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Overexpression of MRX9 impairs processing of RNAs encoding mitochondrial oxidative phosphorylation factors COB and COX1 in yeast

Open AccessPublished:June 29, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102214
      Mitochondrial translation is a highly regulated process, and newly synthesized mitochondrial products must first associate with several nuclear-encoded auxiliary factors to form oxidative phosphorylation complexes. The output of mitochondrial products should therefore be in stoichiometric equilibrium with the nuclear-encoded products to prevent unnecessary energy expense or the accumulation of pro-oxidant assembly modules. In the mitochondrial DNA of Saccharomyces cerevisiae, COX1 encodes subunit 1 of the cytochrome c oxidase and COB the central core of the cytochrome bc1 electron transfer complex; however, factors regulating the expression of these mitochondrial products are not completely described. Here, we identified Mrx9p as a new factor that controls COX1 and COB expression. We isolated MRX9 in a screen for mitochondrial factors that cause poor accumulation of newly synthesized Cox1p and compromised transition to the respiratory metabolism. Northern analyses indicated lower levels of COX1 and COB mature mRNAs accompanied by an accumulation of unprocessed transcripts in the presence of excess Mrx9p. In a strain devoid of mitochondrial introns, MRX9 overexpression did not affect COX1 and COB translation or respiratory adaptation, implying Mrx9p regulates processing of COX1 and COB RNAs. In addition, we found Mrx9p was localized in the mitochondrial inner membrane, facing the matrix, as a portion of it cosedimented with mitoribosome subunits and its removal or overexpression altered Mss51p sedimentation. Finally, we showed accumulation of newly synthesized Cox1p in the absence of Mrx9p was diminished in cox14 null mutants. Taken together, these data indicate a regulatory role of Mrx9p in COX1 RNA processing.

      Keywords

      Abbreviations:

      H (hemagglutinin), LSU (large subunit), mtDNA (mitochondrial DNA)
      Mitochondrial respiratory complexes, the ATP synthase, and the mitoribosome have a genetic hybrid origin; most of their components are encoded by the nuclear genome and a few by the mitochondrial DNA (mtDNA). The synthesis of mitochondrially encoded subunits relies on the mitochondrial gene expression system (
      • Ott M.
      • Amunts A.
      • Brown A.
      Organization and regulation of mitochondrial protein synthesis.
      ); in the yeast model Saccharomyces cerevisiae, mtDNA contains 75 kb and encodes eight polypeptides, with introns present in the genes for COB, COX1, and 21SrRNA (
      • Dujon B.
      Mitochondrial genetics revisited.
      ). Indeed, mitochondrial gene expression is finely regulated, and yeast studies have unveiled several regulatory events in the translation of mitochondrial encoded proteins (
      • Ott M.
      • Amunts A.
      • Brown A.
      Organization and regulation of mitochondrial protein synthesis.
      ,
      • Dujon B.
      Mitochondrial genetics revisited.
      ,
      • Derbikova K.S.
      • Levitsky S.A.
      • Chicherin I.V.
      • Vinogradova E.N.
      • Kamenski P.A.
      Activation of yeast mitochondrial translation: who is in charge?.
      ,
      • Fox T.D.
      Mitochondrial protein synthesis, import, and assembly.
      ). The regulation is necessary to accommodate the mitochondrial products with proteins that are imported from the cytosol avoiding futile energetic expenses in the translation of proteins that need the partnership of others that are being produced elsewhere. Moreover, the mitochondrial products Cox1p, Cox2p, and Cob harbor metal prosthetic groups thereof have to be properly chaperoned to avoid the uncontrolled generation of reactive oxygen species (
      • Khalimonchuk O.
      • Bird A.
      • Winge D.R.
      Evidence for a pro-oxidant intermediate in the assembly of cytochrome oxidase.
      ,
      • Geldon S.
      • Fernández-Vizarra E.
      • Tokatlidis K.
      Redox-mediated regulation of mitochondrial biogenesis, dynamics, and respiratory chain assembly in yeast and human cells.
      ,
      • Veniamin S.
      • Sawatzky L.G.
      • Banting G.S.
      • Glerum D.M.
      Characterization of the peroxide sensitivity of COX-deficient yeast strains reveals unexpected relationships between COX assembly proteins.
      ). A concerted integration of approximately 250 components (
      • Pearce S.F.
      • Rebelo-Guiomar P.
      • D'Souza A.R.
      • Powell C.A.
      • Van Haute L.
      • Minczuk M.
      Regulation of mammalian mitochondrial gene expression: recent advances.
      ) is required for the mitochondrial gene expression. Many of them are spatially associated in higher-order complexes named MIOREX (
      • Kehrein K.
      • Schilling R.
      • Möller-Hergt B.V.
      • Wurm C.A.
      • Jakobs S.
      • Lamkemeyer T.
      • et al.
      Organization of mitochondrial gene expression in two distinct ribosome-containing assemblies.
      ), which consist of the mitoribosomes, factors involved in the mitoribosome assembly, proteins involved in mRNA and DNA metabolism, translational activators, chaperones, proteases, and proteins with unknown function; some of these factors have already more than one function described in mitochondrial RNA metabolism (
      • Barros M.H.
      • Tzagoloff A.
      Aep3p-dependent translation of yeast mitochondrial ATP8.
      ,
      • Hillman G.A.
      • Henry M.F.
      The yeast protein Mam33 functions in the assembly of the mitochondrial ribosome.
      ,
      • De Silva D.
      • Fontanesi F.
      • Barrientos A.
      The DEAD box protein Mrh4 functions in the assembly of the mitochondrial large ribosomal subunit.
      ,
      • Mayorga J.P.
      • Camacho-Villasana Y.
      • Shingú-Vázquez M.
      • García-Villegas R.
      • Zamudio-Ochoa A.
      • García-Guerrero A.E.
      • et al.
      A novel function of Pet54 in regulation of Cox1 synthesis in Saccharomyces cerevisiae mitochondria.
      ,
      • Mays J.N.
      • Camacho-Villasana Y.
      • Garcia-Villegas R.
      • Perez-Martinez X.
      • Barrientos A.
      • Fontanesi F.
      The mitoribosome-specific protein mS38 is preferentially required for synthesis of cytochrome c oxidase subunits.
      ); their multifunctional properties facilitate regulatory loop processes that coordinate mitochondrial gene expression with protein complexes assembly (
      • Fontanesi F.
      Mechanisms of mitochondrial translational regulation.
      ,
      • Kabala A.M.
      • Binko K.
      • Godard F.
      • Charles C.
      • Dautant A.
      • Baranowska E.
      • et al.
      Assembly-dependent translation of subunits 6 (Atp6) and 9 (Atp9) of ATP synthase in yeast mitochondria.
      ,
      • Rak M.
      • Tzagoloff A.
      F1-dependent translation of mitochondrially encoded Atp6p and Atp8p subunits of yeast ATP synthase.
      ,
      • Salvatori R.
      • Kehrein K.
      • Singh A.P.
      • Aftab W.
      • Möller-Hergt B.V.
      • Forne I.
      • et al.
      Molecular wiring of a mitochondrial translational feedback loop.
      ,
      • Seshadri S.R.
      • Banarjee C.
      • Barros M.H.
      • Fontanesi F.
      The translational activator Sov1 coordinates mitochondrial gene expression with mitoribosome biogenesis.
      ).
      Here, we started investigating whether the overexpression of 32 proteins of the MIOREX would unbalance the mitochondrial gene expression system resulting in deleterious effects on mitochondrial translation. Indeed, the overexpression of some components was deleterious and MRX9 in particular called our attention due to its effects on Cox1p translation. Cox1p is a 12-transmembrane domain spanning integral IMM protein part of cytochrome c oxidase (COX), the terminal electron acceptor in the respiratory electron transport chain. This catalytic core subunit contains the two heme groups a and a3 and a copper ion in the CuB center (
      • Fox T.D.
      Mitochondrial protein synthesis, import, and assembly.
      ). Afterward, we noticed that Mrx9p excess was also toxic to COB expression. Cytochrome b is the central core of the bc1 complex, embedded via eight transmembrane spans in the IMM harboring two heme b centers (
      • Fox T.D.
      Mitochondrial protein synthesis, import, and assembly.
      ).
      COX1 is transcribed as a polycistronic transcript containing ATP6/ATP8 of ATP synthase (
      • Manon S.
      • Guerin M.
      Modifications of the relative proteolipid composition in the ATP synthase of a respiratory competent mutant of Saccharomyces cerevisiae.
      ). RNA processing requires splicing to generate the mature COX1 transcript without introns, a process assisted by maturases encoded by ORFs located within some of the introns (
      • Lazowska J.
      • Jacq C.
      • Slonimski P.P.
      Sequence of introns and flanking exons in wild-type and box3 mutants of cytochrome b reveals an interlaced splicing protein coded by an intron.
      ) and nuclear genes such as MSS116, PET54, and COX24 (
      • Barros M.H.
      • Myers A.M.
      • Van Driesche S.
      • Tzagoloff A.
      COX24 codes for a mitochondrial protein required for processing of the COX1 transcript.
      ,
      • Valencik M.L.
      • Kloeckener-Gruissem B.
      • Poyton R.O.
      • McEwen J.E.
      Disruption of the yeast nuclear PET54 gene blocks excision of mitochondrial intron aI5 beta from pre-mRNA for cytochrome c oxidase subunit I.
      ,
      • Séraphin B.
      • Simon M.
      • Boulet A.
      • Faye G.
      Mitochondrial splicing requires a protein from a novel helicase family.
      ). Translation of the COX1 transcript is controlled by Pet309p, Mam33p, Mrx8p, and Mss51p (
      • Decoster E.
      • Simon M.
      • Hatat D.
      • Faye G.
      The MSS51 gene product is required for the translation of the COX1 mRNA in yeast mitochondria.
      ,
      • Manthey G.M.
      • McEwen J.E.
      The product of the nuclear gene PET309 is required for translation of mature mRNA and stability or production of intron-containing RNAs derived from the mitochondrial COX1 locus of Saccharomyces cerevisiae.
      ,
      • Roloff G.A.
      • Henry M.F.
      Mam33 promotes cytochrome c oxidase subunit I translation in Saccharomyces cerevisiae mitochondria.
      ,
      • Verma Y.
      • Mehra U.
      • Pandey D.K.
      • Kar J.
      • Pérez-Martinez X.
      • Jana S.S.
      • et al.
      MRX8, the conserved mitochondrial YihA GTPase family member, is required for de novo Cox1 synthesis at suboptimal temperatures in Saccharomyces cerevisiae.
      ); the pentatricopeptide protein, Pet309p binds to the 5′UTR of the COX1 messenger and recruits Mss116p a DEAD-box helicase, which unwinds the 5′ UTR and facilitate the access of Mss51p to the initiation complex (
      • Manthey G.M.
      • McEwen J.E.
      The product of the nuclear gene PET309 is required for translation of mature mRNA and stability or production of intron-containing RNAs derived from the mitochondrial COX1 locus of Saccharomyces cerevisiae.
      ,
      • De Silva D.
      • Poliquin S.
      • Zeng R.
      • Zamudio-Ochoa A.
      • Marrero N.
      • Perez-Martinez X.
      • et al.
      The DEAD-box helicase Mss116 plays distinct roles in mitochondrial ribogenesis and mRNA-specific translation.
      ). Afterward, Mss51p participates in the assembly process of the Cox1p module (
      • Barrientos A.
      • Zambrano A.
      • Tzagoloff A.
      Mss51p and Cox14p jointly regulate mitochondrial Cox1p expression in Saccharomyces cerevisiae.
      ,
      • Fontanesi F.
      • Clemente P.
      • Barrientos A.
      Cox25 teams up with Mss51, Ssc1, and Cox14 to regulate mitochondrial cytochrome c oxidase subunit 1 expression and assembly in Saccharomyces cerevisiae.
      ,
      • McStay G.P.
      • Su C.H.
      • Tzagoloff A.
      Stabilization of Cox1p intermediates by the cox14p-coa3p complex.
      ), it is sequestered by the Coa3p–Cox14p complex away from COX1 mRNA; a new translation cycle only starts when the biogenesis of the Cox1 module proceeds and Mss51p is released (
      • McStay G.P.
      • Su C.H.
      • Tzagoloff A.
      Stabilization of Cox1p intermediates by the cox14p-coa3p complex.
      ,
      • Pierrel F.
      • Bestwick M.L.
      • Cobine P.A.
      • Khalimonchuk O.
      • Cricco J.A.
      • Winge D.R.
      Coa1 links the Mss51 post-translational function to Cox1 cofactor insertion in cytochrome c oxidase assembly.
      ). Because of the loop control exerted by Mss51p, COX assembly mutants present diminished COX1 translation with the concealed of Mss51p by Coa3p–Cox14p (
      • Barrientos A.
      • Zambrano A.
      • Tzagoloff A.
      Mss51p and Cox14p jointly regulate mitochondrial Cox1p expression in Saccharomyces cerevisiae.
      ,
      • Fontanesi F.
      • Clemente P.
      • Barrientos A.
      Cox25 teams up with Mss51, Ssc1, and Cox14 to regulate mitochondrial cytochrome c oxidase subunit 1 expression and assembly in Saccharomyces cerevisiae.
      ,
      • McStay G.P.
      • Su C.H.
      • Tzagoloff A.
      Stabilization of Cox1p intermediates by the cox14p-coa3p complex.
      ,
      • Pierrel F.
      • Bestwick M.L.
      • Cobine P.A.
      • Khalimonchuk O.
      • Cricco J.A.
      • Winge D.R.
      Coa1 links the Mss51 post-translational function to Cox1 cofactor insertion in cytochrome c oxidase assembly.
      ,
      • McStay G.P.
      • Su C.H.
      • Thomas S.M.
      • Xu J.T.
      • Tzagoloff A.
      Characterization of assembly intermediates containing subunit 1 of yeast cytochrome oxidase.
      ,
      • Perez-Martinez X.
      • Broadley S.A.
      • Fox T.D.
      Mss51p promotes mitochondrial Cox1p synthesis and interacts with newly synthesized Cox1p.
      ). Translation of COX1 is bypassed in COX assembly mutants when COX14 is also deleted, and therefore, Mss51p is free to proceed in new translation cycles (
      • Fontanesi F.
      • Clemente P.
      • Barrientos A.
      Cox25 teams up with Mss51, Ssc1, and Cox14 to regulate mitochondrial cytochrome c oxidase subunit 1 expression and assembly in Saccharomyces cerevisiae.
      ,
      • Zambrano A.
      • Fontanesi F.
      • Solans A.
      • de Oliveira R.L.
      • Fox T.D.
      • Tzagoloff A.
      • et al.
      Aberrant translation of cytochrome c oxidase subunit 1 mRNA species in the absence of Mss51p in the yeast Saccharomyces cerevisiae.
      ).
      Likewise, COB is transcribed together with the tRNA-E gene and harbors two to six introns depending on the yeast strain (
      • Lazowska J.
      • Jacq C.
      • Slonimski P.P.
      Sequence of introns and flanking exons in wild-type and box3 mutants of cytochrome b reveals an interlaced splicing protein coded by an intron.
      ,
      • Nobrega F.G.
      • Tzagoloff A.
      Assembly of the mitochondrial membrane system. DNA sequence and organization of the cytochrome b gene in Saccharomyces cerevisiae D273-10B.
      ). Processing of COB primary transcript requires cis-elements such as the maturase p27 encoded by the bI4 intron (
      • De La Salle H.
      • Jacq C.
      • Slonimski P.P.
      Critical sequences within mitochondrial introns: pleiotropic mRNA maturase and cis-dominant signals of the box intron controlling reductase and oxidase.
      ), while the bI5 intron excision is dependent on Cbp2p (
      • Gampel A.
      • Tzagoloff A.
      In vitro splicing of the terminal intervening sequence of Saccharomyces cerevisiae cytochrome b pre-mRNA.
      ). Translation activation of COB mRNA depends on Cbs1p, Cbs2p, Cbp1p, Cbp3p, and Cbp6p, the highest number of activators for a single mitochondrial RNA (
      • Derbikova K.S.
      • Levitsky S.A.
      • Chicherin I.V.
      • Vinogradova E.N.
      • Kamenski P.A.
      Activation of yeast mitochondrial translation: who is in charge?.
      ). In this study, we show some evidence that suggests a role of Mrx9p in mitochondrial RNA metabolism.
      Mrx9p is a poorly described component of the MIOREX (
      • Kehrein K.
      • Schilling R.
      • Möller-Hergt B.V.
      • Wurm C.A.
      • Jakobs S.
      • Lamkemeyer T.
      • et al.
      Organization of mitochondrial gene expression in two distinct ribosome-containing assemblies.
      ); it cosediments with the small subunit of the mitoribosome (SSU) (
      • Möller-Hergt B.V.
      • Carlström A.
      • Stephan K.
      • Imhof A.
      • Ott M.
      The ribosome receptors Mrx15 and Mba1 jointly organize cotranslational insertion and protein biogenesis in mitochondria.
      ) but also was detected close to the polypeptide tunnel exit of the mitoribosome large subunit (LSU) in proximity-dependent biotinylation studies (
      • Singh A.P.
      • Salvatori R.
      • Aftab W.
      • Aufschnaiter A.
      • Carlström A.
      • Forne I.
      • et al.
      Molecular connectivity of mitochondrial gene expression and OXPHOS biogenesis.
      ). Our data support the association of Mrx9p with the mitoribosome and indicates its involvement in the control of COB and COX1 RNA processing and translation.

