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Yeast Mitochondrial Gln-Trna Is Generated by a Gatfab-Mediated Transamidation Pathway Involving Arc1p-Controlled Subcellular

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Yeast Mitochondrial Gln-Trna Is Generated by a Gatfab-Mediated Transamidation Pathway Involving Arc1p-Controlled Subcellular Downloaded from genesdev.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press Yeast mitochondrial Gln-tRNAGln is generated by a GatFAB-mediated transamidation pathway involving Arc1p-controlled subcellular sorting of cytosolic GluRS Mathieu Frechin,1 Bruno Senger,1 Me´lanie Braye´,1 Daniel Kern,1 Robert Pierre Martin,2,4 and Hubert Dominique Becker1,3 1UPR 9002, ‘‘Architecture et Re´activite´ de l’ARN,’’ Universite´ de Strasbourg, CNRS, Institut de Biologie Mole´culaire et Cellulaire, F-67084 Strasbourg Ce´dex, France; 2UMR 7156, ‘‘Ge´ne´tique Mole´culaire, Ge´nomique, Microbiologie,’’ Department of Molecular and Cellular Genetics, CNRS, Universite´ de Strasbourg, 67084 Strasbourg, France It is impossible to predict which pathway, direct glutaminylation of tRNAGln or tRNA-dependent transamidation of glutamyl-tRNAGln, generates mitochondrial glutaminyl-tRNAGln for protein synthesis in a given species. The report that yeast mitochondria import both cytosolic glutaminyl-tRNA synthetase and tRNAGln has challenged the widespread use of the transamidation pathway in organelles. Here we demonstrate that yeast mitochondrial glutaminyl-tRNAGln is in fact generated by a transamidation pathway involving a novel type of trimeric tRNA- dependent amidotransferase (AdT). More surprising is the fact that cytosolic glutamyl-tRNA synthetase (cERS) is imported into mitochondria, where it constitutes the mitochondrial nondiscriminating ERS that generates the Gln mitochondrial mischarged glutamyl-tRNA substrate for the AdT. We show that dual localization of cERS is controlled by binding to Arc1p, a tRNA nuclear export cofactor that behaves as a cytosolic anchoring platform for cERS. Expression of Arc1p is down-regulated when yeast cells are switched from fermentation to respiratory metabolism, thus allowing increased import of cERS to satisfy a higher demand of mitochondrial glutaminyl- tRNAGln for mitochondrial protein synthesis. This novel strategy that enables a single protein to be localized in both the cytosol and mitochondria provides a new paradigm for regulation of the dynamic subcellular distribution of proteins between membrane-separated compartments. [Keywords: Dual localization; tRNA-dependent amidotransferase; tRNAGln; mitochondria; metabolism; Saccharomyces cerevisiae] Supplemental material is available at http://www.genesdev.org. Received December 12, 2008; revised version accepted March 13, 2009. In all living organisms, ribosome-mediated protein syn- indirect pathways invariably involving tRNA-dependent thesis requires the supply of a set of at least 20 perfectly conversion of a precursor mischarged aa-tRNA (Sheppard paired aminoacyl-transfer RNA (aa-tRNA) species, one et al. 2008). These pathways, which usually compensate for each of the canonical amino acids found in proteins. for the absence of a given aaRS, are not oddities of the Although most of the aa-tRNA is made by aminoacyl- translation machinery, since they sometimes constitute tRNA synthetases (aaRS), a family of 20 enzymes that the sole route for synthesis of a given aa-tRNA. Among catalyze direct acylation of each tRNA species with its all aa-tRNA species used for mRNA translation, one, cognate amino acid (Ibba and So¨ ll 2000), it is now clear glutaminyl-tRNAGln (Q-tRNAQ), is of particular interest, that the process of aa-tRNA synthesis is far from univer- not only because it displays the unique feature of being sally conserved. In different organisms, up to four aa- generated by kingdom-specific pathways or enzymes tRNA species are, or can be, generated by two-step (Tumbula et al. 2000), but also because its synthesis is still a matter of debate and remains to be elucidated for the majority of eukaryotic organelles. To date, all eukar- Corresponding authors. 3 yotes studied so far use glutaminyl-tRNA synthetase E-MAIL [email protected]; FAX 33-3-88-60-22-18. Gln 4E-MAIL [email protected]; FAX 33-3-88-41-70-70. (QRS) for the formation of cytosolic glutaminyl-tRNA Q Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.518109. (Q-ctRNA ) (Ibba et al. 2000), whereas the vast majority GENES & DEVELOPMENT 23:1119–1130 Ó 2009 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/09; www.genesdev.org 1119 Downloaded from genesdev.