al., 2014; Pfeffer Mba1 interacts with nascent polypeptide chains as they emerge et al., 2015). This is supported by data showing that the mitoribo- from the mitoribosome (Preuss et al., 2001; Ott et al., 2006; some stays membrane bound upon deletion of MBA1, either alone Gruschke et al., 2010). Owing to the synthetic genetic interaction or in combination with the C-terminal tail of Oxa1 (Ott et al., 2006). and the cofractionation of Mrx15 and Mba1 with the LSU, we Because we found the Mba1-LSU interaction to be rather weak, we hypothesized that Mrx15 could be in contact with nascent chains. To tested whether Mba1 and Mrx15 showed overlapping functions in test this, we performed in organello labeling of mitochondrial trans- ribosome–membrane attachment. We therefore followed ribo- lation products followed by chemical cross-linking and purification some–membrane interaction upon simultaneous deletion of MRX15 of Mrx15. We chose a cross-linker (DFDNB) with a very short linker and MBA1 in a flotation gradient (Figure 3C). In wild-type mitochon - length (0.3 nm) that can only covalently connect molecules that are dria, a fraction of the mitoribosomes cofractionated with the mem- in very close proximity. Autoradiography of the elution fraction brane marker Cbp4, indicating membrane interaction. In contrast, showed multiple species with electrophoretic mobilities above only a minor fraction of the mitoribosome in the mrx15Δmba1Δ mu- 25 kDa (Figure 5A). In a control experiment with mitochondria from tant floated with the membranes. Therefore, we concluded that the wild type, these species were not detected. This indicated that interaction of mitoribosomes with the membrane is decreased upon Mrx15 was cross-linked to nascent chains. Mba1 also cross-linked to simultaneous deletion of Mba1 and Mrx15. nascent chains, which is in line with previous results (Preuss et al., 2001; Gruschke et al., 2010). We concluded that Mrx15, like Mba1, Absence of Mba1 and Mrx15 leads to complex IV deficiency is not only in contact with the LSU but directly interacts with nascent To further characterize the growth defect in the mutants lacking chains emerging from the tunnel exit. Mrx15, Mba1, or both, we determined steady-state levels of mito- Mba1 forms a stable complex with Mdm38 that can be purified chondrial proteins in the strains. Amounts of proteins implicated in (Bauerschmitt et al., 2010). We hypothesized that Mrx15 could also mitochondrial protein biogenesis did not change in the strains (Figure physically interact with Mba1 or Mdm38. Therefore, we purified 4A). Levels of the cytochrome c oxidase (complex IV) subunit Cox2 FLAG-tagged variants of Mrx15 from mitochondrial extracts with were not changed upon loss of Mrx15, but were reduced in the ab- two different detergents, but no stable complex between these pro- sence of Mba1, with a concomitant accumulation of the precursor teins was detected (Figure 5B). Likewise, we did not observe a com- form of Cox2 (pCox2), in line with previous data (Preuss et al., 2001; bined growth defect in the mrx15Δmdm38Δ cells (Figure 5C). Unlike Bauerschmitt et al., 2010). After synthesis and membrane insertion, Mdm38 and Mba1, Mrx15 and Mba1 apparently carry out their the Cox2 precursor is processed into a mature from (mCox2) by pro- overlapping functions without forming a stable complex. We con- teolytic removal of an N-terminal domain by Imp1 and Cox20, thereby cluded that Mrx15, like Mba1, binds to nascent polypeptide chains generating mature, assembly-competent Cox2 (Nunnari et al., 1993; but does not form a stable complex with Mba1 or Mdm38. Jan et al., 2000). Interestingly, simultaneous loss of Mba1 and Mrx15 leads to a further increase in pCox2 levels relative to mCox2. Mrx15 and Mba1 jointly mediate biogenesis Employing Western blot followed by densitometry analyses, we of the Cox2 precursor found that cytochrome bc complex (complex III) and complex IV The direct interaction of Mrx15 with nascent polypeptide chains subunits were accumulating normally in the absence of Mrx15, while prompted us to investigate whether the protein