      Results

      Overexpression of MIOREX components

      The assembly of the OXPHOS complexes depends on a balanced output of their subunits, mitochondrially or nuclearly encoded products. Therefore, mitochondrial translation is subjected to several regulatory factors, which include mitochondria specific translation activators that have been described for all mitochondrial products (
      • Derbikova K.S.
      • Levitsky S.A.
      • Chicherin I.V.
      • Vinogradova E.N.
      • Kamenski P.A.
      Activation of yeast mitochondrial translation: who is in charge?.
      ,
      • Seshadri S.R.
      • Banarjee C.
      • Barros M.H.
      • Fontanesi F.
      The translational activator Sov1 coordinates mitochondrial gene expression with mitoribosome biogenesis.
      ), and sophisticated feedback regulatory loops for ATP6/8, COB, COX1, and VAR1 translation (
      • Fontanesi F.
      Mechanisms of mitochondrial translational regulation.
      ,
      • Rak M.
      • Tzagoloff A.
      F1-dependent translation of mitochondrially encoded Atp6p and Atp8p subunits of yeast ATP synthase.
      ,
      • Salvatori R.
      • Kehrein K.
      • Singh A.P.
      • Aftab W.
      • Möller-Hergt B.V.
      • Forne I.
      • et al.
      Molecular wiring of a mitochondrial translational feedback loop.
      ,
      • Seshadri S.R.
      • Banarjee C.
      • Barros M.H.
      • Fontanesi F.
      The translational activator Sov1 coordinates mitochondrial gene expression with mitoribosome biogenesis.
      ). A new category of translation regulators includes the ATP6/8 transcript repressor Smt1p (
      • Rak M.
      • Su C.H.
      • Xu J.T.
      • Azpiroz R.
      • Singh A.M.
      • Tzagoloff A.
      Regulation of mitochondrial translation of the ATP8/ATP6 mRNA by Smt1p.
      ). We hypothesized that overexpression of SMT1(MRX5) and other uncharacterized repressors would unbalance the translation of the targeted mitochondrial product. Therefore, we have cloned 32 components of the MIOREX complex (
      • Kehrein K.
      • Schilling R.
      • Möller-Hergt B.V.
      • Wurm C.A.
      • Jakobs S.
      • Lamkemeyer T.
      • et al.
      Organization of mitochondrial gene expression in two distinct ribosome-containing assemblies.
      ) under the control of the GAL10 promoter and evaluated the mitochondrial translation properties of the transformed yeast strain grown in galactose. As a proof of concept, the overexpression of SMT1 (alias MRX5) resulted in a destitute translation of Atp6p and Atp8p (Fig. 1A). In this sense, we have prioritized the overexpression of components with unknown functions to gain insights into their role in the MIOREX complex. After evaluating the MRX genes, AIM17, AIM18, AIM19, AIM25, AIM32, AIM34, MIN10, FMP10, and FMP30 overexpression (Fig. 1, A and B), we noticed a pleiotropic diminishment of mitochondrial products in the strains containing an excess of Aim19p, Aim32p, Aim34p, and Fmp30p accompanied by an elevated rate of petites formation, which can be speculated as a general impairment in the mitochondrial protein synthesis and its consequences in the mtDNA stability (
      • Myers A.M.
      • Pape L.K.
      • Tzagoloff A.
      Mitochondrial protein synthesis is required for maintenance of intact mitochondrial genomes in Saccharomyces cerevisiae.
      ). On the other hand, FMP10 overexpression leads to high petite formation but the accumulation of mitochondrial products seems normal. Growths of these overexpressing strains were also evaluated (Fig. S1), and they presented different rates of impairments, particularly AIM32, which after overnight growth on galactose, resulted in its spots revealing few viable cells. Aim32p is involved in redox quality control, its deletion results in an increased accumulation of proteins with aberrant disulfide linkages (
      • Zhang D.
      • Dailey O.R.
      • Simon D.J.
      • Roca-Datzer K.
      • Jami-Alahmadi Y.
      • Hennen M.S.
      • et al.
      Aim32 is a dual-localized 2Fe-2S mitochondrial protein that functions in redox quality control.
      ). Overexpression of SMT1(MRX5) and MRX9 also resulted in slower growth on galactose and ethanol–glycerol media (Fig. S1).
      Figure thumbnail gr1
      Figure 1Overexpression of MIOREX components and analyses of mitochondrial translation properties. A and B, newly synthesized mitochondrial translation products were analyzed from the indicated strains as described in the section. The radiolabeled bands corresponding to the mitochondrial gene products are marked in the margins as indicated: subunits 1 (Cox1), subunit 2 (Cox2), subunit 3 (Cox3) of cytochrome c oxidase; subunit 6 (Atp6), subunit 8 (Atp8), and subunit 9 (Atp9) of ATP synthase; cytochrome b subunit (Cyt b) of ubiquinol cytochrome c reductase; the mitoribosome Var1 protein. Membranes were also probed against antiporin antibodies for loading control. The number of petites that arose in the experiments is indicated in the bottom of the panels.
      We also tested the overexpression of some characterized components of the mitochondrial translation machinery to evaluate its effects on the output of mitochondrial products or even its toxicity to the mitochondrial translation. Except for CBS2, the overexpression of the tested genes here did not significantly alter the profile of mitochondrial products. Overexpression of COB translation activators CBS1 and CBS2 (
      • Rödel G.
      Two yeast nuclear genes, CBS1 and CBS2, are required for translation of mitochondrial transcripts bearing the 5'-untranslated COB leader.
      ) resulted in different outcomes (Fig. 1B), while CBS1 excess did not significantly change the profile of mitochondrial newly synthesized products, CBS2 excess resulted in the higher petite formation and lower accumulation of newly synthesized Cox1p. It was reported before that Cbs2p forms a complex with Cox1p translation activator Pet309 (
      • Krause K.
      • Lopes de Souza R.
      • Roberts D.G.
      • Dieckmann C.L.
      The mitochondrial message-specific mRNA protectors Cbp1 and Pet309 are associated in a high-molecular weight complex.
      ), therefore extra Cbs2p may sequester Pet309 away from COX1 mRNA processing and translation activation or interfere in Pet309p association with the DEAD-box helicase Mss116p (
      • Manthey G.M.
      • McEwen J.E.
      The product of the nuclear gene PET309 is required for translation of mature mRNA and stability or production of intron-containing RNAs derived from the mitochondrial COX1 locus of Saccharomyces cerevisiae.
      ,
      • De Silva D.
      • Poliquin S.
      • Zeng R.
      • Zamudio-Ochoa A.
      • Marrero N.
      • Perez-Martinez X.
      • et al.
      The DEAD-box helicase Mss116 plays distinct roles in mitochondrial ribogenesis and mRNA-specific translation.
      ). Cbs1p participates in the loop control of Cob assembly and translation (
      • Salvatori R.
      • Kehrein K.
      • Singh A.P.
      • Aftab W.
      • Möller-Hergt B.V.
      • Forne I.
      • et al.
      Molecular wiring of a mitochondrial translational feedback loop.
      ). AEP1 overexpression, but not AEP2, resulted in a significant increment in petite production probably due to its impact on ATP9 expression, although levels of Atp9p seem normal (Fig. 1C) (
      • Payne M.J.
      • Finnegan P.M.
      • Smooker P.M.
      • Lukins H.B.
      Characterization of a second nuclear gene, AEP1, required for expression of the mitochondrial OLI1 gene in Saccharomyces cerevisiae.
      ). On the other hand, overexpression of PET54, PET122, and PET494 (
      • McMullin T.W.
      • Fox T.D.
      COX3 mRNA-specific translational activator proteins are associated with the inner mitochondrial membrane in Saccharomyces cerevisiae.
      ) did not alter COX3 translation. Pet54p is also involved in COX1 intron processing and translation (
      • Mayorga J.P.
      • Camacho-Villasana Y.
      • Shingú-Vázquez M.
      • García-Villegas R.
      • Zamudio-Ochoa A.
      • García-Guerrero A.E.
      • et al.
      A novel function of Pet54 in regulation of Cox1 synthesis in Saccharomyces cerevisiae mitochondria.
      ,
      • Valencik M.L.
      • Kloeckener-Gruissem B.
      • Poyton R.O.
      • McEwen J.E.
      Disruption of the yeast nuclear PET54 gene blocks excision of mitochondrial intron aI5 beta from pre-mRNA for cytochrome c oxidase subunit I.
      ). Atp22p excess, the ATP6 activator (
      • Zeng X.
      • Hourset A.
      • Tzagoloff A.
      The Saccharomyces cerevisiae ATP22 gene codes for the mitochondrial ATPase subunit 6-specific translation factor.
      ), resulted in a small elevation of petite formation.
      MRX9 called our attention due to a diminishment of Cox1p (Fig. 1A) product accompanied by the appearance of an additional aberrant product. The appearance of novel mitochondrial products has been reported, the one named mp15, for instance, is found in COX1 defective mutants in the absence of Mss51p and its expression depends on COX1 intron composition (
      • Barrientos A.
      • Zambrano A.
      • Tzagoloff A.
      Mss51p and Cox14p jointly regulate mitochondrial Cox1p expression in Saccharomyces cerevisiae.
      ,
      • Zambrano A.
      • Fontanesi F.
      • Solans A.
      • de Oliveira R.L.
      • Fox T.D.
      • Tzagoloff A.
      • et al.
      Aberrant translation of cytochrome c oxidase subunit 1 mRNA species in the absence of Mss51p in the yeast Saccharomyces cerevisiae.
      ).

