Cytosolic Events in the Biogenesis of Mitochondrial Proteins
2020; Elsevier BV; Volume: 45; Issue: 8 Linguagem: Inglês
10.1016/j.tibs.2020.04.001
ISSN1362-4326
AutoresYury S. Bykov, Doron Rapaport, Johannes M. Herrmann, Maya Schuldiner,
Tópico(s)ATP Synthase and ATPases Research
ResumoMitochondrial proteins synthetized in the cytosol can be targeted to mitochondria at different stages of gene expression: as mRNAs, ribosome-nascent chain complexes, or complete precursor proteins.While almost all proteins use the same entry gate to the mitochondria, before and after it they can embark on different targeting and import pathways.Delays in mitochondrial protein import or mistargeting to other organelles affect cellular homeostasis; hence, cells have evolved specific mechanisms to sense and counteract such situations.Cytosolic chaperones promote mitochondrial protein import under normal conditions, as well as play a major role in stress response pathways associated with mitochondrial protein import defects. While targeting of proteins synthesized in the cytosol to any organelle is complex, mitochondria present the most challenging of destinations. First, import of nuclear-encoded proteins needs to be balanced with production of mitochondrial-encoded ones. Moreover, as mitochondria are divided into distinct subdomains, their proteins harbor a number of different targeting signals and biophysical properties. While translocation into the mitochondrial membranes has been well studied, the cytosolic steps of protein import remain poorly understood. Here, we review current knowledge on mRNA and protein targeting to mitochondria, as well as recent advances in our understanding of the cellular programs that respond to accumulation of mitochondrial precursor proteins in the cytosol, thus linking defects in targeting-capacity to signaling. While targeting of proteins synthesized in the cytosol to any organelle is complex, mitochondria present the most challenging of destinations. First, import of nuclear-encoded proteins needs to be balanced with production of mitochondrial-encoded ones. Moreover, as mitochondria are divided into distinct subdomains, their proteins harbor a number of different targeting signals and biophysical properties. While translocation into the mitochondrial membranes has been well studied, the cytosolic steps of protein import remain poorly understood. Here, we review current knowledge on mRNA and protein targeting to mitochondria, as well as recent advances in our understanding of the cellular programs that respond to accumulation of mitochondrial precursor proteins in the cytosol, thus linking defects in targeting-capacity to signaling. The eukaryotic cell emerged following an integration between an archaeon and its bacterial symbiont. The host cell and the symbiont, which became a mitochondrion, have coevolved for so long that they are completely dependent on each other [1.Roger A.J. et al.The origin and diversification of mitochondria.Curr. Biol. 2017; 27: R1177-R1192Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar]. On one hand, modern cells rely on mitochondria for efficient production of ATP and vital cofactors, such as iron-sulfur clusters and heme. On the other hand, mitochondria have given up their autonomy for production of lipids and proteins to their host cells. This interdependence creates a constant crosstalk between mitochondria, the cytosol, and all other organelles through the flux of metabolites, involvement in common signaling pathways, and transport of newly synthetized proteins destined to mitochondria, herein termed 'precursor proteins', or 'precursors' [2.Eisenberg-Bord M. Schuldiner M. Mitochatting – if only we could be a fly on the cell wall.Biochim. Biophys. Acta, Mol. Cell Res. 2017; 1864: 1469-1480Crossref PubMed Scopus (0) Google Scholar]. Importing around 1000 different proteins into various mitochondrial locations is one of the most challenging tasks. The first step of targeting (see Glossary) involves the recognition and directional movement of the protein (or its encoding mRNA) from the cytosol to the surface of the correct organelle. Next, the protein can be handed over to the machinery responsible for its integration into the membranes or passage into the aqueous compartments of the organelle by translocation through protein channels or pores. The entire process of both targeting and translocation to mitochondria constitute mitochondrial protein import. The proteins that need to be imported to different compartments contain specific targeting signals that are recognized by both the targeting and translocation machineries (Box 1).Box 1Import Signals of Mitochondrial PrecursorsTwo bounding membranes of mitochondria create a variety of possible topologies for imported proteins (Figure I).Soluble Matrix ProteinsThese constitute ~50% of the mitochondrial proteome [146.Vögtle F.-N. et al.Landscape of submitochondrial protein distribution.Nat. Commun. 2017; 8: 290Crossref PubMed Scopus (41) Google Scholar], with the majority harboring an N terminal cleavable mitochondrial targeting sequence (MTS; shown in red in Figure I). The MTS (10–100 amino acids long, positively charged peptide with a propensity to form an amphipathic α-helix [117.Vögtle F.-N. et al.Global analysis of the mitochondrial N-proteome identifies a processing peptidase critical for protein stability.Cell. 2009; 139: 428-439Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar,147.von Heijne G. Mitochondrial targeting sequences may form amphiphilic helices.EMBO J. 1986; 5: 1335-1342Crossref PubMed Google Scholar]) is necessary and sufficient to direct proteins to the matrix. In addition to the MTS, some matrix proteins can contain internal MTS-like sequences (iMTSLs) that can bind OM receptors and help to keep precursors in an import-competent state [136.Backes S. et al.Tom70 enhances mitochondrial preprotein import efficiency by binding to internal targeting sequences.J. Cell Biol. 2018; 217: 1369-1382Crossref PubMed Scopus (9) Google Scholar]. However, a number of matrix proteins do not have a cleavable MTS and the signals they possess are poorly studied [148.Woellhaf M.W. et al.Import of ribosomal proteins into yeast mitochondria.Biochem. Cell Biol. 2014; 92: 489-498Crossref PubMed Scopus (4) Google Scholar,149.Longen S. et al.The disulfide relay of the intermembrane space oxidizes the ribosomal subunit Mrp10 on its transit into the mitochondrial matrix.Dev. Cell. 2014; 28: 30-42Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. Furthermore, some MTS-containing proteins can be translocated into mitochondria without their MTS [24.Weill U. et al.Genome-wide SWAp-Tag yeast libraries for proteome exploration.Nat. Methods. 2018; 15: 617-622Crossref PubMed Scopus (20) Google Scholar].Inner Membrane (IM) ProteinsThese consist of three groups: (i) mitochondrially encoded proteins synthesized on mitochondrial ribosomes that are inserted from the matrix side (not shown); (ii) metabolite carrier family proteins (shown in green) with a characteristic topology consisting of three modules (numbered 1–3), each of which is a hairpin with two TMDs and a matrix-exposed loop (each of these modules can contain the targeting information and the three modules cooperate to ensure efficient targeting [150.Wiedemann N. et al.The three modules of ADP/ATP carrier cooperate in receptor recruitment and translocation into mitochondria.EMBO J. 2001; 20: 951-960Crossref PubMed Scopus (161) Google Scholar]); and (iii) other proteins with various topologies that usually contain an MTS (red).Intermembrane Space (IMS) ProteinsThere are about 100 different proteins in the IMS, many of which are rather small (8–25 kDa) and contain conserved cysteine motifs that allow oxidative folding (shown in orange). Mia40 is a conserved oxidoreductase in the IMS that mediates the folding of these proteins and therefore their retention in the IMS [151.Chacinska A. et al.Essential role of Mia40 in import and assembly of mitochondrial intermembrane space proteins.EMBO J. 2004; 23: 3735-3746Crossref PubMed Scopus (300) Google Scholar]. Larger