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Originally published In Press as doi:10.1074/jbc.M508281200 on September 14, 2005

J. Biol. Chem., Vol. 280, Issue 46, 38186-38192, November 18, 2005
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Loss of Editing Activity during the Evolution of Mitochondrial Phenylalanyl-tRNA Synthetase*

Hervé Roy{ddagger}, Jiqiang Ling§, Juan Alfonzo{ddagger}§, and Michael Ibba{ddagger}§1

From the {ddagger}Department of Microbiology and the §Biochemistry Program, The Ohio State University, Columbus, Ohio 43210-1292

Received for publication, July 28, 2005 , and in revised form, September 13, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Accurate selection of amino acids is essential for faithful translation of the genetic code. Errors during amino acid selection are usually corrected by the editing activity of aminoacyl-tRNA synthetases such as phenylalanyl-tRNA synthetases (PheRS), which edit misactivated tyrosine. Comparison of cytosolic and mitochondrial PheRS from the yeast Saccharomyces cerevisiae suggested that the organellar protein might lack the editing activity. Yeast cytosolic PheRS was found to contain an editing site, which upon disruption abolished both cis and trans editing of Tyr-tRNAPhe. Wild-type mitochondrial PheRS lacked cis and trans editing and could synthesize Tyr-tRNAPhe, an activity enhanced in active site variants with improved tyrosine recognition. Possible trans editing was investigated in isolated mitochondrial extracts, but no such activity was detected. These data indicate that the mitochondrial protein synthesis machinery lacks the tyrosine proofreading activity characteristic of cytosolic translation. This difference between the mitochondria and the cytosol suggests that either organellar protein synthesis quality control is focused on another step or that translation in this compartment is inherently less accurate than in the cytosol.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The aminoacyl-tRNA synthetases (aaRS)2 are a ubiquitous and essential protein family required for protein synthesis (1–3). Structurally and functionally the aaRSs are divided into two unrelated but biochemically analogous groups, class I and class II (4–6). The aaRSs attach amino acids to the 3'-ends of tRNA containing the corresponding anticodon sequence, and the resulting aminoacyl-tRNAs (aa-tRNAs) are used as substrates for ribosomal translation of mRNA. The accuracy of aa-tRNA synthesis is generally assured by the existence of aaRSs specific for each particular amino acid-tRNA pair. Cognate tRNA recognition and discrimination of non-cognate RNAs are achieved by sequence-specific direct and indirect readout of the numerous combinations of bases present in tRNAs (7–10). The relative structural simplicity and inherent similarity between the amino acid substrates makes their accurate recognition and discrimination more challenging. Although some amino acids such as cysteine and tyrosine are different enough to allow their specific recognition by a particular aaRS (11, 12), others such as valine and isoleucine are less easily distinguished. For example the class I aaRS isoleucyl-tRNA synthetase (IleRS) is only able to poorly discriminate against valine, which has a misactivation rate of about 1:200 compared with the cognate substrate isoleucine. Despite this significant rate of misactivation and misaminoacylation, the accuracy of translation is not compromised because of the existence of an intrinsic proofreading and editing mechanism in IleRS that specifically hydrolyzes both misactivated Val-AMP and misaminoacylated Val-tRNAIle (13, 14). In addition to IleRS, many other class I and class II aaRSs also employ editing to prevent release of non-cognate aa-tRNA and subsequent loss of translational accuracy (reviewed in Refs. 15 and 16). With a few notable exceptions (17), editing generally occurs in specialized domains distal from the active site such as the class I-specific CP1 region of IleRS and leucyl-, and valyl-tRNA synthetases. The editing domains of class II aaRSs are more diverse than their class I counterparts and include the "HXXXH" domain found in both alanyl- (18) and threonyl-tRNA synthetases (ThrRS) (19), an unrelated domain in archaeal ThrRS (20, 21), the Ybak-like domain in prolyl-tRNA synthetase (ProRS) (22–24), and the B3/B4 domain of phenylalanyl-tRNA synthetase (PheRS) (25).