together with mitochondria from mba1Δ mutants had diminished amounts of both Mba1 plays a role in protein insertion. Therefore, we radiolabeled 2390 | B. V. Möller-Hergt et al. Molecular Biology of the Cell FIGURE 4: Mrx15 and Mba1 are required for complex IV accumulation. (A) Steady-state levels of mitochondrial proteins in all indicated strains. Cells were grown in galactose to logarithmic phase, and Western blots probed for proteins necessary for mitochondrial protein biogenesis and complex IV accumulation. (B) Mitochondrial extracts of indicated strains were probed for subunits of complex III (CytB, Qcr7), complex IV (Cox1, Cox13), and complex V (Atp4, F1β). Signal intensities of three independent experiments were quantified and normalized. Significance of observed changes was assessed by a Student’s t test. (C) BN-PAGE of digitonin-solubilized mitochondria from indicated strains. Separated protein complexes were analyzed by Western blotting against subunits of complex III (Rip1), complex IV (Cox1), and complex V (Atp4) (D) Complex III and IV activity measurement. Activity of complex III was followed by measuring cytochrome c reduction, complex IV activity by measuring cytochrome c oxidation at 550 nm. CIII, complex III; CIV, complex IV; CV, complex V; n.s., p > 0.05; **, p ≤ 0.01. mitochondrial translation products in vivo in a pulse–chase experi- enriched, only a smear of nascent chains and some mitoribosomes ment (Figure 6A). Mitochondrial protein synthesis and stability were were copurified (Figure 6C). This indicates that Mrx15, in contrast to not affected in the mrx15Δ mutant compared with the wild type. As Mba1, which interacts specifically with Cox2 (Lorenzi et al., 2016), reported previously (Preuss et al., 2001), the MBA1 deletion mutants does not bind posttranslationally to mitochondrially encoded pro- showed an accumulation of pCox2, and this was enhanced in the teins. Taken together, these results suggest that the observed com- mrx15Δmba1Δ mutant. To quantify this effect, we determined the plex IV deficiency and the respiratory growth defect in the pCox2/mCox2 ratio in both mutants after a 15-min pulse and 90-min mrx15Δmba1Δ mutant are caused by hampered Cox2 biogenesis chase from five independent experiments (Figure 6B). The pCox2/ due to disturbances of ribosome–membrane interactions required mCox2 ratio after 15 min of protein synthesis and a 90-min chase for efficient cotranslational insertion combined with a specific defect was significantly higher in the mrx15Δmba1Δ mutant, revealing an in Cox2 maturation that is carried out by Mba1 (Lorenzi et al., 2016). increased accumulation of the pCox2 variant when both Mrx15 and Mba1 were absent. To check whether Mrx15 interacts with specific DISCUSSION mitochondrial translation products, we purified Mrx15-containing In this study, we identified Mrx15, which helps to tether the mitori - complexes after in organello labeling of mitochondrial translation bosome to the inner membrane for efficient cotranslational inser - products from digitonin lysates. While Mrx15-FLAG could be clearly tion. On the basis of results presented in this study, we propose the Volume 29 October 1, 2018 Mrx15 acts as mitoribosome receptor | 2391 study of the human mitoribosome estab- lished that mL45 is the single contact site to the inner membrane, while the general architecture of the membrane–mitoribo- some contact is conserved between yeast and human (Englmeier et al., 2017). Because homologous proteins of Mrx15 are found only in fungi, it is tempting to speculate that Mrx15 substitutes Mba1 as a permanently bound mitoribosome receptor. Therefore, Mrx15 could occupy a similar binding site on the ribosome as Mba1, ensuring correct mi- toribosome–membrane attachment while Mba1 is escorting Cox2 toward respiratory chain assembly. Permanent membrane attachment of ri- bosomes appears to be specifically impor - FIGURE 5: Mrx15 interacts with nascent chains but does not form a stable complex with Mba1. tant for mitochondrial gene expression. A (A) Cross-linking to nascent mitochondrial polypeptide chains. Mitochondrial