IMS proteins are usually made as precursors, with bipartite signals that consist of an N terminal MTS (red) followed by a TMD (cyan). Following arrest in the IM, these proteins (yellow-green) are cleaved C terminally of the TMD and released into the IMS [152.Glick B.S. et al.Cytochromes c1 and b2 are sorted to the intermembrane space of yeast mitochondria by a stop-transfer mechanism.Cell. 1992; 69: 809-822Abstract Full Text PDF PubMed Google Scholar].Outer Membrane (OM) ProteinsIntegral proteins of the OM can be embedded in different orientations and have a variable number of transmembrane α-helices or β-strands (shown in blue). β-Barrel proteins in eukaryotes can be found only in mitochondrial or chloroplast OMs (light blue). The signals that direct α-helical proteins to the OM are usually associated with the TMD (dark blue) and its flanking regions, but some of them can also contain a special N terminal targeting sequence [11.Ellenrieder L. et al.Biogenesis of mitochondrial outer membrane proteins, problems and diseases.Biol. Chem. 2015; 396: 1199-1213Crossref PubMed Scopus (19) Google Scholar,153.Sinzel M. et al.Mcp3 is a novel mitochondrial outer membrane protein that follows a unique IMP-dependent biogenesis pathway.EMBO Rep. 2016; 17: 965-981Crossref PubMed Scopus (20) Google Scholar]. β-Barrel proteins are targeted via a hydrophobic β-hairpin structural element [122.Jores T. et al.Characterization of the targeting signal in mitochondrial β-barrel proteins.Nat. Commun. 2016; 7: 1-16Crossref Scopus (29) Google Scholar]. Two bounding membranes of mitochondria create a variety of possible topologies for imported proteins (Figure I). Soluble Matrix Proteins These constitute ~50% of the mitochondrial proteome [146.Vögtle F.-N. et al.Landscape of submitochondrial protein distribution.Nat. Commun. 2017; 8: 290Crossref PubMed Scopus (41) Google Scholar], with the majority harboring an N terminal cleavable mitochondrial targeting sequence (MTS; shown in red in Figure I). The MTS (10–100 amino acids long, positively charged peptide with a propensity to form an amphipathic α-helix [117.Vögtle F.-N. et al.Global analysis of the mitochondrial N-proteome identifies a processing peptidase critical for protein stability.Cell. 2009; 139: 428-439Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar,147.von Heijne G. Mitochondrial targeting sequences may form amphiphilic helices.EMBO J. 1986; 5: 1335-1342Crossref PubMed Google Scholar]) is necessary and sufficient to direct proteins to the matrix. In addition to the MTS, some matrix proteins can contain internal MTS-like sequences (iMTSLs) that can bind OM receptors and help to keep precursors in an import-competent state [136.Backes S. et al.Tom70 enhances mitochondrial preprotein import efficiency by binding to internal targeting sequences.J. Cell Biol. 2018; 217: 1369-1382Crossref PubMed Scopus (9) Google Scholar]. However, a number of matrix proteins do not have a cleavable MTS and the signals they possess are poorly studied [148.Woellhaf M.W. et al.Import of ribosomal proteins into yeast mitochondria.Biochem. Cell Biol. 2014; 92: 489-498Crossref PubMed Scopus (4) Google Scholar,149.Longen S. et al.The disulfide relay of the intermembrane space oxidizes the ribosomal subunit Mrp10 on its transit into the mitochondrial matrix.Dev. Cell. 2014; 28: 30-42Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. Furthermore, some MTS-containing proteins can be translocated into mitochondria without their MTS [24.Weill U. et al.Genome-wide SWAp-Tag yeast libraries for proteome exploration.Nat. Methods. 