The editing domains of aaRSs are normally found in the same subunit as the active site, the only known exceptions being PheRS and certain examples of ProRS and ThrRS. PheRS is normally an ({alpha}{beta})2 heterotetramer, with the active site located in the {alpha}-subunit and tRNA binding sites in both subunits. Recent studies in bacteria revealed that hydrolysis of misaminoacylated Tyr-tRNAPhe occurs at an editing site in the {beta}-subunit ~40 Å from the active site (25). This editing site, at the boundary of the B3 and B4 subdomains, is highly conserved in bacterial PheRSs and aligns with a divergent domain conserved in eukaryotic and archaeal sequences that is believed to participate in proofreading. It is less clear whether mitochondrial PheRSs also have the potential to edit misacylated tRNAs, as they are monomers and thus lack the conventional ({alpha}{beta})2 oligomeric form (26, 27). Mitochondrial PheRS sequences are most closely related to the bacterial type, being chimeras of the {alpha}-subunit (with an insertion between motifs 2 and 3) and the C-terminal tRNA anticodon binding domain (B8) of the {beta}-subunit (26). Despite their similarity to bacterial PheRSs, the mitochondrial versions do not contain regions analogous to the known editing domain (25). Although it was originally suggested that mitochondrial PheRSs were active in editing (28), later studies questioned the purity of the enzymes used for these studies (26). Here we describe an investigation of amino acid specificity and editing by mitochondrial and cytosolic PheRSs from the yeast Saccharomyces cerevisiae. Both enzymes display a lack of specificity toward Phe and are able to bind Tyr. The cytosolic enzyme is shown to be less specific than its mitochondrial counterpart but contains an editing site in the {beta}-subunit that specifically edits misacylated Tyr-tRNAPhe. The mitochondrial enzyme is deprived of such an activity and is also able to synthesize the misaminoacylated species Tyr-tRNAPhe. Editing activity toward Tyr-tRNAPhe is also absent from mitochondrial extracts, indicating the lack of this quality control step in vivo. These findings are discussed in the context of other recent studies, which together with the data presented here, raise the possibility that mitochondrial protein synthesis may be less accurate than its cytosolic counterpart.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Strains, Plasmids, and General Methods—Plasmids carrying cytosolic or mitochondrial yeast tRNAPhe genes for T7 runoff transcription were gifts from O. Uhlenbeck (Northwestern University). The strains Escherichia coli XL1-Blue/pQE31-FRS-ec (producing His6-tagged E. coli PheRS) and E. coli BL21/pQE32-FRS-sc (producing His6-tagged cytosolic S. cerevisiae PheRS) were gifts from D. A. Tirrell (California Institute of Technology). E. coli BL21-RIL/pET16b producing His6-tagged mitochondrial S. cerevisiae PheRS was a gift from R. A. Zimmermann (University of Massachusetts). All His6-tagged proteins were purified to >99% purity on nickel-nitrilotriacetic acid-agarose (Qiagen) by standard procedures. The specific activities of the purified proteins (nmol of Phe attached/mg/min) were: wild-type cytoplasmic PheRS, 22; D243A cytoplasmic PheRS, 12; wild-type mitochondrial PheRS, 89; and A333G mitochondrial PheRS, 73. Point mutations were introduced into the pheST genes (encoding the PheRS {alpha}- and {beta}-subunits, respectively) by PCR with two self-complementary 33-mer oligonucleotides that carried the appropriate mutations. Reactions were performed with the QuikChange site-directed mutagenesis kit (Stratagene). Introduction of the desired mutations was monitored by sequencing of the resulting genes. Commercial L-Tyr (Sigma) was shown to be free of phenylalanine (Phe) contamination by pyrophosphate exchange before and after recrystallization of the amino acid as described previously (29). LB and M9 media were prepared as described previously (30). All buffers were adjusted to the correct pH with NaOH unless otherwise indicated. Immunoblotting was performed as described previously (31) using rabbit polyclonal antibodies raised against yeast cytosolic PheRS (AnimalPharm, Healdsburg, CA). tRNATrp and tryptophanyl-tRNA synthetase were prepared as described previously (31).

Preparation of in Vitro Transcribed tRNAPhe—In vitro T7 RNA polymerase runoff transcription reactions were conducted according to standard procedures (32). After ethanol precipitation, tRNA transcripts were resuspended by heating in 10 mM Hepes (pH 7.2), 1 mM EDTA, and 7 M urea, loaded on a Resource Q 6 anion exchange column (Amersham Biosciences) and eluted with a gradient of 0–1 M NaCl in the loading buffer. Fractions containing tRNA were pooled and desalted on a PD-10 column (Amersham Biosciences) against 10 mM Hepes (pH 7.2). The transcripts were ethanol precipitated, washed with 80% ethanol, dried, resuspended in 10 mM Hepes, pH 7.2, and 2 mM MgCl2, and finally refolded by incubation for 1 min at 75 °C followed by slow cooling down to room temperature.