translation recent study of mitoribosome assembly in products were radiolabeled in wild-type mitochondria or mitochondria containing a FLAG- yeast found that the mitoribosomal LSU is tagged variant of Mrx15. After 15 min of labeling, the chemical cross-linker DFDNB was added already attached to the membrane during to covalently link molecules in direct physical contact. The different reactions were stopped, and assembly (Zeng et al., 2018). This study Mrx15 cross-linking products were purified with a FLAG resin. Mba1 and cross-linked molecules found mitoribosome–membrane attach- were iummunoprecipitated employing a PA resin and an Mba1 antibody. As a control, the ment even upon simultaneous deletion of reactions were performed without a FLAG tag or an Mba1 antibody. (B) Purification of FLAG- MBA1, MDM38, and OXA1. Therefore mito- tagged Mrx15 variants. Mitochondria were lysed in two detergents (Triton X-100, digitonin) and Mrx15 purified by employing a FLAG resin. Western blots of the purification were probed ribosome–membrane attachment likely for FLAG, Mba1, Mdm38, and Aco1. (C) Serial-dilution growth test on fermentable (YPD) mainly depends on 21S rRNA incorporation and nonfermentable (YPG) carbon sources of indicated strains. E, elution; NB, not bound; into the LSU. This notion is supported by T, total; WT, wild type. data showing that mutations in 21S rRNA can alter mitoribosome–membrane attach- following model (Figure 6D): Mba1 and Mrx15 bind to the LSU of ment (Spithill et al., 1978). According to data presented here, mito- the mitoribosome and interact with nascent chains emerging from ribosomes from the double-deletion mutant of MBA1 and MRX15 the exit tunnel. Both proteins tether the LSU to the inner mitochon- were still partly membrane attached, suggesting that the 21S rRNA drial membrane to ensure proper alignment of the LSU with the in- of LSU could connect to the inner mitochondrial membrane via the sertion site. Mba1 and Mrx15 are in proximity to nascent chains to expansion segment E29 (Pfeffer et al., 2015). ensure efficient membrane insertion by the insertase Oxa1. Upon Combined absence of Mba1 and Mrx15 provokes a strong respi- completion of Cox2 synthesis, Mba1 dissociates from the mitoribo- ratory growth defect, which at least partly reflects the strong com - some to escort the newly synthesized protein toward downstream plex IV deficiency that exhibits a specific accumulation of the Cox2 assembly processes, while Mrx15 remains ribosome bound. precursor. Cox2 precursor accumulation could be caused by a gen- In the simultaneous absence of Mrx15 and Mba1, a large part of eral defect of membrane insertion or the disruption of shuttling the mitoribosomes is liberated from the inner membrane, pointing newly synthesized Cox2 to the maturation complex consisting of to a critical function of both proteins as ribosome–membrane teth- Cox20 and Imp1/2 (Lorenzi et al., 2016). A general insertion defect ers. However, Mrx15 and Mba1 greatly differ in their mitoribosome- in the mrx15Δmba1Δ cells that exacerbates the insertion defect of binding efficiencies. While the integral inner membrane protein mba1Δ cells would be revealed as a pleiotropic defect in the assem- Mrx15 stayed ribosome bound in the presence of magnesium and at bly of all OXPHOS complexes, as has been observed in mutants moderate ionic strength, we did not detect a permanent interaction affected in common processes for synthesis and insertion of the between the peripheral membrane protein Mba1 and the LSU of the mitochondrially encoded membrane proteins (Altamura et al., 1996; mitoribosome under these conditions. This suggests that Mrx15 is a Prestele et al., 2009; Bauerschmitt et al., 2010; Kuzmenko et al., constant ribosome tether, while Mba1 dynamically interacts with the 2016; Ostojic et al., 2016; Suhm et al., 2018). This, however, is not ribosome. This is in line with a recent study demonstrating a post- the case in the mrx15Δmba1Δ cells. Furthermore, our data, which translational function of Mba1 for complex IV biogenesis (Lorenzi indicate that MRX15 does not show