2018; 15: 617-622Crossref PubMed Scopus (20) Google Scholar]. Inner Membrane (IM) Proteins These consist of three groups: (i) mitochondrially encoded proteins synthesized on mitochondrial ribosomes that are inserted from the matrix side (not shown); (ii) metabolite carrier family proteins (shown in green) with a characteristic topology consisting of three modules (numbered 1–3), each of which is a hairpin with two TMDs and a matrix-exposed loop (each of these modules can contain the targeting information and the three modules cooperate to ensure efficient targeting [150.Wiedemann N. et al.The three modules of ADP/ATP carrier cooperate in receptor recruitment and translocation into mitochondria.EMBO J. 2001; 20: 951-960Crossref PubMed Scopus (161) Google Scholar]); and (iii) other proteins with various topologies that usually contain an MTS (red). Intermembrane Space (IMS) Proteins There are about 100 different proteins in the IMS, many of which are rather small (8–25 kDa) and contain conserved cysteine motifs that allow oxidative folding (shown in orange). Mia40 is a conserved oxidoreductase in the IMS that mediates the folding of these proteins and therefore their retention in the IMS [151.Chacinska A. et al.Essential role of Mia40 in import and assembly of mitochondrial intermembrane space proteins.EMBO J. 2004; 23: 3735-3746Crossref PubMed Scopus (300) Google Scholar]. Larger IMS proteins are usually made as precursors, with bipartite signals that consist of an N terminal MTS (red) followed by a TMD (cyan). Following arrest in the IM, these proteins (yellow-green) are cleaved C terminally of the TMD and released into the IMS [152.Glick B.S. et al.Cytochromes c1 and b2 are sorted to the intermembrane space of yeast mitochondria by a stop-transfer mechanism.Cell. 1992; 69: 809-822Abstract Full Text PDF PubMed Google Scholar]. Outer Membrane (OM) Proteins Integral proteins of the OM can be embedded in different orientations and have a variable number of transmembrane α-helices or β-strands (shown in blue). β-Barrel proteins in eukaryotes can be found only in mitochondrial or chloroplast OMs (light blue). The signals that direct α-helical proteins to the OM are usually associated with the TMD (dark blue) and its flanking regions, but some of them can also contain a special N terminal targeting sequence [11.Ellenrieder L. et al.Biogenesis of mitochondrial outer membrane proteins, problems and diseases.Biol. Chem. 2015; 396: 1199-1213Crossref PubMed Scopus (19) Google Scholar,153.Sinzel M. et al.Mcp3 is a novel mitochondrial outer membrane protein that follows a unique IMP-dependent biogenesis pathway.EMBO Rep. 2016; 17: 965-981Crossref PubMed Scopus (20) Google Scholar]. β-Barrel proteins are targeted via a hydrophobic β-hairpin structural element [122.Jores T. et al.Characterization of the targeting signal in mitochondrial β-barrel proteins.Nat. Commun. 2016; 7: 1-16Crossref Scopus (29) Google Scholar]. Protein translocation into mitochondria is well studied and it is clear that many different pathways exist to assist the correct translocation and integration of proteins to their mitochondrial subcompartments. All of them start at the translocase of the outer membrane (TOM) complex. The TOM complex consists of the β-barrel protein Tom40 that forms a translocation pore, receptor proteins Tom20 and Tom70 that recognize targeting signals on the precursors, Tom22 that can recognize precursors and also plays a structural role, and the small subunits Tom5, 6, and 7 that mediate complex assembly [3.Dekker P.J.T. et al.Preprotein translocase of the outer mitochondrial membrane: molecular dissection and assembly of the general import pore complex.Mol. Cell. Biol. 1998; 18: 6515-6524Crossref PubMed Scopus (181) Google Scholar, 4.Shiota T. et al.Molecular architecture of the active mitochondrial protein gate.Science. 2015; 349: 1544-1548Crossref PubMed Scopus (79) Google Scholar, 5.Brix J. et al.Differential recognition of preproteins by the purified cytosolic domains of the mitochondrial import receptors Tom20, Tom22, and Tom70.J. Biol. 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Transport of proteins into mitochondria.Protein J. 2019; 38: 330-342Crossref PubMed Scopus (17) Google Scholar, 16.Dukanovic J. Rapaport D. Multiple pathways in the integration of proteins into the mitochondrial outer membrane.Biochim. Biophys. Acta Biomembr. 