      Characterization of mrx9 null mutant and MRX9 overexpression

      Albeit MRX9 overexpression results in a lower accumulation of newly synthesized COX1, its deletion did not result in any growth deficiency on nonfermentable carbon sources media or changes in the mitochondrial translation properties in comparison to the WT cells (Fig. 2, A and B).
      Figure thumbnail gr2
      Figure 2MRX9 mutants phenotypes. A, growth properties of mrx9Δ mutant spotted on rich glucose (YPD) and rich nonfermentable ethanol–glycerol (YPEG) media; plates were photographed after 2 days of growth. B, mitochondrial newly synthesized products from the WT and mrx9Δ mutant were identified as in . C, steady-state level of the indicated mitochondrial proteins in the WT were compared to the strain overexpressing MRX9 (+MRX9) and the mrx9Δ null mutant, Coq3p and Coq4p are components of the CoQ synthome (
      • Tzagoloff A.
      • Akai A.
      • Needleman R.B.
      Assembly of the mitochondrial membrane system. Characterization of nuclear mutants of Saccharomyces cerevisiae with defects in mitochondrial ATPase and respiratory enzymes.
      ), Cob and Rip1p of the respiratory complex III (CIII), Cox1p and Cox2p—complex IV (CIV), Atp6—complex V (CV), uL24 is a component of the 54S subunit of the mitoribosome (LSU), Prx1 is a soluble mitochondrial protein, and Porin is a component of the mitochondrial outer membrane. Bands intensities were evaluated and compared using the histogram for pixels densitometry of the Adobe Photoshop program. D, enzymatic activity of the ubiquinol cytochrome c reductase (CIII) measured by the rate of cytochrome c reduction, and the activity of the cytochrome c oxidase (CIV) measured by the rate of reduced cytochrome c oxidation. Data points were obtained from three independent experiments ∗p< 0.05 versus WT.
      Considering the observed deficit in the accumulation of newly synthesized COX1 under MRX9 excess condition, we evaluated whether MRX9 overexpression or mrx9 deletion would impact the steady-state level of Cox1p and other mitochondrial proteins (Fig. 2C). The mrx9 deletion did not change the rate of accumulation of newly synthesized Cox1p (Fig. 2B), and its steady-state levels was roughly equal to that seen in the WT mitochondria. In contrast, the MRX9 overexpression resulted in three times less Cox1p than the WT; Cox2p level was also diminished to a similar rate; all the other tested mitochondrial proteins did not show any significant change in the tested strains (Fig. 2C). Accordingly, the enzymatic activity of cytochrome c oxidase was diminished in the mitochondria isolated from cells overexpressing MRX9, while the NADH ubiquinol cytochrome c reductase activity elevated in the null mutant (Fig. 2, C and D). Altogether, our data indicate that MRX9 excess alters COX biogenesis and activity.
      Paromomycin has been used to evaluate the quality of the mitochondrial translation process by inducing translational misreading and proteotoxic stress in S. cerevisiae mitochondria (
      • Suhm T.
      • Kaimal J.M.
      • Dawitz H.
      • Peselj C.
      • Masser A.E.
      • Hanzén S.
      • et al.
      Mitochondrial translation efficiency controls cytoplasmic protein homeostasis.
      ,
      • Vargas Möller-Hergt B.
      • Carlström A.
      • Suhm T.
      • Ott M.
      Insertion defects of mitochondrially encoded proteins burden the mitochondrial quality control system.
      ). The strain overexpressing MRX9 showed an increased resistance of paromomycin compared to the WT and the mrx9 null mutant strains (Fig. S2). Similar increments in paromomycin resistance were observed before with mitoribosome mutants (
      • Santos B.
      • Zeng R.
      • Jorge S.F.
      • Ferreira-Junior J.R.
      • Barrientos A.
      • Barros M.H.
      Functional analyses of mitoribosome 54S subunit devoid of mitochondria-specific protein sequences.
      ).
      We also tested whether mrx9 deletion would impact growth or Cox1p translation in the COX mutants: shy1, cox14, mss51, pet111, pet309, and pet494. COX assembly mutants usually present a destitute translation of COX1, mostly because Mss51p stays trapped by Cox14p–Coa3p complex in the Cox1p assembly module and cannot reinitiate a new translation cycle (
      • Barrientos A.
      • Zambrano A.
      • Tzagoloff A.
      Mss51p and Cox14p jointly regulate mitochondrial Cox1p expression in Saccharomyces cerevisiae.
      ,
      • Fontanesi F.
      • Clemente P.
      • Barrientos A.
      Cox25 teams up with Mss51, Ssc1, and Cox14 to regulate mitochondrial cytochrome c oxidase subunit 1 expression and assembly in Saccharomyces cerevisiae.
      ). An exception is the cox14 mutant, which is defective in COX assembly (
      • Glerum D.M.
      • Koerner T.J.
      • Tzagoloff A.
      Cloning and characterization of COX14, whose product is required for assembly of yeast cytochrome oxidase.
      ), with a high accumulation of newly synthesized Cox1p and epistatic over other mutations affecting Cox1p module assembly (
      • Barrientos A.
      • Zambrano A.
      • Tzagoloff A.
      Mss51p and Cox14p jointly regulate mitochondrial Cox1p expression in Saccharomyces cerevisiae.
      ,
      • Pierrel F.
      • Bestwick M.L.
      • Cobine P.A.
      • Khalimonchuk O.
      • Cricco J.A.
      • Winge D.R.
      Coa1 links the Mss51 post-translational function to Cox1 cofactor insertion in cytochrome c oxidase assembly.
      ). However, the cox14 null mutant is not epistatic over genes involved in COX1 5′UTR control expression (
      • Mays J.N.
      • Camacho-Villasana Y.
      • Garcia-Villegas R.
      • Perez-Martinez X.
      • Barrientos A.
      • Fontanesi F.
      The mitoribosome-specific protein mS38 is preferentially required for synthesis of cytochrome c oxidase subunits.
      ,
      • Barros M.H.
      • Myers A.M.
      • Van Driesche S.
      • Tzagoloff A.
      COX24 codes for a mitochondrial protein required for processing of the COX1 transcript.
      ,
      • Zambrano A.
      • Fontanesi F.
      • Solans A.
      • de Oliveira R.L.
      • Fox T.D.
      • Tzagoloff A.
      • et al.
      Aberrant translation of cytochrome c oxidase subunit 1 mRNA species in the absence of Mss51p in the yeast Saccharomyces cerevisiae.
      ). Here, we showed that mrx9 deletion in cox14Δ cells impacts negatively in COX1 translation, suggesting Mrx9p is involved with COX1 5′ UTR control (Fig. 3A), and the released Mss51p did not optimally perform new cycles of COX1 translation if Mrx9p is not present, even in the absence of Cox14p. The transformation of the double mutant mrx9Δcox14Δ with pG82/T1, a multicopy plasmid harboring MSS51 and COX24 (
      • Barros M.H.
      • Myers A.M.
      • Van Driesche S.
      • Tzagoloff A.
      COX24 codes for a mitochondrial protein required for processing of the COX1 transcript.
      ), did not rescue the accumulation of Cox1p to the cox14Δ single mutant level (Fig. S3).
      Figure thumbnail gr3
      Figure 3Comparison of mitochondrial protein synthesis in COX mutants harboring or not mrx9 deletion and overexpressing MRX9. A, the indicated strains containing the mrx9Δ deletion (+) and the original strains (−) were grown in rich galactose media and cells labeled for the analyses of mitochondrially encoded newly synthesized products as indicated in the methods sections. B, newly synthesized mitochondrial products from COX mutants transformants harboring the pMRX9-GAL (+) were also analyzed from cells grown in galactose-rich media The respective mitochondrial products were identified as in . Growth of mam33 mutant transformed or not with MRX9 was also compared to the WT strains. Membranes were also probed against antiporin antibodies for loading control.
      Deletion of shy1 results in respiratory growth impairment with a residual COX activity that can be rescued by an extra copy of MSS51 (
      • Mashkevich G.
      • Repetto B.
      • Glerum D.M.
      • Jin C.
      • Tzagoloff A.
      SHY1, the yeast homolog of the mammalian SURF-1 gene, encodes a mitochondrial protein required for respiration.
      ,
      • Barrientos A.
      • Korr D.
      • Tzagoloff A.
      Shy1p is necessary for full expression of mitochondrial COX1 in the yeast model of Leigh's syndrome.
      ); pet111 and pet494 were included as control of defective COX2 and COX3 translation activation (
      • Fox T.D.
      Mitochondrial protein synthesis, import, and assembly.
      ,
      • Zeng X.
      • Hourset A.
      • Tzagoloff A.
      The Saccharomyces cerevisiae ATP22 gene codes for the mitochondrial ATPase subunit 6-specific translation factor.
      ). The mrx9 deletion in the shy1 null mutant worsens its slow growth on respiratory selective media, which can only be observed after 7 days of incubation (Fig. S4).
      MRX9 overexpression was also tested in mss51, pet309, cox14, mam33, and cox24 null mutants, which except for cox14 and mam33 are defective in COX1 translation in respiratory conditions. In these transformed mutants, MRX9 excess was toxic to mitochondrial translation in the cox24Δ background (Fig. 3B). The toxicity was not enough to cause an exacerbated mtDNA loss, and the transformed mutants were still able to spontaneously revert the respiratory deficient phenotype, similarly to the untransformed cox24Δ mutant (
      • Barros M.H.
      • Myers A.M.
      • Van Driesche S.
      • Tzagoloff A.
      COX24 codes for a mitochondrial protein required for processing of the COX1 transcript.
      ). In the mam33 mutant, MRX9 overexpression leads to destitute COX1 translation reflecting in the absence of any noticeable growth on ethanol–glycerol media. MAM33 was firstly characterized as required for COX1 translation and adaptation to the respiratory metabolism (
      • Roloff G.A.
      • Henry M.F.
      Mam33 promotes cytochrome c oxidase subunit I translation in Saccharomyces cerevisiae mitochondria.
      ). Therefore, it seems that MRX9 excess is still toxic to cells devoid either of Cox24p or Mam33p. Absence or excess of Mrx9p did not alter Cox24p endogenous level (Fig. S5). In these assays we observed again that extra MRX9 intensified the accumulation of an uncommon peptide in the mss51 mutant overexpressing MRX9; the higher accumulation of this peptide occurs independently of Mss51p presence, but in the cox14Δ, or pet309Δ backgrounds, Mrx9p excess did not lead to its synthesis. A rational explanation for that are the increment of free Mss51p in cox14Δ cells and the poor stability of COX1 transcripts in pet309Δ (
      • Manthey G.M.
      • McEwen J.E.
      The product of the nuclear gene PET309 is required for translation of mature mRNA and stability or production of intron-containing RNAs derived from the mitochondrial COX1 locus of Saccharomyces cerevisiae.
      ). Similar accumulation properties of mp15, an uncommon peptide, was described before in mss51 mutant cells (
      • Zambrano A.
      • Fontanesi F.
      • Solans A.
      • de Oliveira R.L.
      • Fox T.D.
      • Tzagoloff A.
      • et al.
      Aberrant translation of cytochrome c oxidase subunit 1 mRNA species in the absence of Mss51p in the yeast Saccharomyces cerevisiae.
      ), which strengthens the hypothesis that the aberrant peptide detected in the presence of GAL-MRX9 overexpression and mp15 have the same nature.
      We also evaluate whether the mrx9 deletion would have an additive effect on mitoribosome mutants. Recently, we described new LSU mitoribosome mutants, some of them with poor growth on respiratory media (
      • Santos B.
      • Zeng R.
      • Jorge S.F.
      • Ferreira-Junior J.R.
      • Barrientos A.
      • Barros M.H.
      Functional analyses of mitoribosome 54S subunit devoid of mitochondria-specific protein sequences.
      ); considering the proximity of Mrx9p with the LSU tunnel exit components, particularly uL23, mL57, and uL29 (
      • Singh A.P.
      • Salvatori R.
      • Aftab W.
      • Aufschnaiter A.
      • Carlström A.
      • Forne I.
      • et al.
      Molecular connectivity of mitochondrial gene expression and OXPHOS biogenesis.
      ), we hypothesized that Mrx9p may play a role in the mitoribosome biogenesis. If this was the case, then its removal should have an additive effect over the slow respiratory growth of uL16, uL23, or uL29 mutants (
      • Santos B.
      • Zeng R.
      • Jorge S.F.
      • Ferreira-Junior J.R.
      • Barrientos A.
      • Barros M.H.
      Functional analyses of mitoribosome 54S subunit devoid of mitochondria-specific protein sequences.
      ); likewise the aforementioned phenotype of shy1 mrx9 double mutant, mrx9 removal marginally worsens the respiratory growth of the uL23 mutant (Fig. S6).

