Identification of a GatCAB-like AdT in yeast. (A) Growth phenotypes of the pet112Δ and pet112-TAP strains obtained in fermentation (YPD) and respiratory (YPGly) conditions after 48 h of incubation at 30°C. Origin and engineering of the strains are described in the Materials and Methods and Supplemental Table 1. (B, lanes 2,3) G250 Colloidal Blue-stained SDS-PAGE separation of the Pet112-TAP interactants purified by TAP. (Lane 1) Molecular weight standards. The three putative mitochondrial proteins, identified by mass spectrometry, are highlighted. (C) Coomassie Blue-stained SDS-PAGE of the overexpressed S. cerevisiae GatFAB AdT purified on an immobilized Co²⁺ column. The band labeled with an asterisk (*) corresponds to contaminating E. coli prolyl-isomerase (25 kDa; Mukherjee et al. 2003). (D) Autoradiogram of the TLC plate analyzing the conversion of 10 μM tRNA^Asn-bound [¹⁴C]aspartate ([¹⁴C]D) into [¹⁴C]asparagine ([¹⁴C]N) catalyzed by 0.1 μM pure S. cerevisiae GatFAB AdT in the presence (+) or absence (−) of glutamine as an amide group donor. Aspartylation and transamidation reactions of the Thermus thermophilus tRNA^Asn were performed as described previously (Bailly et al. 2007).

Mitochondrial localization and activity of the S. cerevisiae GatFAB AdT. (A) Visualization of the mitochondrial localization of the C-terminally GFP-tagged GatA, GatB, and GatF subunits by fluorescence microscopy. Yeast gatA-GFP, gatB-GFP, and gatF-GFP strains were logarithmically grown on synthetic medium as described in the Materials and Methods. Living cells were stained with Red MitoTracker and examined with a phase interference microscope as described in the Materials and Methods. GFP fluorescence can be overlapped with the Red MitoTracker staining of mitochondria. Note that a fraction of Ymr293c localizes also in the cytosol in agreement with a recent report on a possible additional function of this protein in this compartment (Federovitch et al. 2008). (B) Autoradiogram of the TLC plate showing the conversion of yeast mitochondrial tRNA-bound [¹⁴C]E into [¹⁴C]Q catalyzed by a yeast mitochondrial extract. Coupled aminoacylation and transamidation of 16 μg of unfractionated mitochondrial tRNA, using 10 μg of a tRNA-free mitochondrial protein extract, was performed in 50 μL of transamidation buffer supplemented with 20 μM of [¹⁴C]E and with (+) or without (−) 1 mM of glutamine (amide group donor). After 40 min of incubation, samples were treated as described in the Materials and Methods.

Identification of the missing nondiscriminating ERS and of the AdT tRNA^Q substrate. (A) Aminoacylation curves of unfractionated _mtRNA with _mERS and subsequent addition of _mERS (-○-) or _cERS (-◆-) when the aminoacylation plateau with _mERS was reached (16 min, dashed line). Autoradiogram of the TLC plate analyzing transamidation by purified yeast GatFAB AdT of [¹⁴C]E-_mtRNA (40 μL) generated at plateau level by _mERS (inset i) or generated at plateau level by combined charging with _mERS and _cERS (inset ii). (B) Identification by Northern blotting of the tRNA^Q and tRNA^E species present in our unfractionated _mtRNA preparations. 15 μg of unfractionated _mtRNA (mi) was hybridized with probes specific for mitochondrial-encoded tRNA^Q (_mtR^Q), tRNA^E (_mtR^E), and nuclear-encoded tRNA^Q (_ctR^Q) and tRNA^K (tRK1) shown previously to be imported into mitochondria. As a control, 15 μg of unfractionated _ctRNA (cy) was probed with the same probes. (C) Charging curves of purified _mtRNA^Q and _mtRNA^E by _cERS (respectively, -◆- and -●-) and _mERS (respectively, -◇- and -○-). (Inset) Transamidation of the pure mischarged [¹⁴C]E-_mtRNA^Q by pure yeast GatFAB AdT.

A noncanonical MTS located after the first 190 residues of _CERS is responsible for its mitochondrial import. (A) Schematic representation of the different _cERS variants. (B) Growth phenotypes on fermentative (YPD) or respiratory (YPGly) media of haploid S. cerevisiae cersfl, cersΔ90, cersΔ190, and cersΔ199 strains (see Supplemental Table 1). (C) Multiple alignment of S. cerevisiae _cERS with other eukaryotic _cERSs and glutamyl-prolyl-tRNA synthetases (EPRS). (Sce) S. cerevisiae, (Spo) Schizosaccharomyces pombe, (Dme) Drosophila melanogaster, (Ath) A. thaliana, (Hsa) Homo sapiens. The region analyzed corresponds to the residues 190–199 of the S. cerevisiae _cERS. Black shading denotes 100% amino acid identity, while gray signifies at least 80% homology (amino acid conservation in at least four out of the five proteins).

_cERS is maintained in the cytosol by binding to Arc1p, a cytosolic retention platform, whose expression is down-regulated in response to a switch from fermentation to respiration. (A) Immunodetection, by Western blot, of _cERS in purified cytosolic (cyt) and mitochondrial (mit) protein extracts of ARC1 (wild type, RS354) or arc1Δ strains. Amounts of total protein and cross-contamination of each mitochondrial extract were controlled using antibodies against Hsp70 (Hsp70) as a mitochondrial marker and against phosphoglycerokinase (PGK) as a cytosolic marker. (B) Analysis of the subcellular localization of N_cERS-GFP in ARC1 (wild type, RS354) or arc1Δ yeast strains transformed with pRS315/Nter_cERS-GFP. Living cells were stained with Red MitoTracker and examined with a confocal microscope as described in the Materials and Methods. Cellular areas where fluorescence of NcERS-GFP overlaps with the Red MitoTracker staining of mitochondria are indicated by white arrowheads. (C) Western blot quantification of import of N_cERS-GFP in mitochondria of ARC1 (wild type, RS354) or arc1Δ strains. Amounts of total protein and level of cytosolic contamination of each mitochondrial extract were controlled using anti-Hsp70 (Hsp70) and anti-PGK (PGK) antibodies, respectively. (D) Western blot quantification of the variation in expression, by wild-type yeast strain (ARC1), of Arc1p, _cERS, and _cMRS during fermentation (YPD) or respiration (YPGly). Histograms representing quantifications of the relative amounts of _cERS (A), N_cERS-GFP (C), and Arc1p (D) present in extracts were performed with ImageJ software.

Subcellular localization, ligand binding, and activities of the proteins and RNA involved in S.cerevisiae Q-_mtRNA^Q formation. In S. cerevisiae, the mitochondrial (m) tRNA^Q is glutamylated by a fraction of the cytosolic (c) ERS imported into mitochondria. The mitochondrial E-tRNA^Q is then transamidated into Q-tRNA^Q by a mitochondrial GatFAB AdT. Formation of the mitochondrial E-tRNA^E is mediated solely by the mitochondrial ortholog (_mERS) of the _cERS. A portion of the _cERS is maintained in the cytosol by binding to Arc1p that additionally by capturing the cytosolic MRS hinders its nuclear addressing. Both enzymes are essential to the cytosolic translation since they catalyze formation of the cytosolic E-tRNA^E and M-tRNA^M, respectively.