2011; 1808: 971-980Crossref PubMed Scopus (0) Google Scholar], they will not be addressed here. By contrast to translocation, targeting steps occurring in the cytosol cannot be studied as easily and hence have been less investigated and understood. Moreover, studies using gene deletion or downregulation were not successful in deciphering these steps, potentially due to redundancy of targeting factors [17.Krumpe K. et al.Ergosterol content specifies targeting of tail-anchored proteins to mitochondrial outer membranes.Mol. Biol. Cell. 2012; 23: 3927-3935Crossref PubMed Scopus (61) Google Scholar]. Until now, few insights have been gleaned regarding targeting events of mitochondrial proteins while conceptually they can, and probably do, happen at multiple different stages of gene expression: before translation, during translation, and after translation (Figure 1). Yet, to date, it is still not clear for most mitochondrial proteins whether their targeting signal can be recognized directly by a receptor on mitochondria or whether they require a dedicated targeting factor. Moreover, dedicated targeting factors, such as the signal recognition particle (SRP) of the secretory pathway, have not been identified for mitochondrial proteins. What is emerging clearly though is that cells have evolved a variety of quality control mechanisms that survey the protein on its way to mitochondria and activate various signaling and stress responses to deal with accumulation of precursor proteins in the cytosol. With new available biochemical, cell biological, and genetic tools, we believe that the time is ripe to revisit some of the major open questions in the field of mitochondrial protein targeting with the aim of reaching a detailed mechanistic understanding of these processes, at a level similar to that obtained for translocation. A path of a protein to its destination organelle can start even before translation by localization of its encoding mRNA. Usually, such mRNA targeting is mediated by a special sequence located in its untranslated regions (UTRs). Several mRNAs were directly shown to use this mechanism for mitochondrial targeting in Saccharomyces cerevisiae (from hereon called yeast) [18.Olivas W. Parker R. The Puf3 protein is a transcript-specific regulator of mRNA degradation in yeast.EMBO J. 2000; 19: 6602-6611Crossref PubMed Google Scholar, 19.Margeot A. et al.In Saccharomyces cerevisiae, ATP2 mRNA sorting to the vicinity of mitochondria is essential for respiratory function.EMBO J. 2002; 21: 6893-6904Crossref PubMed Scopus (79) Google Scholar, 20.Margeot A. et al.Why are many mRNAs translated to the vicinity of mitochondria: a role in protein complex assembly?.Gene. 2005; 354: 64-71Crossref PubMed Scopus (43) Google Scholar, 21.Garcia M. et al.Mitochondrial presequence and open reading frame mediate asymmetric localization of messenger RNA.EMBO Rep. 2010; 11: 285-291Crossref PubMed Scopus (40) Google Scholar, 22.Gadir N. et al.Localization of mRNAs coding for mitochondrial proteins in the yeast Saccharomyces cerevisiae.RNA. 2011; 17: 1551-1565Crossref PubMed Scopus (76) Google Scholar]. In yeast, targeting of mRNAs via their 3′- or 5′-UTRs is probably not absolutely required for the mitochondrial targeting of the encoded protein because generic 5′- and 3′-UTRs used in yeast whole-genomic libraries do not systematically perturb protein localization to mitochondria [23.Huh W.-K. et al.Global analysis of protein localization in budding yeast.Nature. 2003; 425: 686-691Crossref PubMed Scopus (2982) Google Scholar,24.Weill U. et al.Genome-wide SWAp-Tag yeast libraries for proteome exploration.Nat. Methods. 2018; 15: 617-622Crossref PubMed Scopus (20) Google Scholar]. Still, in yeast, there is a well-studied RNA-binding protein that is associated with mitochondria, Pumilio-homology domain family protein 3 (Puf3). Puf3 specifically binds mRNAs encoding proteins with mitochondria-related functions and modulates mitochondrial recruitment of some of them by binding a 3′-UTR motif [22.Gadir N. et al.Localization of mRNAs coding for mitochondrial proteins in the yeast Saccharomyces cerevisiae.RNA. 2011; 17: 1551-1565Crossref PubMed Scopus (76) Google Scholar,25.Gerber A.P. et al.Extensive association of functionally and cytotopically related mRNAs with Puf family RNA-binding proteins in yeast.PLoS Biol. 2004; 2e79Crossref PubMed Scopus (462) Google Scholar, 26.Saint-Georges Y. et al.Yeast mitochondrial biogenesis: a role for the PUF RNA-binding protein Puf3p in mRNA localization.PLoS One. 2008; 3e2293Crossref PubMed Scopus (147) Google Scholar, 27.Zhu D. et al.A 5′ cytosine binding pocket in Puf3p specifies regulation of mitochondrial mRNAs.