      MRX9 overexpression delays the adaptation to the respiratory metabolism

      S. cerevisiae is a Crabtree-positive cell that presents repression of aerobic catabolism in the presence of glucose (
      • De Deken R.H.
      The crabtree effect: a regulatory system in yeast.
      ). When the glucose is exhausted from the media, the yeast cells start to transit from the fermentative metabolism to the respiratory metabolism with drastic changes in the expression of tricarboxylic acid cycle enzymes and components of the mitochondrial electron transport chain (
      • DeRisi J.L.
      • Iyer V.R.
      • Brown P.O.
      Exploring the metabolic and genetic control of gene expression on a genomic scale.
      ). It was recently demonstrated that mitoribosomal protein synthesis occurs at high levels during glucose fermentation, which depends on bL27 mitochondria-specific C-terminal extension and its binding partner Mam33p (
      • Anderson J.M.
      • Box J.M.
      • Stuart R.A.
      The mitospecific domain of Mrp7 (bL27) supports mitochondrial translation during fermentation and is required for effective adaptation to respiration.
      ), as mentioned before; it is also required for COX1 translation and adaptation to the diauxic shift (
      • Roloff G.A.
      • Henry M.F.
      Mam33 promotes cytochrome c oxidase subunit I translation in Saccharomyces cerevisiae mitochondria.
      ). As it was already described for mam33 mutants, we investigated whether, besides Cox1p synthesis deficiency, the overexpression of Mrx9p would also impact the transition from fermentative to respiratory metabolism.
      To a better accompaniment of Mrx9p levels, a hemagglutinin (HA)-tagged version of MRX9 was cloned under the control of different promoters not repressed by glucose and its expression was compared. Curiously, the steady-state level of the Mrx9p-HA product was higher when expressed under the control of the endogenous promoter in comparison to the version under the control of the alcohol dehydrogenase ADH promoter, which was previously shown to be stronger than a weak version of the cytochrome c gene, CYC1, promoter (
      • Mumberg D.
      • Müller R.
      • Funk M.
      Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds.
      ). The MRX9-HA under the control of the translation elongation factor 1α (TEF1) promoter and the glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter resulted in higher contents of MRX9-HA. In cells grown in galactose media, the transformant harboring the GAL10-MRX9 fusion accumulate more Mrx9-HA than the GPD-MRX9 or TEF-MRX9 constructs (Fig. 4A).
      Figure thumbnail gr4
      Figure 4Evaluation of MRX9 overexpression and adaptation to the respiratory metabolism. A, Mrx9p-HA content obtained from mitochondria isolated from the indicated strains grown in galactose media. Mitochondrial protein extracts were separated on SDS-PAGE for membrane transfer and immunodecoration with anti-HA. Ponceau staining is shown below. B, growth properties of the indicated strains spotted on rich glucose (YPD) and rich nonfermentable ethanol–glycerol (EG) media from cells at exponential fermentative growth. The EG plates were photographed after 1 day and 2 days as indicated; the YPD plate was photographed after 2 days of growth. C, mitochondrial newly synthesized products of the indicated strains were labeled from glucose-grown cells at exponential phase. The indicated products on the left are the same described in . D, growth of GPD-MRX9 transformants were also assessed from culture cells pregrown on glucose or galactose and spotted on YPD and YPEG at exponential logarithmic phase. Plates were photographed after 1 day at 30 °C. E, mitochondrial translation properties from glucose or galactose grown cells as indicated. The signals “−” and “+” indicate the presence of the correspondent plasmid in the strain. The respective mitochondrial products were identified as in .
      The differential expression of MRX9-HA under these different promoters allowed the evaluation of the adaptation of the transformants to the diauxic shift and the respiratory metabolism. In this experiment, cells were grown to Abs ∼1,0 at 600 nm and then spotted on glucose and ethanol–glycerol rich media. The faster the adaptation to the respiratory metabolism, the faster they will grow on ethanol–glycerol. After 1 day of growth, we were able to see that the cells expressing the GPD-MRX9-HA construct have a clear delay in their respiratory growth, which was also confirmed by the analyses of the growth curve of this transformed strain in ethanol–glycerol liquid media (Fig. 4B). A slightly lower growth rate was also noticed in cells expressing the TEF-MRX9-HA construct. The mrx9 deletion, however, did not significantly change the adaptation to the respiratory media (Fig. 4B). We also tested whether the GPD-MRX9 transformed cells have delayed adaptation to the respiratory metabolism rather than a respiratory deficiency itself; therefore, we compared growth on ethanol–glycerol media from fermentative cells pregrown on glucose and spotted at their logarithmic growth versus cells already adapted to the respiratory metabolism after pregrowth on galactose. When pregrown in galactose, cells harboring the GPD-MRX9 construct show similar growth on ethanol–glycerol media in comparison to untransformed cells (Fig. 4D).
      MRX9-HA expression was also evaluated in cells at logarithmic growth in glucose, that is, fermentative condition and in ethanol–glycerol media. In these conditions, the TEF-MRX9-HA and GPD-MRX9-HA transformants showed a high yield of Mrx9p-HA, and its unprocessed version is also accumulated in both growth conditions. In glucose-grown cells, the unprocessed Mrx9-HAp signal is stronger, probably due to a mitochondrial import limitation present in glucose fermentative growth conditions. Accordingly, the Mrx9p-HA level with the endogenous promoter was three times higher in respiratory conditions, while the porin level was roughly 20 times stronger in cells growing in the respiratory condition (Fig. S7), indicating that MRX9 endogenous expression is less repressed in glucose than porin and other mitochondrial proteins (
      • DeRisi J.L.
      • Iyer V.R.
      • Brown P.O.
      Exploring the metabolic and genetic control of gene expression on a genomic scale.
      ).
      The delay in the adaptation to the respiratory metabolism caused by MRX9 overexpression was also confirmed when we assessed newly synthesized mitochondrial gene products in glucose fermentative cells at exponential growth (Fig. 4C). Under this condition, cells expressing the GPD-MRX9-HA fusion resulted in a poor accumulation of mitochondrial products, while as for the cells harboring the TEF-MRX9-HA fusion, the mitochondrial translation is also diminished. The deletion of mrx9 did not significantly alter the accumulation of mitochondrial products in comparison to the WT cells (Fig. 4C).
      Considering the deleterious effect of MRX9 excess on COX1 translation, we tried to circumvent it by overexpressing PET309 in a multicopy plasmid and the couple MSS51– COX24, initially cloned in the same multicopy plasmid pG82/T1 (
      • Barros M.H.
      • Myers A.M.
      • Van Driesche S.
      • Tzagoloff A.
      COX24 codes for a mitochondrial protein required for processing of the COX1 transcript.
      ). Neither PET309 nor MSS51/COX24 multicopy plasmids were able to ameliorate the growth rate of GPD-MRX9 transformants on ethanol–glycerol when pregrown culture was conducted in glucose (Fig. 4D). Analyses of newly synthesized mitochondrial product of these transformants strains, in the same growth conditions described previously, indicate that in fermenting cells COX1 synthesis is the most reduced product when GPD-MRX9 construct is present. Again, extra PET309 or MSS51/COX24 did not significantly change the translation properties caused by Mrx9p excess (Fig. 4E). Probably because of the level of MRX9 expression, in galactose grown cells the GPD-MRX9 construct was less toxic to COX1 translation (Fig. 4E), in agreement with the presented growth results (Fig. 4D). Altogether, these results strongly support the idea that the deficit in COX1 translation caused by Mrx9p excess retards the adaptation to the respiratory metabolism.
      Finally, we compared the effects of the GPD-MRX9 overexpression in the mam33Δ background, under fermentative and respiratory conditions. As previously reported, Mam33p is required for proper Cox1p translation in fermentative conditions (
      • Roloff G.A.
      • Henry M.F.
      Mam33 promotes cytochrome c oxidase subunit I translation in Saccharomyces cerevisiae mitochondria.
      ); the MRX9 excess did not change the accumulation of newly synthesized mitochondrial products in comparison with the untransformed mam33Δ, except that in respiratory conditions the uncommon peptide between Atp6p and Atp8p products were once again accumulated with the presence of extra Mrx9p. Curiously, the accumulation of newly synthesized Cob and Atp9p seems augmented in the double mutant mam33Δ mrx9Δ, the effect on Atp9p is difficult to interpret due to its electrophoretic properties, but it seems that Mrx9p also regulates Cob translation (Fig. S8).