ATP-PPi Exchange Reaction—The reaction was carried out at 37 °C in a medium containing 100 mM Na-Hepes (pH 7.2), 30 mM KCl, 10 mM MgCl2, 2 mM NaF, 2 mM ATP, 2 mM [32P]PPi (1 cpm/pmol), various amounts of Phe (1.5–300 µM) and Tyr (0.2 to 7 mM), and 13 nM cytosolic PheRS or 10–70 nM mitochondrial enzyme. After 1–5 min, 25 µlofthe reaction were removed and added to a solution containing 1% charcoal, 5.6% HClO4, and 75 mM PPi. The radiolabeled ATP bound to the charcoal was filtered through a 3MM Whatman filter disc under vacuum and washed three times with 5 ml of water and once with 5 ml of ethanol. The filters were dried, and the radioactivity was counted by liquid scintillation counting (Ultima Gold, Packard Instrument Co.).

Aminoacylation Assay with Radiolabeled Amino Acids—Aminoacylation was performed in 100 mM Na-Hepes (pH 7.2), 30 mM KCl, 2 mM ATP, 10 mM MgCl2, 25 µM L-[14C]Phe (280 cpm/pmol), 5 µM tRNA transcript, and 10–100 nM yeast PheRS. 15-µl aliquots were spotted on 3MM filter disks (Whatman), washed three times in 10% trichloroacetic acid, and dried. The amount of radioactivity retained was determined by liquid scintillation counting. One unit of PheRS corresponded to the amount of enzyme necessary to catalyze the formation of 1 nmol of Phe-tRNAPhe min–1 mg–1 protein at 37 °C.

Aminoacylation Assay with Radiolabeled tRNA—Synthesis of tRNA 32P-labeled pA76 transcripts were performed essentially as described previously (33) except that the CCA-3'-end was removed prior to labeling. Briefly, the CCA-3'-terminal nucleotides of the tRNA were first removed by treatment of 20 µM tRNA transcript with 73 µg/ml Crotalus atrox venom (Sigma) in a buffer containing 40 mM sodium Gly (pH 9.0) and 10 mM magnesium acetate. The mix was incubated for 2 h at 21 °C and phenol/chloroform extracted and ethanol precipitated and finally desalted by gel filtration through a Sephadex G 25 column (Amersham Biosciences). The CCA-3'-end of the tRNA was reconstituted and radiolabeled by incubation for 10 min at 37 °C with 0.5 µM snake venom-treated tRNA in 50 mM Na-Gly (pH 9.0), 10 mM MgCl2, 10 µM CTP, 9 µM ATP, 1 µM [{alpha}-32P]ATP with 3 µg/ml E. coli tRNA-terminal nucleotidyltransferase (34) in a final volume of 20 µl. The reaction was stopped by the addition of 1 volume of phenol, and the resulting mixture was gel filtered twice through a G25 column. As described previously (33), the aminoacylation reaction was performed in a 10-µl aminoacylation medium (see above) containing 5 mM cold amino acids or Phe analogues, 5 µM transcript, and a trace of radiolabeled tRNA. After 15 min of incubation an aliquot was removed and incubated for 30 min at room temperature with P1 RNase. The liberated [{alpha}-32P]AMP and aminoacyl-[{alpha}-32P]AMP were separated by TLC on polyethyleneimine cellulose and visualized as described previously (33).

Post-transfer Editing Assay—The cytosolic or mitochondrial Tyr-tRNAPhe and Phe-tRNAPhe (S. cerevisiae transcripts) were prepared as described previously (25) in an aminoacylation reaction containing 30 µM [3H]Tyr (180 cpm/pmol), 0.5 µM mitochondrial PheRS A333G, and 5 µM corresponding in vitro transcribed tRNAPhe. Comparison to the total tRNA concentration then allowed us to estimate that purified Phe-tRNAPhe yields were about 20% (i.e. these preparations also contained 80% uncharged tRNA), whereas Tyr-tRNAPhe yields were 15% (85% uncharged), within the typical range expected for aminoacyl-tRNA preparations. Post-transfer editing reaction mixtures contained 100 mM Na-Hepes (pH 7.2), 30 mM KCl, 10 mM MgCl2, 1 µM [3H]Tyr-tRNAPhe, and a catalytic amount of PheRS (5 nM cytosolic or 5 nM to 2 µM mitochondrial yeast PheRS). The mixture was incubated at 37 °C, and the post-transfer editing reaction was followed by measuring the remaining radiolabeled aa-tRNA in aliquots of 15 µl after 0–12 min of incubation as described for the aminoacylation assay (see above). The amount of cytosolic or mitochondrial crude extract from S. cerevisiae used in the editing assay was determined according to the PheRS specific activity measured for the extracts, 0.78 nmol/min/mg of protein and 0.19 nmol/min/mg of protein, respectively.

