Volume 26, Issue 5, Pages 625-637 (June 2007) Evidence for an Adaptation Mechanism of Mitochondrial Translation via tRNA Import from the Cytosol  Piotr Kamenski, Olga Kolesnikova, Vanessa Jubenot, Nina Entelis, Igor A. Krasheninnikov, Robert P. Martin, Ivan Tarassov  Molecular Cell  Volume 26, Issue 5, Pages 625-637 (June 2007) DOI: 10.1016/j.molcel.2007.04.019 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Cloverleaf Structures of Cytosolic and Mitochondrial tRNAsLys of S. cerevisiae Molecular Cell 2007 26, 625-637DOI: (10.1016/j.molcel.2007.04.019) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 tRK1 Directing Activity of the N-Terminal Region of Pre-Msk1p (A) In silico predicted spatial organization (topological diagram) of pre-Msk1p. Structural elements are annotated according to the E. coli enzyme (Onesti et al., 1995, 2000). Active site elements of the C-terminal domain, which are conserved in all class II aaRS, are in black; the N-terminal anticodon binding domain with characteristic OB fold is in gray; the hinge region and “insertions sequences” are in white. Rectangles indicate the helices (H), the arrows indicate β elements. The arrow indicates the border of the truncated N-terminal version of pre-Msk1p. (B) RNA import into isolated yeast mitochondria. The upper panel represents the autoradiograph of the denaturating PAGE-separated RNAs protected in the in vitro import assay. The left path corresponds to 2% of the input. All reactions were performed in the presence of IDPs isolated from the WΔM strain. Recombinant proteins added (50 ng) are indicated above: Nter, the N-terminal domain of pre-Msk1p (amino acids 1–245); pre-Msk1p, full-sized pre-Msk1p. The error bars of the quantification diagram represent ±SEM value and result from three independent experiments, the import directing efficiency of the IDP (from WΔM) + pre-Msk1p mixture was taken as 1. (C) In vivo import of tRK1 driven by the N-terminal part of pre-Msk1p. (Left) Growth of the recombinant strain expressing the N-terminal domain of pre-Msk1p instead of the full-size molecule on glucose (YPD)- and glycerol (YPEG)-containing media. WΔM does not express any pre-Msk1p; WΔM(pNRS) expresses only the N-terminal domain of pre-Msk1p. WΔM(pMRS) is expressing the full-sized pre-Msk1p. Cultivation was at 30°C for 3 days. Serial dilutions (1:10) for each strain are presented. (Right) Northern hybridization of promitochondrial (WΔM and WΔM[pNRS]) or mitochondrial (WΔM[pMRS]) RNAs with tRK1-, tRK2-, and mitochondrial tRNALeu (tRL)-specific probes. The absence of signal with the tRK2 probe demonstrates that mitochondrial preparations were not contaminated with cytosolic tRNAs. The absence of the signal with the tRL probe reflects the loss of mtDNA. Molecular Cell 2007 26, 625-637DOI: (10.1016/j.molcel.2007.04.019) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 Functional Replacement of the MSK1 Gene by Its A. gossypii Ortholog (A) Alignment of mitochondrial lysyl-tRNA synthetases of S. cerevisiae (Msk1p) and A. gossypii (AshRS). Conserved residues are in red, semiconserved in green, those with similar hydrophobicity in blue, and nonaligned in black. The arrow indicates the border of the truncated N-terminal version of pre-Msk1p. (B) Respiratory phenotypes of AshRS expressing WΔM strains WΔM(pAshRS) and WΔM(pAshRS,pNRS) at 30°C or 37°C on YPEG medium. (C) Growth curves of the recombinant strains in liquid YPEG. (D) Oxygen consumption of the AshRS expressing WΔM strains at 30°C or 37°C on YPGal medium. The error bars of the quantification diagram represent ±SEM value and result from three independent measures. (E) Northern hybridization of mitochondrial RNA from AshRS expressing strains (as in Figure 2A). At the bottom, tRK1 import quantification diagram. The error bars result from at least two independent experiments. The ratio between the signals corresponding to tRK1 and the mtDNA-expressed tRL served to evaluate tRK1 import efficiency. The import level in WΔM(pMRS) was taken as 1. Molecular Cell 2007 26, 625-637DOI: (10.1016/j.molcel.2007.04.019) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 Stability and Aminoacylation of tRK3 in AshRS-Expressing Strains (A) Northern hybridization. The strains and temperature of cultivation are indicated at the top, the hybridization probes at the left: tRK3, tRNAGlu (tRE) and tRNAGln (tRQ). tr3, 20 ng T7 transcript of the cloned tRK3 gene possessing the same sequence as the mature tRK3. The graph at the bottom shows the ratios between the tRK3 and tRE/Q signals; the ratios in WΔM(pMRS) strain are taken as 1. (B) Analysis of tRK3 aminoacylation by northern hybridization of RNA isolated and separated in acid conditions. tRL, hybridization with mitochondrial tRL-specific probe is used as a reference. OH-da, tRNA from WΔM(pMRS) strain deacylated in basic conditions. Positions of aminoacylated (aa) and deacylated (da) forms are indicated. 