      Mrx9p intramitochondrial localization and distribution

      To gain a better topology localization of the Mrx9p protein, we followed the Mrx9p-HA solubilization properties after sonication with or without carbonate, as well its distribution in mitochondria and mitoplasts subjected to proteinase K digestion. Mrx9p is a hydrophobic inner membrane protein facing the matrix compartment that is recovered in the membrane fraction following disruption of mitochondria by sonic irradiation in the presence of carbonate (Fig. 5A) and resistant to proteinase K in mitochondria and in mitoplasts, the latter lacking an intact outer membrane (Fig. 5B). Conversion of mitochondria to mitoplasts by hypotonic shock was confirmed from the substantial loss of the soluble intermembrane marker cytochrome b2 and supported by the decrease of Sco1p as a result of the proteinase K treatment of mitoplasts but not mitochondria. Sco1p is an inner membrane protein facing the intermembrane space (
      • Glerum D.M.
      • Shtanko A.
      • Tzagoloff A.
      SCO1 and SCO2 act as high copy suppressors of a mitochondrial copper recruitment defect in Saccharomyces cerevisiae.
      ). The matrix protein α-ketoglutarate dehydrogenase (
      • Repetto B.
      • Tzagoloff A.
      Structure and regulation of KGD2, the structural gene for yeast dihydrolipoyl transsuccinylase.
      ) was protected against proteinase K in mitochondria and mitoplasts confirming the intactness of the inner membrane in the mitoplasts.
      Figure thumbnail gr5
      Figure 5Mrx9p is in the mitochondrial inner membrane facing the matrix side. A, mitochondrial proteins isolated from cells expressing the Mrx9p-HA constructs (Mit) were sonicated and centrifuged, obtaining supernatant (S) and pellet (SMP). The pellet was suspended in carbonate salt and centrifuged again, with a new super (CS) and pellet (CP). The distribution of Mrx9p-HA in the different fractions was assessed using an anti-HA antibody; the solubilization was also checked using an antibody against the matrix soluble Kgd2p and the inner membrane protein Sco1p. B, mitochondria containing the Mrx9-HA (Mit) were osmotically challenged with Hepes buffer generating mitoplasts devoid of the outer membrane and the intermembrane compartment (Mp). Mitochondria and mitoplasts were digested (+) or not (−) with proteinase K to test the availability of the indicated tested proteins to digestion.
      Mrx9p is a component of the MIOREX complex (
      • Kehrein K.
      • Schilling R.
      • Möller-Hergt B.V.
      • Wurm C.A.
      • Jakobs S.
      • Lamkemeyer T.
      • et al.
      Organization of mitochondrial gene expression in two distinct ribosome-containing assemblies.
      ); it cosediments with the small subunit of the mitoribosome (SSU) (
      • Möller-Hergt B.V.
      • Carlström A.
      • Stephan K.
      • Imhof A.
      • Ott M.
      The ribosome receptors Mrx15 and Mba1 jointly organize cotranslational insertion and protein biogenesis in mitochondria.
      ), and it is also close to the polypeptide tunnel exit of the mitoribosome LSU in proximity-dependent biotinylation studies (
      • Singh A.P.
      • Salvatori R.
      • Aftab W.
      • Aufschnaiter A.
      • Carlström A.
      • Forne I.
      • et al.
      Molecular connectivity of mitochondrial gene expression and OXPHOS biogenesis.
      ). Here, we evaluate the distribution of Mrx9-HAp in the WT strain and also assessed the mitoribosome assembly status in the null mutant mrx9Δ and in the strain overexpressing MRX9-HA under the control of the GAL10 promoter. Mitochondria were isolated and mitoribosomes extracted in the presence of 0.5 mM MgCl2 (
      • De Silva D.
      • Fontanesi F.
      • Barrientos A.
      The DEAD box protein Mrh4 functions in the assembly of the mitochondrial large ribosomal subunit.
      ) either using Triton, a stringent detergent, or digitonin, a mild detergent. The extracts were subjected to sedimentation in a sucrose gradient as previously described (
      • Guedes-Monteiro R.F.
      • Ferreira-Junior J.R.
      • Bleicher L.
      • Nóbrega F.G.
      • Barrientos A.
      • Barros M.H.
      Mitochondrial ribosome bL34 mutants present diminished translation of cytochrome c oxidase subunits.
      ). We used the bL31 antibody to follow the sedimentation of the 54S subunit and anti-mS37 to follow the 37S subunit (Fig. 6).
      Figure thumbnail gr6
      Figure 6The Mrx9-HA sedimentation pattern and the assembly of the mitoribosome 54S and 37S subunits were assessed from the strain expressing MRX9-HA construct with its endogenous promoter (WT/MRX9-HA) and with the GAL10 promoter (+GAL/MRX9-HA) and in the mrx9 null mutant (mrx9Δ). Mitochondrial proteins were obtained from galactose grown cells and extracted in the presence of 0.5 mM MgCl2 and the indicated detergent (0.8% Triton – A, 0.8% digitonin B). The extracts were sedimented in a linear sucrose gradient (1M to 0.3 M as represented by the gray bar). Fractions were collected from the bottom (
      • Ott M.
      • Amunts A.
      • Brown A.
      Organization and regulation of mitochondrial protein synthesis.
      ) to top (
      • Mays J.N.
      • Camacho-Villasana Y.
      • Garcia-Villegas R.
      • Perez-Martinez X.
      • Barrientos A.
      • Fontanesi F.
      The mitoribosome-specific protein mS38 is preferentially required for synthesis of cytochrome c oxidase subunits.
      ). The Western blots were assayed for anti-HA, anti-bL31 (54S component), anti-mS37 (37S component), anti-Mss51p, and anti-Prx1p (soluble—sedimentation control). Prx1p is shown in two bands U (unprocessed) and P (processed), which are dependent on Oct1p/Imp processing state (
      • Kaur J.
      • Stuart R.A.
      Truncation of the Mrp20 protein reveals new ribosome-assembly subcomplex in mitochondria.
      ). The structure of the mitoribosome (74S), its large (54S), and small subunit (37S) are represented on the bottom of panels (A and B), based on the sedimentation properties of bL31 and mS37.
      In the tested strains (WT+ MRX9HA, mrx9Δ, WT+MRX9HA-GAL10), bL31 sediments at fractions 1 and 2 at the bottom of the sucrose gradient, fraction 1 is probably enriched with the assembled mitoribosome and fraction 2 with the 54S subunit (Fig. 6). The mS37, and therefore 37S subunit, is enriched in fractions 3, 4, and 5 in all tested strains. In both cases, the sedimentation properties of bL31 and mS37 did not change significantly in Triton or digitonin conditions (Fig. 6B). On the contrary, for Mrx9-HA, the two detergents lead to distinct outcomes; with Triton, the Mrx9-HA distribution peaks in the lighter fractions 10 and 11 (Fig. 6A); with digitonin Mrx9-HA, it was spread from fractions 1 to 10 (Fig. 6B). These results indicate that Mrx9p is associated with the mitoribosome subunits as previously suggested (
      • Kehrein K.
      • Schilling R.
      • Möller-Hergt B.V.
      • Wurm C.A.
      • Jakobs S.
      • Lamkemeyer T.
      • et al.
      Organization of mitochondrial gene expression in two distinct ribosome-containing assemblies.
      ,
      • Möller-Hergt B.V.
      • Carlström A.
      • Stephan K.
      • Imhof A.
      • Ott M.
      The ribosome receptors Mrx15 and Mba1 jointly organize cotranslational insertion and protein biogenesis in mitochondria.
      ,
      • Singh A.P.
      • Salvatori R.
      • Aftab W.
      • Aufschnaiter A.
      • Carlström A.
      • Forne I.
      • et al.
      Molecular connectivity of mitochondrial gene expression and OXPHOS biogenesis.
      ) but its association is lost in stringent extraction conditions. In the strain overexpressing MRX9-HA using digitonin, the Mrx9-HA product is also found in heavier fractions but it peaks in fraction 7 (Fig. 6B).
      As shown before (Figs. 1 and 3), yeast cells overexpressing MRX9 presented a deficiency in COX1 translation and the exacerbated accumulation of an aberrant translation product that resembles the reported mp15 (
      • Zambrano A.
      • Fontanesi F.
      • Solans A.
      • de Oliveira R.L.
      • Fox T.D.
      • Tzagoloff A.
      • et al.
      Aberrant translation of cytochrome c oxidase subunit 1 mRNA species in the absence of Mss51p in the yeast Saccharomyces cerevisiae.
      ). Therefore, we hypothesized that if mp15 and the aforementioned product are the same, then MRX9 excess is affecting Mss51p accessibility for translation activation and elongation control (
      • Perez-Martinez X.
      • Broadley S.A.
      • Fox T.D.
      Mss51p promotes mitochondrial Cox1p synthesis and interacts with newly synthesized Cox1p.
      ). Although Mss51p was not found in the MIOREX complexes (
      • Ott M.
      • Amunts A.
      • Brown A.
      Organization and regulation of mitochondrial protein synthesis.
      ,
      • Kehrein K.
      • Schilling R.
      • Möller-Hergt B.V.
      • Wurm C.A.
      • Jakobs S.
      • Lamkemeyer T.
      • et al.
      Organization of mitochondrial gene expression in two distinct ribosome-containing assemblies.
      ), its association to the mitoribosome has been reported in some special high salt conditions (
      • Singh A.P.
      • Salvatori R.
      • Aftab W.
      • Aufschnaiter A.
      • Carlström A.
      • Forne I.
      • et al.
      Molecular connectivity of mitochondrial gene expression and OXPHOS biogenesis.
      ). Here, we investigated Mss51p distribution in the tested sucrose gradients; with the WT levels of Mrx9-HA, Mss51p is found in heavier fractions (f1 and f2) coincident to the distribution of the mitoribosome subunit bL31 and to its sedimentation in another study (
      • Mays J.N.
      • Camacho-Villasana Y.
      • Garcia-Villegas R.
      • Perez-Martinez X.
      • Barrientos A.
      • Fontanesi F.
      The mitoribosome-specific protein mS38 is preferentially required for synthesis of cytochrome c oxidase subunits.
      ). Two other peaks of Mss51p distribution were also detected: one in fraction 9 and the second in fraction 12 (Fig. 6B). The fraction 9 peak may correspond to the 420 KDa Cox1 module assembly (
      • Fontanesi F.
      • Soto I.C.
      • Horn D.
      • Barrientos A.
      Mss51 and Ssc1 facilitate translational regulation of cytochrome c oxidase biogenesis.
      ) and the second, in fraction 12, to the reported 120 KDa complex hypothesized to be responsible for, or source of, Mss51p function on COX1 translation (
      • Pierrel F.
      • Bestwick M.L.
      • Cobine P.A.
      • Khalimonchuk O.
      • Cricco J.A.
      • Winge D.R.
      Coa1 links the Mss51 post-translational function to Cox1 cofactor insertion in cytochrome c oxidase assembly.
      ,
      • Fontanesi F.
      • Soto I.C.
      • Horn D.
      • Barrientos A.
      Mss51 and Ssc1 facilitate translational regulation of cytochrome c oxidase biogenesis.
      ).
      In mrx9Δ mitochondria digitonin extraction, Mss51p is no longer found together with bL31; it shows a peak again in fraction 9 and a new peak in a lighter fraction (f12-f13). Finally, when MRX9-HA is overexpressed, Mss51p is mostly found in the lighter complex, and a small amount peaks in fraction 7, which is coincident with Mrx9p-HA distribution. Indeed, biotin proximity studies suggested an interaction of Mss51p with Mrx9p (
      • Singh A.P.
      • Salvatori R.
      • Aftab W.
      • Aufschnaiter A.
      • Carlström A.
      • Forne I.
      • et al.
      Molecular connectivity of mitochondrial gene expression and OXPHOS biogenesis.
      ). The Mss51p concentrated in the lighter fraction can be interpreted as an accumulation of translationally active Mss51p complex hampered to proceed in new COX1 translation cycles by the Mrx9p excess. In pulled-down experiments, we were unable to detect a copurification of a tagged version of Mrx9 and Mss51p (not shown).
      Anti-Prx1 was used as a sedimentation control of lighter fractions; Prx1p is a soluble mitochondrial protein, subjected to Oct1p and Imp2p processing, present in the matrix and in the intermembrane space (
      • Gomes F.
      • Palma F.R.
      • Barros M.H.
      • Tsuchida E.T.
      • Turano H.G.
      • Alegria T.G.P.
      • et al.
      Proteolytic cleavage by the inner membrane peptidase (IMP) complex or Oct1 peptidase controls the localization of the yeast peroxiredoxin Prx1 to distinct mitochondrial compartments.
      ), and an association of Prx1p with mitoribosome subcomplexes has been reported (
      • Kaur J.
      • Stuart R.A.
      Truncation of the Mrp20 protein reveals new ribosome-assembly subcomplex in mitochondria.
      ). In the mrx9Δ strain gradient, some Prx1p is also found spread in the bottom fractions, indicating a possible interaction with such subcomplexes in these cells. It was proposed that Prx1p might prevent oxidative damage during the assembly of ribosomal subcomplexes (
      • Kaur J.
      • Stuart R.A.
      Truncation of the Mrp20 protein reveals new ribosome-assembly subcomplex in mitochondria.
      ). The major Prx1p band in the panels can be interpreted as the Prx1p processed by Oct1p (
      • Gomes F.
      • Palma F.R.
      • Barros M.H.
      • Tsuchida E.T.
      • Turano H.G.
      • Alegria T.G.P.
      • et al.
      Proteolytic cleavage by the inner membrane peptidase (IMP) complex or Oct1 peptidase controls the localization of the yeast peroxiredoxin Prx1 to distinct mitochondrial compartments.
      ), the lighter upper band as the unprocessed product. In some panels, a smaller Prx1p band is also detected and it has been interpreted as a degradation product.