Preparation of Cytosolic and Mitochondrial Fractions of S. cerevisiae— S. cerevisiae strain W303 was grown in rich medium containing 2% galactose, and mitochondria were isolated as described previously (35). Spheroplasts were prepared by Zymolyase-20T (ICN) treatment and broken by three passes through an EmulsiFlex®-C5 (AVESTIN) in 0.6 M sorbitol (Fluka), 10 mM Tris-HCl (pH 7.4), and 1 mM phenylmethylsulfonyl fluoride. Mitochondria were purified by centrifugation and extensive washing in the same buffer as described previously (36). The cytosolic fraction obtained during this procedure was clarified by ultracentrifugation at 100,000 x g. Highly purified organelles were obtained by centrifugation at 100,000 x g (Beckman SW41) in a PercollTM step gradient (Amersham Biosciences) (40% PercollTM in 0.6 M sorbitol (Fluka), 10 mM Tris-HCl (pH 7.4), overlaid with 20% PercollTM in the same buffer). These procedures yielded cytosolic and mitochondrial fractions with less than 0.5% cross-contamination as judged by Western blot analysis with antibodies specific for proteins in the different compartments as described elsewhere (37). Mitochondria were suspended in aminoacylation buffer (see above) followed by sonication for 1 min at 50% output with a Sonifier 450 (Branson) equipped with a microprobe. The resulting extract was centrifuged at 100,000 x g for 1 h (S100) to precipitate the membrane fractions. The resulting soluble extracts were used for aminoacylation assays.

Phylogenetic Analyses—179 PheRS {alpha}-subunit sequences (92 from eubacteria, 23 from archaea, and 35 and 29 cytosolic and mitochondrial eukaryotic sequences, respectively) were aligned with the ClustalX program version 1.83 (38). The alignment process was guided with the known three-dimensional structure of PheRS of Thermus thermophilus and refined manually. The trees were generated by applying the neighbor-joining method with the program ClustalX or the Phylip package version 6.63 (39) to a set of 100 bootstrap replicates of the ungapped alignment. Maximum likelihood analysis was also applied to the same alignment with the program Puzzle version 5.2 (40) where more than 50 x 106 quartets were analyzed. The trees were rooted using the nine known sequences of o-phosphoseryl-tRNA synthetase found in methanogenic archaea that were previously identified as paralogues of the {alpha}-subunit of PheRS (41).



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  		FIGURE 1.
Specific deacylation of Tyr-tRNAPhe by yeast cytosolic PheRS. Reactions were performed with 1 µM Tyr-tRNAPhe prepared from in vitro transcribed yeast cytosolic tRNAPhe (circles) or yeast mitochondrial tRNAPhe (triangles) with the addition of 5 nM cytosolic PheRS (filled symbols) or in the absence of enzyme (open symbols). Values shown are the means of three independent experiments, with error bars representing ± 1 S.D. The possible contribution of nonspecific nuclease activity to the observed deacylation was excluded by the finding that cytosolic PheRS did not significantly deacylate Phe-tRNAPhe after its synthesis (see for example Fig. 3C).

 