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 20192-20197Crossref PubMed Scopus (0) Google Scholar]. It was therefore suggested that Puf3 promotes the targeting of mitochondrial mRNAs to the mitochondrial surface for proximal translation. Indeed, a case study of Cox17 [28.Beers J. et al.Purification, characterization, and localization of yeast Cox17p, a mitochondrial copper shuttle.J. Biol. Chem. 1997; 272: 33191-33196Crossref PubMed Scopus (0) Google Scholar,29.Cavallaro G. Genome-wide analysis of eukaryotic twin CX9C proteins.Mol. BioSyst. 2010; 6: 2459-2470Crossref PubMed Scopus (63) Google Scholar] showed that its 3′-UTR confers localization to mitochondria in a Puf3-dependent way [18.Olivas W. Parker R. The Puf3 protein is a transcript-specific regulator of mRNA degradation in yeast.EMBO J. 2000; 19: 6602-6611Crossref PubMed Google Scholar]. While the above seems to suggest a role for Puf3 in mRNA and therefore protein targeting, there is still a debate regarding its role. Most studies report that Puf3 is localized to the cytosol [23.Huh W.-K. et al.Global analysis of protein localization in budding yeast.Nature. 2003; 425: 686-691Crossref PubMed Scopus (2982) Google Scholar, 24.Weill U. et al.Genome-wide SWAp-Tag yeast libraries for proteome exploration.Nat. Methods. 2018; 15: 617-622Crossref PubMed Scopus (20) Google Scholar, 25.Gerber A.P. et al.Extensive association of functionally and cytotopically related mRNAs with Puf family RNA-binding proteins in yeast.PLoS Biol. 2004; 2e79Crossref PubMed Scopus (462) Google Scholar,30.Sheth U. Parker R. Decapping and decay of messenger RNA occur in cytoplasmic processing bodies.Science. 2003; 300: 805-808Crossref PubMed Scopus (865) Google Scholar]. Only one work reported that Puf3 colocalizes with mitochondria where it can bind Mdm12 and Arp2 [31.García-Rodríguez L.J. et al.Puf3p, a Pumilio family RNA binding protein, localizes to mitochondria and regulates mitochondrial biogenesis and motility in budding yeast.J. Cell Biol. 2007; 176: 197-207Crossref PubMed Scopus (0) Google Scholar]. This finding pointed to a possible connection of mitochondrial protein-coding RNA regulation to mitochondria–endoplasmic reticulum (ER) contact sites and motility. Both deletion and overexpression of Puf3 do not significantly affect yeast growth on respiratory media, as would have been expected for an important factor affecting mitochondrial protein import, but do cause slower growth on this media at elevated temperatures [31.García-Rodríguez L.J. et al.Puf3p, a Pumilio family RNA binding protein, localizes to mitochondria and regulates mitochondrial biogenesis and motility in budding yeast.J. Cell Biol. 2007; 176: 197-207Crossref PubMed Scopus (0) Google Scholar]. The phosphorylation state and stability of Puf3 is modulated by carbon source availability [31.García-Rodríguez L.J. et al.Puf3p, a Pumilio family RNA binding protein, localizes to mitochondria and regulates mitochondrial biogenesis and motility in budding yeast.J. Cell Biol. 2007; 176: 197-207Crossref PubMed Scopus (0) Google Scholar, 32.Lee C.