a genetic interaction with the et al., 2016), whereby Mba1 shuttles Cox2 from synthesis to its as- C-terminal domain of Oxa1, the insertase, or Mdm38, are in line sembly factor Cox20 for maturation and respiratory chain assembly. with a context-specific function Mrx15 exerts together with Mba1 Cryo-EM tomography has revealed the organization of mitoribo- during Cox2 biogenesis. A special aspect of Cox2 biogenesis is that somes in isolated organelles, where Mba1 occupies a position con- the protein contains by far the largest and most charged soluble necting the tunnel exit with the insertion site. In the absence of Mba1, domains of all mitochondrially encoded proteins, and these do- a specific density was absent, but the general arrangement of the ri - mains need to be translocated over the inner membrane (Herrmann bosome relative to the inner membrane was unchanged (Pfeffer et al., 1995). A disturbed ribosome–membrane contact could et al., 2015), suggesting the presence of other ribosome tethers like specifically impact the translocation of the highly hydrophilic Cox2 the Mrx15 protein identified here. In contrast to yeast Mba1, its mam - domains, particularly when connected with the absence of the shut- malian homologue mL45 is an integral and firmly bound subunit of tling factor Mba1 (Lorenzi et al., 2016) that presumably removes the mitoribosomal LSU (Greber et al., 2014b). A recent tomographic pCox2 from the insertion site. 2392 | B. V. Möller-Hergt et al. Molecular Biology of the Cell In addition to the clear energetic advantages of tethering ribo- somes to membranes to facilitate cotranslational protein insertion, the firm interaction of the mitoribosome and the inner mitochondrial membrane allows further specialization in how translation is orga- nized. For example, translational activators regulate translation of specific client mRNA in yeast mitochondria (Fox, 2012). This class of proteins is firmly membrane bound and essential for translation in yeast mitochondria. Because of these characteristics, it was sug- gested that a main function of translational activators is to localize the synthesis of the hydrophobic translation products to the inner membrane (McMullin and Fox, 1993; Fox, 1996). Moreover, by gath- ering several translational activators controlling synthesis of the three cytochrome oxidase subunits (Naithani et al., 2003), localized assembly sites are established that likely facilitate joining of the sub- units. These sites of translation and subsequent biogenesis are not evenly distributed in the inner membrane, as assembly of each OX- PHOS complex preferentially occurs at different subcompartments within the inner membrane (Stoldt et al., 2018), highlighting the in- tricate organization of mitochondrial gene expression. We have previously found mitoribosomes to interact with many proteins to organize biogenesis of the organellar-encoded proteins. These interactions give rise to large assemblies called MIOREX complexes (Kehrein et al., 2015) that unite many steps of mitochon- drial gene expression, including mRNA maturation, translation, as- sembly, and turnover. Here, employing an alternative strategy to identify and classify components interacting with the mitoribosome provided support for this general concept. Specifically, at least parts of mRNA maturation occur on the LSU, as revealed by its interaction with the COB mRNA splicing factor Cbp2. It remains an exciting task to develop strategies to further categorize the interactome of the yeast mitoribosome and to establish how the different processes are organized on the mitoribosome. MATERIALS AND METHODS Yeast strains and growth media All yeast strains used in this study (Supplemental Table 1) were iso- genic to either the wild-type strains W303a or CW04. MRX15 was disrupted with a kanamycin resistance cassette. The MBA1 coding sequence was replaced with an HIS3 selection cassette. FLAG- or PA-tagged variants of Mrx15 were created by replacing the endog- enous stop codon with a FLAG sequence or a PA sequence employ- ing a TRP1 or HIS3 selection cassette. Cells were grown