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  		FIGURE 2.
Comparison of the {beta}-subunits of eubacterial and eukaryotic PheRSs. A, modular structure of the PheRS {beta}-subunit. B, sampling of an alignment of the B3/B4 domain regions. Positions previously identified as being involved in the editing activity of E. coli PheRS are indicated with black arrows. The equivalent positions found in the yeast {beta}-subunit are indicated with white arrows. The sequence alignment (44 eubacterial and 44 eukaryotic sequences) was shaded with the program Bioedit. Gray boxes indicate 60% conservation according to similarity matrix (PAM250)-based shading. Black boxes indicate residues identical in more than 60% of the sequences.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The {beta}-Subunit of Cytosolic PheRS Contains a Post-transfer Editing Site—Previous studies (42, 43) suggested that yeast cytosolic PheRS could hydrolyze Tyr-tRNAPhe, as recently directly demonstrated for the E. coli enzyme (25). Tyr-tRNAPhe was synthesized and added to a deacylation reaction containing yeast cytosolic PheRS. Cytosolic PheRS was able to specifically deacylate Tyr-tRNAPhe derived from both cytosolic and mitochondrial tRNAs, indicating the presence of trans editing activity (Fig. 1). Amino acid sequences from 179 PheRSs were aligned to investigate whether this activity in the yeast enzyme could be attributed to a catalytic site in the {beta}-subunit, as is the case in E. coli. Examination of the PheRS {beta}-subunit sequence alignment allowed identification of domains B1, B3, and B4 (B2 is absent from eukaryotes), but B3/B4 residues which are involved in bacterial editing were not well conserved in their eukaryotic counterparts (Fig. 2). The average percentage amino acid identities observed within eubacterial and eukaryotic B3/B4 ungapped domain alignments were 42 and 57%, respectively, whereas there was far less similarity when eukaryotic and eubacterial B3/B4 domains were compared with each other (15% identity). No conservation was seen at the small residues Thr354 and Ala356, changes in which ablated editing by E. coli PheRS. Moderate conservation was observed at His265 and Glu334 (His158 and Asp243, respectively, in yeast PheRS) suggesting that those residues might contribute to the editing activity of the cytosolic enzyme. Based on this analysis, cytosolic PheRS variants were produced containing the replacements {beta}H158A and {beta}D243A. Although the {beta}H158A replacement had no effect (data not shown), introduction of {beta}D243A led to a loss in Tyr-tRNAPhe deacylation activity (Fig. 3A) similar to that observed for E. coli variants defective in editing (25). The loss of editing but not synthetic activity resulting from the {beta}D243A replacement was confirmed by the ability of this PheRS variant to synthesize Tyr-tRNAPhe (Fig. 3B). The misaminoacylation activity of PheRS {beta}D243A was specific for yeast cytosolic tRNAPhe, as mitochondrial tRNAPhe was not a substrate for tyrosylation. This confirms previous indications that mitochondrial tRNAPhe could not be phenylalanylated by cytosolic PheRS (Fig. 3C). This discrepancy between the aminoacylation and editing capacities of mitochondrial tRNAPhe, together with the absence of an obvious editing domain, prompted us to investigate the organellar PheRS in more detail.

Selectivity of the Active Sites of Mitochondrial and Cytosolic PheRS— The specificity of the active sites of both yeast PheRSs toward Phe and Tyr was investigated by determining the catalytic constants for steady-state amino acid activation from ATP-PPi exchange kinetics (TABLE ONE). The mitochondrial PheRS was four times more efficient than the cytosolic PheRS with respect to Phe activation. This was mostly because of the 7-fold weaker apparent affinity of the cytosolic enzyme for its substrate compared with the mitochondrial enzyme. This tendency was reversed when Tyr was the substrate. Because Tyr exhibits poor solubility we were unable to saturate the mitochondrial enzyme. Instead, the kcat/Km was estimated at sub-saturating concentrations of Tyr. Although the cytosolic enzyme was twice as efficient for Tyr activation as its mitochondrial counterpart, for both enzymes the relative efficiency of activation of Phe was only 1–2 orders of magnitude higher than for Tyr. To determine whether such a poor apparent selectivity was because of the contamination of Tyr by a trace of Phe we assessed the purity of the Tyr substrate by mass spectrometry (data not shown). Even after several cycles of heating and cooling, the Tyr used for our experiments was found to be stable and free of any trace of Phe.


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  		TABLE ONE
Steady-state kinetic constants for ATP-[32P]PPi exchange for cytosolic and mitochondrial PheRS from yeast

 



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  		FIGURE 3.
Disruption of post-transfer editing by yeast cytosolic PheRS. A, post-transfer editing of Tyr-tRNAPhe by wild-type and variant PheRSs. Reactions were performed with 1 µM cytosolic Tyr-tRNAPhe in the presence of 5 nM enzyme. B, tyrosylation of tRNAPhe by cytosolic PheRS. Reactions were performed with 10 µM cytosolic tRNA or 3.5 µM mitochondrial tRNA and 0.1 µM PheRS. C, phenylalanylation of mitochondrial tRNAPhe. Reactions were performed with 3.5 µM in vitro transcribed tRNAPhe and 0.1 µM PheRS. Circle, wild-type cytosolic PheRS; triangle, editing-defective ({beta}D243A) cytosolic PheRS; square, mitochondrial PheRS; diamond, without enzyme; filled symbols, cytosolic tRNA; open symbols, mitochondrial tRNA.