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press Frechin et al. of prokaryotes use the two-step transamidation pathway can be rescued by expression of a mitochondrially tar- in which two enzymes are working in tandem. First, geted version of the Bacillus subtilis GatB subunit (Kim a nondiscriminating glutamyl-tRNA synthetase (ND- et al. 1997). This suggests that a mitochondrial AdT could ERS) generates a mischarged Glu-tRNAGln (E-tRNAQ) well exist and be responsible for Q-tRNAQ formation in (Lapointe et al. 1986), then, the glutamate-charged yeast mitochondria. However, Rinehart et al. (2005) tRNAQ is transamidated into glutamine by a tRNA- demonstrated that the yeast mitochondrial glutamyl- dependent amidotransferase (AdT). The AdT enzymes tRNA synthetase (mERS) is incapable of charging with catalyzing this process differ between bacteria and ar- glutamate any of the tRNAQ species present in yeast chaea. Bacteria solely use a heterotrimeric enzyme called mitochondria. This constitutes a strong argument against the GatCAB AdT (Curnow et al. 1997; Becker and Kern the existence of a mitochondrial transamidation pathway 1998), while archaea utilize an archaeal-specific hetero- in yeast. Indeed, if there is no ND-ERS in mitochondria, dimeric GatDE AdT (Tumbula et al. 2000). then the mischarged E-tRNAQ, which is the obligatory The way Q-tRNAQ is generated in organelles is far from substrate intermediate for the AdT, cannot be generated. uniform among eukaryotes and is unknown for many Consequently, with no means of supplying the AdT with organisms. The first report on organellar Q-tRNAQ syn- its substrate, the existence of a mitochondrial tRNA- thesis came from experiments with barley chloroplast dependent transamidation pathway makes no sense. Yet, extracts, which unambiguously showed that Q-tRNAQ the S. cerevisiae genome encodes a mitochondrial GatB synthesis proceeds by transamidation of a mischarged ortholog. The logical deduction one would make is that glutamyl-tRNAGln (E-tRNAQ) (Scho¨ n et al. 1988). In the yeast mitochondrial GatB protein, Pet112p, has de- addition, this work suggested that plant and mouse viated from its canonical function in tRNA-dependent mitochondria use the same transamidation pathway to transamidation and exhibits an essential alternative mito- Gln Q form mitochondrial glutaminyl-tRNA (Q-mtRNA ), chondrial function. We therefore designed a series of based on the result that QRS activity could not be experiments to identify the functional role of this protein. detected in any of the mitochondrial extracts that were Using a systems-based approach and despite expect- assayed. The general idea that organellar and especially ations to the contrary, we characterized a novel type of mitochondrial Q-tRNAQ synthesis uniformly proceeds trimeric AdT in yeast mitochondria. Biochemical, ge- via the transamidation pathway was later reinforced by netic, and subcellular localization experiments unambig- accumulating sequence data coming from whole-genome uously prove that this AdT is mitochondrial and is Q sequencing projects. All eukaryotic genomes sequenced responsible for Q-mtRNA formation. We confirm that so far are deprived of the gene encoding a mitochondrial yeast mERS is indeed a discriminating ERS and cannot Q QRS (mQRS). With the exception of a few protozoans, produce E-mtRNA . However, to our surprise, we found they concomitantly all display a gene for a GatB ortholog, that the cytosolic ERS (cERS) can synthesize the mis- named PET112, which is always predicted to encode charged E-tRNAQ substrate for the AdT. In agreement a mitochondrial protein. Since the presence of a gatB with this result, we found that a fraction of cERS localizes gene in a given prokaryotic genome invariably signifies to mitochondria and is in fact the missing nondiscrimi- the presence of an AdT in the corresponding organism, nating ERS. By trying to understand how the cERS can the presence of mitochondrial GatB homologs (Pet112p) be dual localized, we found that the major portion of cERS in eukaryotes should predict the existence of a mitochon- is sequestered in the cytoplasm by binding to Arc1p, drial AdT. Recent studies with Arabidopsis thaliana a protein that serves as a cytoplasmic anchoring platform validated this assumption by showing that mitochondrial for cERS but also for cytosolic methionyl-tRNA synthe- Q and chloroplastic Q-tRNA are actually made by a unique tase (cMRS). Finally, we also found that the level of Arc1p dual-targeted GatCAB AdT (Pujol et al. 2008) that trans- expression is decreased when the cells are switched from amidates both mitochondrial and chloroplastic E-tRNAQ fermentation to respiratory metabolism, which results in Q formed by a unique mischarging ERS also addressed to increased cERS import to allow higher levels of Q-mtRNA both cellular compartments. This paradigm is, however, formation for elevated mitochondrial protein synthesis. contradicted by two studies reporting the existence of We propose that Arc1p constitutes a relay in the molecular a mQRS in trypanosomatidae (Nabholz et al. 1997; pathway that allows yeast cells to switch from fermenta- Rinehart et al. 2004). Likewise, a recent study reported tion to respiration.
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