      MRX9 excess in mtDNA intronless strains

      The mitochondrial COX1 gene is transcribed as a polycistronic precursor RNA containing COX1 with variable but multiple introns (
      • Manon S.
      • Guerin M.
      Modifications of the relative proteolipid composition in the ATP synthase of a respiratory competent mutant of Saccharomyces cerevisiae.
      ,
      • Bonitz S.G.
      • Coruzzi G.
      • Thalenfeld B.E.
      • Tzagoloff A.
      • Macino G.
      Assembly of the mitochondrial membrane system. Physical map of the Oxi3 locus of yeast mitochondrial DNA.
      ). Considering that the uncommon mp15 peptide synthesis depends on the constitution of COX1 introns (
      • Zambrano A.
      • Fontanesi F.
      • Solans A.
      • de Oliveira R.L.
      • Fox T.D.
      • Tzagoloff A.
      • et al.
      Aberrant translation of cytochrome c oxidase subunit 1 mRNA species in the absence of Mss51p in the yeast Saccharomyces cerevisiae.
      ), we tested how Mrx9p excess would affect mitochondrial translation in mtDNA intronless strain. Contrary to our previous results, in the intronless background, overexpression of MRX9 did not result in impairments in COX1 translation or a diauxic shift delay. Cells expressing GPD-MRX9 fusion were grown on glucose and spotted at the exponential growth phase on respiratory ethanol–glycerol media, forcing the respiratory adaptation. While in the mtDNA intronless background, the overexpression of MRX9 did not cause any growth delay; in the strain harboring introns, a slow growth is observed, confirming previous results (Fig. 7A). The Mrx9p-HA levels derived from the GPD-MRX9 fusion were similar independently of the intron presence or media composition (Fig. S9). Moreover, in galactose grown cells transformed with the GAL-MRX9 construct, the accumulation of newly synthesized mitochondrial products is not affected in the strain devoid of mitochondrial introns, but, once again, COX1 translation is impaired with Mrx9p excess in the strain harboring introns (Fig. 7B). Mitochondrial products were also analyzed from cells grown in glucose at exponential fermentative growth; similarly to Figure 4D, the GPD-MRX9 expression resulted in a significant reduction of the mitochondrial products but not in the intronless strain (Fig. 7C). Curiously, MRX9 overexpression leads to a higher accumulation of newly synthesized Cob in the intronless strains either in fermentative cells with the GPD-MRX9 construct and in galactose grown cells with the GAL-MRX9 fusion (Fig. 7, B and C).
      Figure thumbnail gr7
      Figure 7MRX9 excess toxicity is dependent on the presence of mitochondrial introns. A, glucose-grown cells of the indicated strains were spotted on rich glucose media (YPD) and rich ethanol–glycerol media (YPEG) and photographed after 2 days at 30 °C. B and C, mitochondrial newly synthesized products were labeled from the indicated strains harboring (+) or not (-) the indicated MRX9 plasmid. Cells at exponential logarithmic growth (B in glucose, C in galactose) were incubated in the presence of methionine-cysteine 35S mixture and the mitochondrial products identified as in . D, schematic representation of the W303-1B yeast strain mitochondrial primary RNA transcripts. COB and COX1 exons are represented with capital letters and the introns with lowercases letters, double arrows positioned the probes employed in the Northern blots as follows: COX2 entire coding sequence, COB a 23 nucleotide primer positioned just after the first ATG (, (
      • Guedes-Monteiro R.F.
      • Franco L.V.R.
      • Moda B.S.
      • Tzagoloff A.
      • Barros M.H.
      5' processing of Saccharomyces cerevisiae mitochondrial tRNAs requires expression of multiple genes.
      )); COX1 exon a4 entire sequence. Scale and graphics were based on a previous review (
      • Dieckmann C.L.
      • Staples R.R.
      Regulation of mitochondrial gene expression in Saccharomyces cerevisiae.
      ). E, mitochondrial RNAs from the indicated strains were extracted and separated in a 1% agarose gels with a BsteII digest of λ phage serving as a size standard. The ethidium-bromide stained gel on the left is representative of one extraction, the base pairs sizes of the standard fragments are indicated on the right. RNAs were transferred to a nylon membrane, hybridized to 3′end biotin-conjugated probes for COB, COX1, and COX2 depicted in (D). The asterisk ∗ between 3675 and 2323 indicates a faint band of a partially processed COX1.
      Processing of the mtDNA genes harboring introns COX1 and COB were also evaluated in the mrx9 null mutant and in the strain overexpressing MRX9. Northern blots from isolated mitochondria were probed against COB, COX1, and COX2 probes. COX2 mRNA levels were similar in the tested strains, but mature COB and COX1 mRNAs were poorly accumulated and unprocessed intermediates detected in both cases (Fig. 7D). Therefore, we can conclude that MRX9 excess interferes in the processing of COX1 and COB primary transcripts and that is likely the main cause of the phenotypes previously described here. The diminishment of these transcripts can be either a direct effect of the Mrx9p excess on the mRNAs or due to a titration on splicing factors. COB and COX1 RNAs processing and translation control share some proteins in common, such as the bI4 maturase, Mss116p, Mrs1p, and the COB translation factor Cbs2p, which is also necessary for bI4 maturase function (
      • Dujon B.
      Mitochondrial genetics revisited.
      ,
      • De La Salle H.
      • Jacq C.
      • Slonimski P.P.
      Critical sequences within mitochondrial introns: pleiotropic mRNA maturase and cis-dominant signals of the box intron controlling reductase and oxidase.
      ,
      • Huang H.R.
      • Rowe C.E.
      • Mohr S.
      • Jiang Y.
      • Lambowitz A.M.
      • Perlman P.S.
      The splicing of yeast mitochondrial group I and group II introns requires a DEAD-box protein with RNA chaperone function.
      ,
      • Muroff I.
      • Tzagoloff A.
      CBP7 codes for a co-factor required in conjunction with a mitochondrial maturase for splicing of its cognate intervening sequence.
      ).
      The overexpression of MRX9 was also tested on the expression of mitochondrial ARG8m at the COX1, COX2, COB, and ATP9 loci of mtDNA in the arg8Δ mutant strain. COX2 and ATP9 are mitochondrial intronless genes. ARG8m is a version of nuclear ARG8 but modified to accommodate for the differences in the genetic code of yeast mitochondria (
      • Steele D.F.
      • Butler C.A.
      • Fox T.D.
      Expression of a recoded nuclear gene inserted into yeast mitochondrial DNA is limited by mRNA-specific translational activation.
      ) (Fig. 8). Overexpression of GAL-MRX9 in cox2Δ, and atp9Δ in minimal galactose media did not impair growth in the absence of arginine, indicating that the reporter gene ARG8m is being sufficiently produced and MRX9 excess is not toxic to its expression in galactose. However, in the study of the 5′ UTR of cox1Δ and cobΔ strains, the ARG8m expression was not sufficient to warrant optimal growth when MRX9 is overexpressed. In the glucose spots, the GAL-MRX9 construct is shut off, and the transformant strain grows as much as the originally deleted cox1Δ and cobΔ strains. These results strengthen the hypothesis that Mrx9p interferes in 5′UTR activation or processing of these two mitochondrial genes.
      Figure thumbnail gr8
      Figure 8MRX9 excess impairs proper activation of the reporter ARG8m under control of COB and COX1 5′ UTR. Mitochondrial mutants strains cox1Δ (cox1::ARG8), cox2Δ (cox2::ARG8), cobΔ (cob::ARG8), and atp9Δ (atp::ARG8) and the same strains transformed with the GAL-MRX9 fusion (+MRX9) were grown overnight in rich galactose media. Cells were spotted on minimal galactose (GAL) and minimal glucose without supplementation (GLU) and supplemented with arginine (GAL + Arg) (GLU + ARG). On the right, a depicted panel of the respective disrupted gene. Plates were photographed after 3 days of growth at 30 °C.