-D. Tu B.P. Glucose-regulated phosphorylation of the PUF protein Puf3 regulates the translational fate of its bound mRNAs and association with RNA granules.Cell Rep. 2015; 11: 1638-1650Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 33.Miller M.A. et al.Carbon source-dependent alteration of Puf3p activity mediates rapid changes in the stabilities of mRNAs involved in mitochondrial function.Nucleic Acids Res. 2014; 42: 3954-3970Crossref PubMed Scopus (25) Google Scholar]. Maybe the most intriguing is the observation that upon PUF3 deletion, when the mitochondrial association of COX17 mRNA is lost, protein translocation is not affected but rather, stabilization of the mRNA occurs [18.Olivas W. Parker R. The Puf3 protein is a transcript-specific regulator of mRNA degradation in yeast.EMBO J. 2000; 19: 6602-6611Crossref PubMed Google Scholar]. Moreover, the PUF domain alone is sufficient to promote COX17 mRNA degradation, suggesting a role for mRNA turnover regulation rather than targeting for this specific mRNA. But what about all other mitochondrial proteins and their transcripts? Whole-genome studies in yeast revealed different effects of Puf3 deletion on mRNA abundance, translation efficiency, and mitochondrial proteome abundance, highlighting its complex regulatory role [26.Saint-Georges Y. et al.Yeast mitochondrial biogenesis: a role for the PUF RNA-binding protein Puf3p in mRNA localization.PLoS One. 2008; 3e2293Crossref PubMed Scopus (147) Google Scholar,34.Kershaw C.J. et al.Integrated multi-omics analyses reveal the pleiotropic nature of the control of gene expression by Puf3p.Sci. Rep. 2015; 5: 15518Crossref PubMed Scopus (26) Google Scholar,35.Wang Z. et al.Novel insights into global translational regulation through Pumilio family RNA-binding protein Puf3p revealed by ribosomal profiling.Curr. Genet. 2019; 65: 201-212Crossref PubMed Scopus (5) Google Scholar]. Hence, to date, there is no unequivocal evidence that Puf3-mediated mRNA-localization is a part of the mitochondrial protein targeting process. Instead, Puf3 might help to regulate mRNA stability and translation and thus contribute to coordination of protein synthesis on cytosolic and mitochondrial ribosomes during the assembly of the oxidative phosphorylation (OXPHOS) components. Puf3 is probably not the only factor that can mediate UTR-dependent mRNA targeting to mitochondria but others have not yet been identified and studied. What happens in mammals? A recent RNA-sequencing study using proximity labeling of RNAs with an engineered efficient alkaline peroxidase revealed that, in mammalian cells, there are many mRNAs localized to the mitochondrial outer membrane (OM) and no distinct class of proteins is enriched among them. Part of the RNAs are localized to mitochondria in a microtubule-dependent manner, some are translation-dependent (see later), and others are recruited independently of their translation, via their 3′-UTRs [36.Fazal F.M. et al.Atlas of subcellular RNA localization revealed by APEX-seq.Cell. 2019; 178: 473-490Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar]. Hence, the localization of these mRNAs likely depends on RNA-binding proteins. Interestingly, this latter class of mRNAs was enriched with those encoding mitochondrial ribosome and OXPHOS components that require coordinated import and assembly [36.Fazal F.M. et al.Atlas of subcellular RNA localization revealed by APEX-seq.Cell. 2019; 178: 473-490Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar]. However, the RNA-binding proteins mediating this recruitment have not yet been identified. Moreover, mRNA recruitment to mitochondria is not necessarily directly linked to translation since ribosome profiling experiments in yeast did not find the same RNAs that were localized to mitochondria using microarrays and fluorescent in situ hybridization [22.Gadir N. et al.Localization of mRNAs coding for mitochondrial proteins in the yeast Saccharomyces cerevisiae.RNA. 2011; 17: 1551-1565Crossref PubMed Scopus (76) Google Scholar,37.Garcia M. et al.Mitochondria-associated yeast mRNAs and the biogenesis of molecular complexes.Mol. Biol. Cell. 2006; 18: 362-368Crossref PubMed Google Scholar,38.Williams C.C. et al.Targeting and plasticity of mitochondrial proteins revealed by proximity-specific ribosome profiling.Science. 2014; 346: 748-751Crossref PubMed Scopus (150) Google Scholar]. It is not clear whether translation of mRNAs targeted to mitochondria can also be init
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