at 30°C in YP medium (2% peptone, 1% yeast extract) in the presence of either 2% dextrose, 2% galactose, or 2% glycerol. Isolation of mitochondria Mitochondria were isolated as described previously (Gruschke et al., 2011). For experiments analyzed by mass spectrometry, mi- tochondria were isolated by a sucrose step gradient. The mito- chondrial pellet was resuspended in SHE buffer (20 mM HEPES/ KOH, pH 7.4, 250 mM sucrose, 1 mM EDTA) and loaded on a step polypeptides with Mrx15. After in organello labeling of mitochondrial translation products, a FLAG-tagged variant of Mrx15 was purified, and eluates were analyzed by Western blotting and autoradiography. FIGURE 6: Mrx15 is involved in accumulation of premature Cox2. (D) Model of Mrx15 and Mba1 function in Cox2 membrane insertion. (A) In vivo radiolabeling of all indicated strains. Mitochondrial protein In wild-type cells, Mrx15 and Mba1 bind to the LSU and nascent synthesis of all mutants was followed for 15 min The stability of the chains to ensure ribosome–membrane attachment and proper synthesized polypeptides was chased for 90 min. (B) Quantification of membrane insertion. Upon deletion of both factors, membrane the pCox2 to mCox2 ratio after 15 min of synthesis and a 90-min insertion is hampered, and pCox2 accumulates. Consequently, this chase. Bands of five independent experiments were quantified, and leads to decreased Cox2 maturation, complex IV assembly, and significance between displayed values was assessed by a Student’s respiratory deficiency. E, elution; IMS, intermembrane space; NB, not t test. (C) Copurification of newly synthesized mitochondrial bound; T, total; WT, wild type; n.s., p > 0.05; **, p ≤ 0.01. Volume 29 October 1, 2018 Mrx15 acts as mitoribosome receptor | 2393 gradient. The layers consisted of 1.75, 0.95, 0.68, and 0.45 M su- (Thermo Fisher). When the dye front had migrated one-third of the crose in 20 mM HEPES/KOH (pH 7.4) and 1 mM EDTA. Mitochon- way through the gel, the dark blue cathode buffer was replaced with dria were spun down (134,000 × g, 1 h, 4°C), recovered from the a light blue cathode buffer. Afterward, separated protein complexes 1.74–0.95 interface, and resuspended in SH buffer (20 mM HEPES, were blotted onto polyvinylidene difluoride membranes. pH 7.4, 0.6 M sorbitol). At a concentration of 10 mg/ml, mitochon- dria were snap-frozen in liquid nitrogen. Complex III and IV activity assay For respiratory chain activity measurements, 0.5 mg of mitochondria Sucrose gradients were lysed (50 mM Tris/HCl, pH 7.4, 100 mM KCl, 2.5 MgCl , Isolated mitochondria were lysed (10 mM Tris/HCl, pH 7.4, 10 mM 0.1 mM EDTA, 1X complete, 1 mM PMSF, 2% digitonin) for 10 min KOAc, 0.5 mM Mg(OAc) , 10 mM EDTA, 5 mM β-mercaptoethanol, on ice. Afterward, the lysate was cleared by centrifugation (16,000 × 1% dodecylmaltoside, 1 mM phenylmethylsulfonyl fluoride [PMSF], g, 10 min, 4°C) and diluted 50-fold in 50 mM Tris/HCl (pH 7.4). 1X complete, 0.1 mM spermidine, 5% glycerol), and sucrose gradi- For complex III activity measurements, KCN (1 mM) and cyto- ent experiments were performed as described previously (Kehrein chrome c (0.5 mg/ml) were added to the lysate. The reduction of et al., 2015). Collected samples were precipitated by addition of cytochrome c was monitored by measuring the change in absor- 12% trichloroacetic acid. The resuspended samples were analyzed bance at 550 nm upon addition of quinol (0.08 mg/ml). Activity was by SDS–PAGE and Western blotting or mass spectrometry accord- calculated by determining the slope of the reaction. ing to published procedures (Chiurillo et al., 2016). For complex IV activity measurements, antimycin A (0.2 mg/ml) was added to the lysate. The oxidation of cytochrome c was moni- Carbonate extraction and protease protection tored by measuring the change in absorbance at 550 nm upon ad- Isolated mitochondria (100 µg) were resuspended in 0.1 M Na CO or dition of reduced cytochrome c (0.33 mg/ml). Activity was calcu- 2 3 NaCl and incubated 30 min on ice. Membrane and soluble fractions lated as for complex III. were separated by centrifugation (100,000 × g, 30 min, 4°C), TCA Purification of FLAG-tagged Mrx15 precipitated, and analyzed by SDS–PAGE and Western blotting. Mitochondria from yeast strains expressing FLAG-tagged variants Mitochondria (100 µg) were incubated in SH buffer or 20 mM of Mrx15 were lysed for 10 min on ice (10 mM Tris/HCl, pH 7.4, HEPES/KOH or lysis buffer (SH buffer with 0.2% Triton X-100) for 150 mM NaCl, 1 mM EDTA, 1% digitonin or Triton X-100, 1 mM 30 min at 4°C. Afterward, 0.1 mg/ml proteinase K was added, and PMSF, 1X complete). Subsequently, samples were diluted 1:1 in the reaction was incubated for 20 min at 4°C. Intact mitochondria or dilution buffer (10 mM, Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, mitoplasts were spun down (25,000 × g, 10 min, 4°C). The superna- 0.1% digitonin or Triton X-100, 1 mM PMSF, 1x complete), and the tant and pellet were analyzed by SDS–PAGE and Western blotting. lysate was cleared by centrifugation (16,000 × g, 10 min, 4°C). The cleared lysate was incubated with anti-FLAG M2 affinity gel for 2 h Ribosome sedimentation at 4°C. The affinity gel was washed three times with dilution buffer. Isolated mitochondria were lysed in different buffers (10 mM Tris/HCl, Purified proteins were eluted by incubation for 20 min with dilu - pH 7.4, 10/25/100/250 mM KOAc, 5/5/20/50 mM Mg(OAc) , 0.1 mM tion buffer supplemented with 0.15 µg/µl 3× FLAG peptide. Sam- spermidine, 5% glycerol, 1% DDM, 1 mM PMSF, 1X complete) for ples of all relevant fractions were precipitated by addition of 12% 10 min on ice. The lysate was diluted in one volume of the respective TCA. The resuspended samples were analyzed by SDS–PAGE and lysis buffer without DDM and cleared by centrifugation (16,000 × g, Western blotting. 10 min, 4°C). The cleared lysate was underlaid with half a volume of For testing copurification of newly synthesized mitochondrial sucrose cushion (10 mM Tris/HCl, pH 7.4, 10/25/100/250 mM KOAc, translation products with Mrx15, a FLAG-tagged variant was puri- 5/5/20/50 mM Mg(OAc) , 0.1 mM spermidine, 5% glycerol, 1% fied after in organello labeling (Gruschke et al., 2010). Mitochondria DDM, 1 mM PMSF, 1X complete, 1.2 M sucrose) and centrifuged were lysed after the labeling reaction (10 mM Tris/HCl, pH 7.4, 100 (189,062 × g, 105 min, 4°C). Afterward, the supernatant and pellet mM NaCl, 1 mM EDTA, 2% digitonin, 1 mM PMSF, 1X complete), fractions were separated and analyzed by Western blotting. and the purification was carried out as described earlier. Flotation gradients Miscellaneous methods Isolated mitochondria were resuspended in buffer A (20 mM Tris/ Polyclonal antisera against Mrp1 and Mrx15 were generated by in- HCl, pH 7.4, 25 mM KOAc, 5 mM Mg(OAc) , 1 mM PMSF) and jecting rabbits with recombinantly expressed and purified antigens. treated with three consecutive freeze–thaw cycles. Mitochondria The antibodies against PA and FLAG were obtained from Sigma. were frozen in liquid nitrogen and thawed at 37°C. Afterward, the Labeling of mitochondrial translation products and chemical cross- reaction was resuspended in 10 volumes of 2.5 M sucrose in linking of nascent chains were carried out as previously described 20 mM Tris/HCl (pH 7.4). The suspension was overlaid with one (Gruschke et al., 2010). volume of 2.2, 1.5, and 1 M sucrose and centrifuged (40,000 × g, 16 h, 4°C). The gradient was fractionated, and individual fractions were analyzed by Western blotting. ACKNOWLEDGMENTS We thank members of our laboratories for stimulating discussions BN-PAGE and Western blotting and Marc Wirth and Ignasi Forne (Ludwig Maximilian University of Isolated mitochondria (150 µg) were resuspended in BN-PAGE buffer Munich, Germany) for help with mass spectrometric analyses. This (50 mM Bis-Tris/HCl, pH 7.2, 150 mM NaCl, 2 mM aminohexanoic work was supported by grants from the Swedish Research Coun- acid, 1 mM EDTA, 1X complete, 1 mM PMSF, 1% digitonin or 1% cil, the Carl Tryggers Foundation, and the Knut and Alice Wallen- DDM, 12% glycerol) and lysed 10 min on ice. The lysate was cleared berg Foundation. 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