      Discussion

      Mitochondria OXPHOS complexes can form large structures called supercomplexes (
      • Schägger H.
      • Pfeiffer K.
      Supercomplexes in the respiratory chains of yeast and mammalian mitochondria.
      ), whose formation and stoichiometry is modulated by the metabolic state and energetic demand of the cell (
      • Cogliati S.
      • Cabrera-Alarcón J.L.
      • Enriquez J.A.
      Regulation and functional role of the electron transport chain supercomplexes.
      ). In yeast, the two observed supercomplexes consist of the dimeric bc1 complex associated with one or two cytochrome c oxidase complexes on either side of bc1, while the ATP synthase dimers and oligomers are required for cristae formation (
      • Schägger H.
      • Pfeiffer K.
      Supercomplexes in the respiratory chains of yeast and mammalian mitochondria.
      ,
      • Cogliati S.
      • Cabrera-Alarcón J.L.
      • Enriquez J.A.
      Regulation and functional role of the electron transport chain supercomplexes.
      ,
      • Paumard P.
      • Vaillier J.
      • Coulary B.
      • Schaeffer J.
      • Soubannier V.
      • Mueller D.M.
      • et al.
      The ATP synthase is involved in generating mitochondrial cristae morphology.
      ). Mitochondrial supercomplexes are the final elements of a highly ordered orchestration of events, tightly regulated, involving mtDNA gene expression, mitochondrial protein import, and OXPHOS complexes assembly. Mitochondrial translation control is critical for gene expression; it depends on nuclearly encoded imported proteins to be active. Moreover, translation activations of mitochondrial mRNAs are coupled to complexes assembly by feedback loops control (
      • Fox T.D.
      Mitochondrial protein synthesis, import, and assembly.
      ,
      • Fontanesi F.
      Mechanisms of mitochondrial translational regulation.
      ,
      • Kabala A.M.
      • Binko K.
      • Godard F.
      • Charles C.
      • Dautant A.
      • Baranowska E.
      • et al.
      Assembly-dependent translation of subunits 6 (Atp6) and 9 (Atp9) of ATP synthase in yeast mitochondria.
      ,
      • Rak M.
      • Tzagoloff A.
      F1-dependent translation of mitochondrially encoded Atp6p and Atp8p subunits of yeast ATP synthase.
      ,
      • Salvatori R.
      • Kehrein K.
      • Singh A.P.
      • Aftab W.
      • Möller-Hergt B.V.
      • Forne I.
      • et al.
      Molecular wiring of a mitochondrial translational feedback loop.
      ,
      • Seshadri S.R.
      • Banarjee C.
      • Barros M.H.
      • Fontanesi F.
      The translational activator Sov1 coordinates mitochondrial gene expression with mitoribosome biogenesis.
      ), unveiling a strong functional interplay between the two existing genomes. Moreover, intermediate OXPHOS modules can exert control in the translation of components of a different OXPHOS complex module (
      • Rak M.
      • Tzagoloff A.
      F1-dependent translation of mitochondrially encoded Atp6p and Atp8p subunits of yeast ATP synthase.
      ,
      • Franco L.V.R.
      • Su C.H.
      • Burnett J.
      • Teixeira L.S.
      • Tzagoloff A.
      Atco, a yeast mitochondrial complex of Atp9 and Cox6, is an assembly intermediate of the ATP synthase.
      ,
      • Su C.H.
      • McStay G.P.
      • Tzagoloff A.
      Assembly of the rotor component of yeast mitochondrial ATP synthase is enhanced when Atp9p is supplied by Atp9p-Cox6p complexes.
      ). In this work, we identified Mrx9p as a new component of the regulatory conundrum, ultimately required for the final stoichiometry of the OXPHOS complexes.
      Mrx9p excess toxicity is dependent on the presence of mtDNA introns; it interferes in the processing of COB and COX1 introns during the translation of the respective mRNAs (Fig. 8). Although the evolutionary significance of intervening sequences in yeast mtDNAs is still under debate (
      • Dujon B.
      Mitochondrial genetics revisited.
      ), the removal of all mitochondrial introns results in altered mitochondrial morphology, gene expression, and metabolism impacting growth and lifespan (
      • Rudan M.
      • Bou Dib P.
      • Musa M.
      • Kanunnikau M.
      • Sobočanec S.
      • Rueda D.
      • et al.
      Normal mitochondrial function in Saccharomyces cerevisiae has become dependent on inefficient splicing.
      ). Indeed, the presence of introns provides an additional step of communication between nuclear and mitochondrial genomes, in which nuclearly encoded proteins regulate the expression of mitochondrial genes. For instance, our labeling experiments showed the regulatory effect of COB introns in translation, the strains devoid of introns clearly accumulated more of the newly synthesized Cob.
      Translation of COX1 and COB transcripts, the correct inner membrane insertion of the newly synthesized polypeptides, and the association with assembly factors are dependent on the function of a multitude of proteins, with different levels of regulation, from RNA processing to protein chaperones (
      • Salvatori R.
      • Kehrein K.
      • Singh A.P.
      • Aftab W.
      • Möller-Hergt B.V.
      • Forne I.
      • et al.
      Molecular wiring of a mitochondrial translational feedback loop.
      ,
      • Nobrega F.G.
      • Tzagoloff A.
      Assembly of the mitochondrial membrane system. DNA sequence and organization of the cytochrome b gene in Saccharomyces cerevisiae D273-10B.
      ,
      • Fontanesi F.
      • Soto I.C.
      • Horn D.
      • Barrientos A.
      Assembly of mitochondrial cytochrome c-oxidase, a complicated and highly regulated cellular process.
      ). Indeed, many of these proteins affect both COX1 and COB expression, including Mrs1p, Mss116p, Cbs2p, Nam1p, Nam2p, and even the maturase encoded by bI4 (
      • Dieckmann C.L.
      • Staples R.R.
      Regulation of mitochondrial gene expression in Saccharomyces cerevisiae.
      ). Accordingly, we propose that Mrx9p also belongs to this group of factors affecting the expression of both genes.
      The overexpression experiments assumed the premise that an eventual excess of translation repressor or some sort of downregulator would result in loss of translation of a specific polypeptide. Indeed, we showed that SMT1(MRX5) overexpression resulted in the diminished translation of the ATP6/8 transcript as expected (
      • Rak M.
      • Su C.H.
      • Xu J.T.
      • Azpiroz R.
      • Singh A.M.
      • Tzagoloff A.
      Regulation of mitochondrial translation of the ATP8/ATP6 mRNA by Smt1p.
      ), while the excess of CBS2 and AEP1 were putatively toxic to the gene expression system as suggested by the elevated number of petites, confirming previous results observed for CBS2 (
      • Tzschoppe K.
      • Kohlwein S.D.
      • Rödel G.
      Yeast translational activator Cbs2p: mitochondrial targeting and effect of overexpression.
      ). Overexpression of poorly characterized MIOREX components FMP10, FMP30, and AIM19 (
      • Kehrein K.
      • Schilling R.
      • Möller-Hergt B.V.
      • Wurm C.A.
      • Jakobs S.
      • Lamkemeyer T.
      • et al.
      Organization of mitochondrial gene expression in two distinct ribosome-containing assemblies.
      ) also lead to elevated petite formation and a pleiotropic diminishment of mitochondrial products, and in these cases, the understanding of the toxicity of their gene products may help unveil their specific function. Overexpression of MRX9 was further evaluated using tagged proteins and different promoters that lead to a variable yield of the protein; clearly, Mrx9p excess in the fermentative grown cells was toxic to respiratory adaptation. Therefore, considering the regulatory effect of Mrx9p in COX1 and COB mRNAs, the presence of intervening sequences can be an evolutionary advantage for the optimal adaptation to the respiratory metabolism.
      In our working model (Fig. 9), Mrx9p facilitates the access of translation activators to COB and COX1 5′ UTR and regulates the access of splicing factors to their introns. In the WT strain growing under respiratory conditions, occurs the optimal recruitment of translation activators and the mRNAs are properly processed. Fermentative and respiratory grown cells expressing, respectively, GPD-MRX9 construct and GAL-MRX9 construct present the excess of Mrx9p interfering in the 5′UTR and in intron removal of COB and COX1. If Mrx9p, translation activators, and splicing factors are competing for the same mRNA sites, then extra Mrx9p would ultimately unbalance the proper modular assembly of cytochrome c oxidase and the bc1 complex (
      • Ndi M.
      • Marin-Buera L.
      • Salvatori R.
      • Singh A.P.
      • Ott M.
      Biogenesis of the bc1 complex of the mitochondrial respiratory chain.
      ,
      • Franco L.V.R.
      • Su C.H.
      • Tzagoloff A.
      Modular assembly of yeast mitochondrial ATP synthase and cytochrome oxidase.
      ,
      • Barros M.H.
      • McStay G.P.
      Modular biogenesis of mitochondrial respiratory complexes.
      ,
      • McStay G.P.
      • Su C.H.
      • Tzagoloff A.
      Modular assembly of yeast cytochrome oxidase.
      ), impairing respiratory adaptation (GPD-MRX9) and respiratory growth (GAL-MRX9).
      Figure thumbnail gr9
      Figure 9Model for Mrx9p function in COX1 processing and translation. In the WT, Mrx9p associated with the mitochondrial inner membrane (IMM) and the mitoribosome. COX1 mRNA starts its translation before all intervening sequences (in gray) are processed and concomitantly with the insertion of the nascent polypeptide (Cox1p) in the IMM (
      • Ott M.
      • Amunts A.
      • Brown A.
      Organization and regulation of mitochondrial protein synthesis.
      ,
      • Fox T.D.
      Mitochondrial protein synthesis, import, and assembly.
      ,
      • Möller-Hergt B.V.
      • Carlström A.
      • Stephan K.
      • Imhof A.
      • Ott M.
      The ribosome receptors Mrx15 and Mba1 jointly organize cotranslational insertion and protein biogenesis in mitochondria.
      ). Optimal Cox1p translation requires Pet309p, Mss51p, Mss116p, Mam33p, Mrx8p, and Cox24p (
      • Fox T.D.
      Mitochondrial protein synthesis, import, and assembly.
      ,
      • Barros M.H.
      • Myers A.M.
      • Van Driesche S.
      • Tzagoloff A.
      COX24 codes for a mitochondrial protein required for processing of the COX1 transcript.
      ,
      • Decoster E.
      • Simon M.
      • Hatat D.
      • Faye G.
      The MSS51 gene product is required for the translation of the COX1 mRNA in yeast mitochondria.
      ,
      • Manthey G.M.
      • McEwen J.E.
      The product of the nuclear gene PET309 is required for translation of mature mRNA and stability or production of intron-containing RNAs derived from the mitochondrial COX1 locus of Saccharomyces cerevisiae.
      ,
      • Roloff G.A.
      • Henry M.F.
      Mam33 promotes cytochrome c oxidase subunit I translation in Saccharomyces cerevisiae mitochondria.
      ,
      • Verma Y.
      • Mehra U.
      • Pandey D.K.
      • Kar J.
      • Pérez-Martinez X.
      • Jana S.S.
      • et al.
      MRX8, the conserved mitochondrial YihA GTPase family member, is required for de novo Cox1 synthesis at suboptimal temperatures in Saccharomyces cerevisiae.
      ,
      • De Silva D.
      • Poliquin S.
      • Zeng R.
      • Zamudio-Ochoa A.
      • Marrero N.
      • Perez-Martinez X.
      • et al.
      The DEAD-box helicase Mss116 plays distinct roles in mitochondrial ribogenesis and mRNA-specific translation.
      ). In galactose, expression of nuclearly encoded mitochondrial proteins is not repressed and their steady-state level elevated inside the organelle, including those involved in intron processing (
      • Dieckmann C.L.
      • Staples R.R.
      Regulation of mitochondrial gene expression in Saccharomyces cerevisiae.
      ). Some of them depicted in the panel: Mrs1p, Mss116p, Cox24p, Nam1p, Nam2p, and the mitochondrial bI4 maturase, which function depends on Cbs2p (
      • Muroff I.
      • Tzagoloff A.
      CBP7 codes for a co-factor required in conjunction with a mitochondrial maturase for splicing of its cognate intervening sequence.
      ). The accumulation of these factors intensifies the mitochondrial protein synthesis. When MRX9 is overexpressed (+++MRX9) in glucose (GPD-MRX9 construct), our data show destitute mitochondrial protein synthesis; in galactose (GAL-MRX9 construct), its excess lead to dislocation of Mss51p from the activation complex, interference in the 5′ UTR sequence and impairment in intron processing. Finally, in the null mutant (Δmrx9), Mss51p is not optimally recruited to the 5′ UTR activation complex.
      Improper adaptation to the diauxic shift together with destitute Cox1p translation in fermentative conditions was also observed in mam33Δ mutants together with the loss of optimal COX1 splicing (
      • Roloff G.A.
      • Henry M.F.
      Mam33 promotes cytochrome c oxidase subunit I translation in Saccharomyces cerevisiae mitochondria.
      ); indeed, Mam33p has multiple functions: it chaperones newly imported mitoribosomal proteins to ensure proper assembly of the mitoribosome (
      • Hillman G.A.
      • Henry M.F.
      The yeast protein Mam33 functions in the assembly of the mitochondrial ribosome.
      ), interacts with bL27 mitospecific domain and controls optimal mitochondrial translation under glucose (
      • Anderson J.M.
      • Box J.M.
      • Stuart R.A.
      The mitospecific domain of Mrp7 (bL27) supports mitochondrial translation during fermentation and is required for effective adaptation to respiration.
      ), and together with Mrx6p and Pim1p regulates mtDNA copy number (
      • Göke A.
      • Schrott S.
      • Mizrak A.
      • Belyy V.
      • Osman C.
      • Walter P.
      Mrx6 regulates mitochondrial DNA copy number in Saccharomyces cerevisiae by engaging the evolutionarily conserved Lon protease Pim1.
      ). The phenotypes resemblance between mam33 deletion and MRX9 excess suggests that Mrx9p may interfere in Mam33p function.
      Overexpression of MRX9 in pos-diauxic grown cells diminishes specifically the accumulation of newly synthesized Cox1p as well elicit the appearance of an aberrant translation product, resembling mp15 properties, which is usually found in the absence of translationally active Mss51p and dependent on COX1 intron composition (
      • Barrientos A.
      • Zambrano A.
      • Tzagoloff A.
      Mss51p and Cox14p jointly regulate mitochondrial Cox1p expression in Saccharomyces cerevisiae.
      ,
      • Zambrano A.
      • Fontanesi F.
      • Solans A.
      • de Oliveira R.L.
      • Fox T.D.
      • Tzagoloff A.
      • et al.
      Aberrant translation of cytochrome c oxidase subunit 1 mRNA species in the absence of Mss51p in the yeast Saccharomyces cerevisiae.
      ). The regulatory loop performed by Mss51p (
      • Fontanesi F.
      Mechanisms of mitochondrial translational regulation.
      ) is bypassed in cox14Δ cells (
      • Barrientos A.
      • Zambrano A.
      • Tzagoloff A.
      Mss51p and Cox14p jointly regulate mitochondrial Cox1p expression in Saccharomyces cerevisiae.
      ) but not in cox14Δcox24Δ double mutants (
      • Mays J.N.
      • Camacho-Villasana Y.
      • Garcia-Villegas R.
      • Perez-Martinez X.
      • Barrientos A.
      • Fontanesi F.
      The mitoribosome-specific protein mS38 is preferentially required for synthesis of cytochrome c oxidase subunits.
      ,
      • Barros M.H.
      • Myers A.M.
      • Van Driesche S.
      • Tzagoloff A.
      COX24 codes for a mitochondrial protein required for processing of the COX1 transcript.
      ); similarly, in the cox14Δmrx9Δ double mutant, the bypass is not as efficient as in the cox14Δ mutant. This result suggests that translationally active Mss51p did not efficiently perform new cycles of COX1 translation in the double mutant cox14Δmrx9Δ. In our model, the absence of Mrx9p leads to a suboptimal association of Mss51p with the mitoribosome and the translational activation complex (Fig. 9).
      Moreover, Mrx9p removal or overexpression altered the Mss51p sedimentation pattern, similar to that reported for the cox24Δ mutants (
      • Mays J.N.
      • Camacho-Villasana Y.
      • Garcia-Villegas R.
      • Perez-Martinez X.
      • Barrientos A.
      • Fontanesi F.
      The mitoribosome-specific protein mS38 is preferentially required for synthesis of cytochrome c oxidase subunits.
      ) with a concentration of Mss51p in the lighter fractions of the gradient. Cox24p (mS38) is a component of the mitoribosome (
      • Desai N.
      • Brown A.
      • Amunts A.
      • Ramakrishnan V.
      The structure of the yeast mitochondrial ribosome.
      ) required for proper COX1 splicing and COX1, COX2, and COX3 translation (
      • Mays J.N.
      • Camacho-Villasana Y.
      • Garcia-Villegas R.
      • Perez-Martinez X.
      • Barrientos A.
      • Fontanesi F.
      The mitoribosome-specific protein mS38 is preferentially required for synthesis of cytochrome c oxidase subunits.
      ,
      • Barros M.H.
      • Myers A.M.
      • Van Driesche S.
      • Tzagoloff A.
      COX24 codes for a mitochondrial protein required for processing of the COX1 transcript.
      ) When we tested the overexpression of MRX9 in some COX mutants, it was clearly more harmful to the mitochondrial protein synthesis of cox24Δ null mutant.
      We hypothesized that Mrx9p is part of the regulatory system that the mitochondrial introns exert on gene expression based on the following phenotypes of MRX9 excess toxicity: defective COX1 and COB mRNAs processing; poor arginine complementation by ARG8m placed under the control of COX1 and COB 5′ UTR; the slower adaptation to the respiratory metabolism depends on the presence of mitochondrial introns; interference in Mss51p distribution and availability for new COX1 translation cycles, as also observed in the mrx9 null mutant. Although a direct interaction of a tagged version of Mrx9p with Mss51p was not observed (data not shown), previous studies have indicated their physical proximity (
      • Singh A.P.
      • Salvatori R.
      • Aftab W.
      • Aufschnaiter A.
      • Carlström A.
      • Forne I.
      • et al.
      Molecular connectivity of mitochondrial gene expression and OXPHOS biogenesis.
      ). In the cell map obtained from systematic synthetic genetic array studies (
      • Costanzo M.
      • VanderSluis B.
      • Koch E.N.
      • Baryshnikova A.
      • Pons C.
      • Tan G.
      • et al.
      A global genetic interaction network maps a wiring diagram of cellular function.
      ), Mrx9p function is clustered together with proteins known to have a role in RNA metabolism. In the future, Mrx9p interaction studies are necessary to determine whether the toxicity of Mrx9p excess arose from its effects on splicing factors or directly on RNA primary transcripts.