 
Specificity of Yeast Cytosolic PheRS Editing—To further define the specificity of the editing reaction several amino acids and Phe analogues were tested for their ability to be attached to tRNAPhe by cytosolic PheRS and the editing-defective variant {beta}D243A. The compounds used were the 20 canonical amino acids, three intermediates of Phe metabolism (phenylpyruvate, p-hydroxyphenylpyruvate, and prephenate), and four other Phe analogues (p-fluoro, p-chloro, p-bromo-, and p-amino-Phe) that are known to be charged but not edited by E. coli PheRS (25). In addition to Phe, para-halogenated Phe derivatives, p-amino-Phe, Leu, and Trp were attached to tRNA by both enzymes indicating that all were substrates for the synthetic site of the {alpha}-subunit but not for the editing site of the {beta}-subunit (Fig. 4A). In the case of Trp, this supports previous data from in vivo misincorporation experiments (44). The {beta}D243A variant also synthesized Tyr-tRNAPhe indicating that this particular aminoacyl group is recognized by both the synthetic and editing sites of PheRS (Fig. 4B).

Mitochondrial PheRS Lacks the Capacity to Edit Mischarged tRNAPhe— To test the capacity of yeast mitochondrial PheRS to edit misaminoacylated products, Tyr-tRNAPhe was incubated with the enzyme under conditions previously used to monitor the same activity in bacterial and cytosolic enzymes. No significant hydrolysis of Tyr-tRNAPhe was observed in the presence of excess enzyme, the rate of deacylation being comparable with that seen in the absence of PheRS (Fig. 5A). These data showed that yeast mitochondrial PheRS, in contrast to its cytosolic counterpart, was unable to trans edit exogenous Tyr-tRNAPhe. To probe the possibilities that mitochondrial PheRS either edits solely at the pretransfer step or that it edits Tyr-tRNAPhe only in cis (i.e. when still bound to PheRS), we attempted to tyrosylate tRNAPhe with the wild-type enzyme. Wild-type mitochondrial PheRS was able to stably synthesize Tyr-tRNAPhe, suggesting the absence of effective editing mechanisms in this enzyme (Fig. 5B). To investigate whether mitochondrial PheRS contains a low level of cis editing activity not readily detectable under the standard assay conditions, the amino acid binding pocket was enlarged to better accommodate tyrosine as described previously (45) for the E. coli enzyme. The resulting variant, mitochondrial PheRS A333G, showed considerably improved Tyr-tRNAPhe synthesis compared with the wild type providing further evidence that the organellar protein lacks editing activity (Fig. 5B). We also attempted to monitor pretransfer editing as described previously (25) for the E. coli enzyme but were unable to detect any significant activity for either the cytosolic or mitochondrial enzymes (data not shown).

Trans Editing of Tyr-tRNAPhe Is Confined to Cytosolic Protein Synthesis in Yeast—Although our in vitro studies showed that mitochondrial PheRS could not edit Tyr-tRNAPhe, it is possible that other factors might act in trans after dissociation of the non-cognate aa-tRNA from the enzyme. Such factors could include specific trans editing enzymes (24, 46) or cytosolic PheRS imported into yeast mitochondria as described for other aaRSs (37). Yeast mitochondrial and cytosolic fractions were purified as described previously (37), and the localization of PheRS was examined in each compartment by immunoblotting (Fig. 6A). Cytosolic PheRS was not detectable in mitochondrial extracts, suggesting that trans editing by imported {beta}-subunits would not compensate for the absence of editing activity in the mitochondrial enzyme. The amount of each subcellular fraction required to aminoacylate comparable amounts of tRNAPhe with Phe was quantified (Fig. 6B), and based upon these measurements comparable phenylalanylation activities were then incubated with different Tyr-tRNAPhe species and the rate of deacylation monitored (Fig. 6C). The cytosolic fraction was able to rapidly deacylate Tyr-tRNAPhe, whereas the mitochondrial extract showed no significant difference in the rates of deacylation of Phe-tRNAPhe and Tyr-tRNAPhe. These findings were in agreement with the in vitro data described above and supported the notion that Tyr-tRNAPhe is not subject to proofreading and editing in yeast mitochondria.



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  		FIGURE 4.
Substrate specificity of editing by yeast cytosolic PheRS. TLC analysis of the aminoacylation of tRNAPhe with various amino acids and Phe analogues by the wild-type cytosolic PheRS (A) and the editing-defective variant {beta}D243A PheRS (B). TLC analysis of the aminoacylation of tRNATrp with Trp by tryptophanyl-tRNA synthetase (C). No aa, no amino acid.