100% corresponds to fully aminoacylated tRNAs. (C) Analysis of tRK1 aminoacylation in the mitochondria. tRK2 probe was used to prove the absence of cytosolic contamination. The error bars of the quantification diagrams in (B) and (C) represent the ±SEM value and result from at least two independent experiments. Molecular Cell 2007 26, 625-637DOI: (10.1016/j.molcel.2007.04.019) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 Mitochondrial Translation in Recombinant Strains (A) Autoradiograph of the pulse-chase-labeled mitochondrial proteins separated on the 15% denaturing PAGE. An equal number of cells was taken for translation reactions in each series. Mitochondrial proteins are indicated according to a standard mitochondrial translation pattern (Fox et al., 1991). To confirm that the observed pattern corresponded to the expected polypeptides, western analysis was performed with antibodies against yeast cytochrome b and Cox2p (@Cytb and @Cox2p), at the left. (B) Quantification of the relative amounts of three mitochondrially synthesized proteins: Cox1p, Cox2p, and Var1p (as indicated at the right). The ratio between the signal corresponding to a given protein and the total amount of radioactive polypeptides served for evaluation. The value for the WΔM(pMRS) was taken as 1. (C) Quantification of the overall mitochondrial translation activity. The error bars of the diagrams represent ±SEM value and result from at least two independent experiments (B and C). Molecular Cell 2007 26, 625-637DOI: (10.1016/j.molcel.2007.04.019) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 The Effect of the Elevated Temperature on Mitochondrial tRNAs Modification (A–C) Primer extension experiment. (A) The cloverleaf structure of the yeast mitochondrial tRNALys (tRK3). The primer used is indicated by a gray line, ∗ indicates the anticipated arrest of extension at the modified U34, and ∗∗ indicates the position of the first G after the start of extension and corresponds to the arrest of extension in the presence of ddC. (B) Autoradiograph of the extension products. At the left, the sequence of the RNA is indicated, from the primer to the 5′ end; at the right, asterisks show positions of the arrests. tr3, the T7 transcript of the tRK3 gene used as the control template. “ddNTP,” elongation was performed in the presence of all four ddNTPs. Extensions with yeast RNA were performed in presence of ddC. (C) Quantification of the extension assay. The ratio between the signals corresponding to the product of reverse transcription arrest at the modified base of the anticodon and at the first G was taken as the indication of the modification extent. Results of two independent experiments were quantified. (D) Analysis of thio modification at the position 2 of the wobble uridine by northern hybridization of mitochondrial tRNAs separated in APM-containing gels. In parallel, RNA separations were performed in gels without APM. The retarded diffused zone corresponds to the thiolated (T) version of the tRNA; the band at the bottom corresponds to the nonthiolated version (NT). Longer gels without APM are presented to demonstrate the absence of degradation. The numbers above the autoradiographs correspond to the same samples. (E) Quantification of the APM gels. Similar hybridizations were performed with the probes for tRK3, tRQ (presented in [D]), and tRE (included only in the graph). Percentage of modification was evaluated as a percent of the T signal with respect to T + NT ones. The error bars of the diagram represent ±SEM value and result from two independent experiments. Molecular Cell 2007 26, 625-637DOI: (10.1016/j.molcel.2007.04.019) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 7 Hypothetic Mechanism of tRK1 Involvement in Mitochondrial Translation Both tRK2, decoding the AAA, and tRK1, decoding exclusively AAG lysine codons, are required for cytosolic translation. tRK3, encoded in the mitochondrial DNA, can decode both AAA and AAG codons in mitochondrial mRNAs at 30°C. At 37°C, the cmnm5s2U34 in the wobble position of tRK3 becomes hypomodified, which makes less efficient AAG decoding, without affecting AAA decoding. In these stress conditions, imported tRK1 can cure the deficiency by decoding the AAG codons. Molecular Cell 2007 26, 625-637DOI: (10.1016/j.molcel.2007.04.019) Copyright © 2007 Elsevier Inc. Terms and Conditions