      Experimental procedures

      Yeast strains and growth media

      The genotypes and sources of yeast strains used in this study are listed in Table S1. Yeast strains were maintained in YPD (1% yeast extract, 2% peptone, 2% glucose), YPGal (1% yeast extract, 2% peptone, 2% galactose), YPEG (1% yeast extract, 2% peptone, 2% glycerol, 2% ethanol), minimal glucose (2% glucose, 0.67% yeast nitrogen base without amino acids, supplemented with auxotrophic requirements), and sporulation media (0.5% yeast extract, 1% peptone, 0.05% glucose). All reagents, unless otherwise indicated, were obtained from Sigma–Aldrich.

      Overexpression of MIOREX components

      The MIOREX components AEP1, AEP2, AIM17, AIM18, AIM19, AIM25, AIM32, AIM34, ATP22, CBS1, CBS2, FMP10, FMP30, MAM33, MIN10, MRX1, MRX2(COQ11), MRX3, MRX4, MRX5(SMT1), MRX6, MRX8, MRX9, MRX10, MRX11, MRX12, MRX13, MRX16 PET20, PET54, PET122, PET494, and YLH47 were amplified by PCR with the proper pairs of oligonucleotides listed in Table S2 and cut with the indicated restriction enzyme. The digested PCR products were cloned into YIp351-GAL or YIplac204-GAL plasmids (
      • Zampol M.A.
      • Busso C.
      • Gomes F.
      • Ferreira-Junior J.R.
      • Tzagoloff A.
      • Barros M.H.
      Over-expression of COQ10 in Saccharomyces cerevisiae inhibits mitochondrial respiration.
      ) in which the coding sequence of the cloned gene is under control of the GAL10 promoter.

      Construction of MRX9 alleles

      The MRX9 WT allele was amplified from nuclear DNA with primers MRX9-F0 and MRX9-2 (Table S2); the 2042 bp PCR product was doubled, digested with BamH1 and Sal1, and cloned in pUC18 and YIp35I (
      • Hill J.E.
      • Myers A.M.
      • Koerner T.J.
      • Tzagoloff A.
      Yeast/E. coli shuttle vectors with multiple unique restriction sites.
      ), both previously cut with the same enzymes yielding pUC18/MRX9 and pMRX9/ST1. The pUC18/MRX9 was used as a template for a new PCR cycle with the bidirectional primers MRX9-D1 and MRX9-D2 resulting in a clean deletion of MRX9. The linear product containing MRX9 flanking region in pUC18 was digested with Bcl1 and ligated to HIS3 on a 1 kb BamH1 fragment of DNA. The mrx9::HIS3 null allele was recovered from this plasmid as a 1.7 kb Sal1-BamH1 fragment and was substituted by one-step gene replacement for the WT allele in the yeast strain W303-1B.
      MRX9 C-terminal was fused to the HA-tag by PCR amplification with MRX9-F0 and MRX9-HA primers. This PCR product contains the MRX9 endogenous promoter and it was cloned into YIp352 (
      • Hill J.E.
      • Myers A.M.
      • Koerner T.J.
      • Tzagoloff A.
      Yeast/E. coli shuttle vectors with multiple unique restriction sites.
      ). A second PCR amplification using MRX9-HA and MRX9-1 primers amplified only the MRX9 coding sequence. This PCR product was cloned into YIp352-ADH, YIp351-TEF1, YIp351-GPD, and YIp351-GAL (
      • Zampol M.A.
      • Busso C.
      • Gomes F.
      • Ferreira-Junior J.R.
      • Tzagoloff A.
      • Barros M.H.
      Over-expression of COQ10 in Saccharomyces cerevisiae inhibits mitochondrial respiration.
      ), in which the coding sequence of MRX9-HA allele is under control of the GAL10 promoter, alcohol dehydrogenase promoter (ADH), the translation elongation factor 1α promoter (TEF1), and the GAPDH promoter (GPD) (
      • Mumberg D.
      • Müller R.
      • Funk M.
      Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds.
      ).

      Mitochondrial protein synthesis

      Depending on the experiments, yeast cell cultures were grown in rich galactose media or rich glucose media. Mitochondrial translation products were labeled in whole cells with a mixture of [35S] methionine and [35S] cysteine (7 mCi/mmol) in the presence of cycloheximide. Cells were lysed and protein extracted as previously described (
      • Santos B.
      • Zeng R.
      • Jorge S.F.
      • Ferreira-Junior J.R.
      • Barrientos A.
      • Barros M.H.
      Functional analyses of mitoribosome 54S subunit devoid of mitochondria-specific protein sequences.
      ). Total cellular proteins were separated by SDS-PAGE on 17.5% polyacrylamide or 6M UREA-PAGE on 12.5% polyacrylamide gel systems.

      Mitochondria isolation and fractionation

      Yeast mitochondria were prepared by the method of Herrmann et al. (
      • Herrmann J.M.
      • Foelsch H.
      • Neupert W.
      • Stuart R.A.
      Isolation of yeast mitochondria and study of mitochondrial protein translation.
      ) using Zymolyase 20T, Zymo Research to obtain spheroplasts. Four milligram of mitochondrial preparation were solubilized in 400 μl of an extraction buffer (20 mM Hepes pH 7.4, 25 mM KCl, 0.5 mM PMSF, 0.8% Triton X100 [or 2% Digitonin], 5 mM MgCl2) and were centrifuged at 27,000g for 15 min and applied onto a linear sucrose gradient 0.3 to 1.0 M, with the same constitution of the correspondent extraction buffer. The linear sucrose gradients were centrifuged for 3 h at 40,000 rpm in the 55Ti Beckmann rotor, Beckmann Coulter (
      • De Silva D.
      • Fontanesi F.
      • Barrientos A.
      The DEAD box protein Mrh4 functions in the assembly of the mitochondrial large ribosomal subunit.
      ). Antibodies and sources are listed in Table S3.
      RNA was extracted from the mitochondria at a protein concentration of 5 mg/ml as described (
      • Moda B.S.
      • Ferreira-Júnior J.R.
      • Barros M.H.
      Partial suppression of the respiratory defect of qrs1/her2 glutamyl-tRNA amidotransferase mutants by overexpression of the mitochondrial pentatricopeptide Msc6p.
      ). Total mitochondrial RNAs were separated on a 1% agarose gel. The gel was stained with ethidium bromide, photographed, and the RNAs blotted to a nylon membrane. Following UV crosslinking, the nylon membrane was hybridized overnight at 42 °C with COB and COX1 probes (Table S1) that were 3′ end labeled with a biotin kit (Thermo Scientific) as described elsewhere (
      • Moda B.S.
      • Ferreira-Júnior J.R.
      • Barros M.H.
      Partial suppression of the respiratory defect of qrs1/her2 glutamyl-tRNA amidotransferase mutants by overexpression of the mitochondrial pentatricopeptide Msc6p.
      ).

      Enzymatic assays of mitochondrial respiratory complexes

      Ubiquinol-cytochrome c reductase activity was measured by following the increase in absorbance at 550 nm due to the reduction of cytochrome c in the presence of 10 mM NADH (NCCR). COX was assayed by following the oxidation of ferro-cytochrome at 550 nM (
      • Tzagoloff A.
      • Akai A.
      • Needleman R.B.
      Assembly of the mitochondrial membrane system. Characterization of nuclear mutants of Saccharomyces cerevisiae with defects in mitochondrial ATPase and respiratory enzymes.
      ). GraphPad Prism software was used for graphs and data analyses by Turkey’s statistical test.

      Data availability

      All data discussed here are presented in the manuscript.

      Supporting information

      This article contains supporting information (
      • Marbois B.
      • Gin P.
      • Faull K.F.
      • Poon W.W.
      • Lee P.T.
      • Strahan J.
      • et al.
      Coq3 and Coq4 define a polypeptide complex in yeast mitochondria for the biosynthesis of coenzyme Q.
      ,
      • Franco L.V.R.
      • Su C.H.
      • McStay G.P.
      • Yu G.J.
      • Tzagoloff A.
      Cox2p of yeast cytochrome oxidase assembles as a stand-alone subunit with the Cox1p and Cox3p modules.
      ,
      • Glerum D.M.
      • Tzagoloff A.
      Submitochondrial distributions and stabilities of subunits 4, 5, and 6 of yeast cytochrome oxidase in assembly defective mutants.
      ,
      • Barros M.H.
      • Tzagoloff A.
      Regulation of the heme A biosynthetic pathway in Saccharomyces cerevisiae.
      ,
      • Guedes-Monteiro R.F.
      • Franco L.V.R.
      • Moda B.S.
      • Tzagoloff A.
      • Barros M.H.
      5' processing of Saccharomyces cerevisiae mitochondrial tRNAs requires expression of multiple genes.
      ,
      • Gruschke S.
      • Gröne K.
      • Heublein M.
      • Hölz S.
      • Israel L.
      • Imhof A.
      • et al.
      Proteins at the polypeptide tunnel exit of the yeast mitochondrial ribosome.
      ,
      • Dieckmann C.L.
      • Tzagoloff A.
      Assembly of the mitochondrial membrane system. CBP6, a yeast nuclear gene necessary for synthesis of cytochrome b.
      ,
      • Poon W.W.
      • Barkovich R.J.
      • Hsu A.Y.
      • Frankel A.
      • Lee P.T.
      • Shepherd J.N.
      • et al.
      Yeast and rat Coq3 and Escherichia coli UbiG polypeptides catalyze both O-methyltransferase steps in coenzyme Q biosynthesis.
      ,
      • Jin C.
      • Myers A.M.
      • Tzagoloff A.
      Cloning and characterization of MRP10, a yeast gene coding for a mitochondrial ribosomal protein.
      ,
      • Crivellone M.D.
      • Wu M.A.
      • Tzagoloff A.
      Assembly of the mitochondrial membrane system. Analysis of structural mutants of the yeast coenzyme QH2-cytochrome c reductase complex.
      ).

      Conflicts of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Acknowledgments

      This work was supported by grants and fellowships from Fundação de Amparo a Pesquisa de São Paulo ( FAPESP 2013/07937–8 ; FAPESP 2020/05812–7 ), Conselho Nacional de Desenvolvimento Científico e Tecnológico ( CNPq 302337/2018–0 ) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. Jhulia Chagas, Maria Kfouri and Leticia Franco were fellowship recipients from FAPESP (FAPESP- 2020/05173–8, 2019/23984–2, 2019/02799–2).

      Author contributions

      M. H. B. and L. V. R. F. conceptualization; J. A. C. C. and M. A. K. M. S. methodology; J. A. C. C., M. A. K. M. S., and M. H. B. investigation; M. H. B. and J. A. C. C. writing–original draft; M. H. B. supervision.

      Supporting information

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