 
Phylogenetic Analysis of PheRS—Phylogenetic trees were calculated using two neighbor-joining methods and a character-based maximum likelihood method and rooted with an alignment of o-phosphoseryl-tRNA synthetase sequences. All trees exhibited the same overall topology (Fig. 7), with the mitochondrial/eubacterial sequences forming one major cluster and archaeal/eukaryotic sequences the other. In agreement with previous reports (47, 48), the only exceptions were spirochete PheRS sequences, which clustered with the archaea, and mycoplasmataceae PheRS sequences, which clustered with the mitochondria.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Yeast Mitochondrial PheRS Is an Error-prone Enzyme—Previous studies (26) have shown that although the two PheRSs of yeast differ significantly in their tertiary and quaternary structures, they share similar kinetic parameters for cognate aminoacylation and are thus functionally analogous. The most significant difference between the cytosolic and mitochondrial versions of human PheRS is a lower apparent affinity for tRNA in the organellar enzyme (27). High-resolution crystal structures suggested that the substantial differences between the canonical ({alpha}{beta})2 PheRS and the mitochondrial {alpha}-type might not significantly impact tRNA aminoacylation, as the additional domains in the former were presumed to have alternative non-canonical functions. For example, the helix-turn-helix motif in domain B5 was shown to be a bona fide DNA binding motif in T. thermophilus PheRS, capable of binding looped double-stranded DNA (49). Deletion and mutagenesis of B5 and other domains did not abolish the canonical activity of PheRS, leading to the conclusion that domains 1–5 of the {beta}-subunit are dispensable for efficient aminoacylation (50). One notable exception is the B3/B4 editing domain, which is required in vitro and in vivo for the hydrolysis of non-cognate Tyr-tRNAPhe synthesized by PheRS. No region homologous to B3/B4 is detectable at the amino acid sequence level in mitochondrial PheRS, and the studies described here show that this correlates with a lack of editing activity in the enzyme. Comparison of Phe and Tyr activation by the cytosolic and mitochondrial enzymes showed only modest differences in substrate specificities, indicating that both enzymes have significant capacities for non-cognate aminoacylation. Previous studies (51) have shown that Tyr and Phe are likely present at comparable levels in mitochondria and the cytosol, indicating that the likelihood of non-cognate amino acid activation by PheRS will not be affected by subcellular localization. For the cytosolic enzyme, the existence of an editing mechanism ensures that any errors in amino acid activation do not affect the overall accuracy of the aminoacylation reaction. No such editing mechanism exists, either in cis or trans, during mitochondrial phenylalanylation, and the organellar PheRS can be considered an error-prone enzyme. Other aaRSs, such as certain ProRSs (46, 52) and ThrRSs (21), have also been shown to have natural uncorrected mischarging activities that result in non-cognate aminoacyl-tRNA synthesis. However, in both cases the enzymes are not considered error-prone, as they associate with other proteins that ensure non-cognate aminoacyl-tRNAs are rapidly edited (53). Mitochondrial PheRS is markedly different in that no detectable editing activity is associated with it, suggesting amino acid specificity was lost during evolution from more accurate ancestral proteins.



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  		FIGURE 5.
Non-cognate aminoacylation by yeast mitochondrial PheRS. A, deacylation of Tyr-tRNAPhe. Tyr-tRNAPhe was prepared using in vitro transcribed yeast mitochondrial tRNAPhe and incubated in the presence of 1 µM (•) yeast mitochondrial PheRS or without enzyme (x). B, aminoacylation of the mitochondrial tRNAPhe transcript (3.5 µM) with Tyr (30µM) by the mitochondrial PheRS (2µM). •, wild-type mitochondrial PheRS; {blacktriangleup}, A333G mitochondrial PheRS. The comparatively low final charging level of tRNA (10–30%) likely reflects reactions performed at substantially sub-saturating concentrations of Tyr because of practical considerations.

 



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  		FIGURE 6.
Aminoacylation and post-transfer editing by yeast mitochondrial and cytosolic fractions. A, immunodetection of PheRS in cytosolic and mitochondrial fractions. 20 µg of protein from mitochondrial and cytosolic fractions was subjected to SDS/PAGE, and the PheRSs were subsequently detected by immunoblotting using rabbit anti-yeast cytosolic PheRS antibodies. This allowed detection of mitochondrial PheRS ({alpha}m) and both subunits of the cytosolic PheRS ({alpha}c and{beta}c) as indicated by the corresponding arrows. Lanes: 1, mitochondrial fraction; 2, cytosolic fraction. B, phenylalanylation activity. Aminoacylation of the cytosolic tRNAPhe transcript (5 µM) was performed with Phe (30 µM) by the mitochondrial (160 µg of protein/ml) ({blacktriangleup}) or cytosolic (180 µg of protein/ml) (•) fractions. C, post-transfer editing of Tyr-tRNAPhe. Tyr-tRNAPhe (black) or Phe-tRNAPhe (gray) was prepared using in vitro transcribed yeast cytosolic or mitochondrial tRNAPhe and incubated, respectively, with the same amounts of cytosolic (circles) or mitochondrial (triangles) crude extracts as used in B.

 



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  		FIGURE 7.
Phylogenetic analysis of PheRS {alpha}-subunits (A) and comparison of the organization of eubacterial and mitochondrial PheRS genes (B). Black squares, dark gray squares, and light gray squares indicate nodes found in >95%, >85%, and >65%, respectively, of the trees generated by neighbor-joining or maximum likelihood analysis. A single recombination event (dashed line) involving the eubacterial {alpha}, {beta}-subunit operon might be at the origin of the fused {alpha}, {beta} mitochondrial PheRS.

 
Loss of the Editing Subdomain during Evolution of Mitochondrial PheRS—Previous analyses (47, 48) based upon alignments of PheRS {alpha}-subunit amino acid sequences indicated a canonical phylogeny for this protein family, with the only significant horizontal gene transfer being observed in the spirochetes. The updated analysis described here provides a similar phylogenetic tree composed of monophyletic archaeal/eukaryotic and bacterial clusters, with the mitochondrial PheRSs forming a single, distinct subgroup within the latter (Fig. 7). These data suggest a single bacterial origin for extant mitochondrial PheRSs but provide few clues on the subsequent evolution of the organellar protein. The widespread retention of a discrete full-length {beta}-subunit indicates that editing activity was lost after separation of the mitochondrial proteins. In addition to the absent B3/B4 editing module, mitochondrial PheRSs lack the B5 DNA binding domain, B6/B7 oligomerization domain, and B2 EMAP domain, suggesting that loss of editing may have been part of a broader process whereby numerous functions dispensable for protein synthesis were lost during evolution of the organellar protein. This loss of function is, to date, limited to mitochondria, as chloroplast PheRSs are of the ({alpha}{beta})2 type and contain an intact B3/B4 domain (54).

Editing of Misacylated tRNAs in Mitochondria—The loss of editing function from mitochondrial PheRS is particularly striking because it was accompanied by a significant change in the oligomeric state of the protein. Less dramatic changes were also shown to lead to a loss of editing function from human mitochondrial LeuRS, which is able to stably synthesize Ile-tRNALeu in vitro (55). The editing function of LeuRS is contained in the highly conserved CP1 domain (56, 57), but key residues in this region necessary for hydrolysis of non-cognate aminoacyl-tRNA have diverged during evolution leading to a loss of function from the mitochondrial protein. Although it is unclear whether other factors act in trans to compensate for the lack of editing by mitochondrial LeuRS, the evolutionary trend toward a more error-prone aaRS mirrors that described above for PheRS. This, in turn, raises the question as to whether other mitochondrial aaRSs might also lack the capacity to edit misaminoacylated tRNAs. For example, mitochondrial ThrRS lacks either of the modules known to edit Ser-tRNAThr (19–21) suggesting that this misacylated species might also escape hydrolysis. However, Ser-tRNAThr can be directly hydrolyzed by free-standing editing domains acting in trans emphasizing the need for direct experimental confirmation that particular editing pathways are absent from mitochondria.

The absence of certain aaRS editing reactions from mitochondria suggests that the need for fidelity during aminoacyl-tRNA synthesis may be lower in this compartment than in the cytosol, perhaps accelerating the loss of these energetically costly secondary activities. Loss of the editing domain from mitochondrial PheRS is in stark contrast to the general pattern of synthetase evolution, where modules with new functionality have been systematically integrated into extant proteins (58). This fundamental difference between mitochondrial and cytosolic aaRS evolution and function indicates that either organellar protein synthesis quality control is focused on another step or that translation in this compartment is inherently less accurate. From a physiological stand-point, reduced accuracy during translation might not be as detrimental in the mitochondrial system, where only a small number of proteins are encoded. In the presence of an efficient protein degradation machinery, it is not unlikely that the bulk of the mitochondrial quality control occurs post-translationally at the level of protein stability as has been recently shown (see for example Ref. 59 and references therein). In this respect, only folded proteins that can join an active respiratory complex are protected from degradation. Mistakes in any protein subunit resulting from reduced translational accuracy can be easily accommodated by very rapid protein degradation. This model could be easily re-enforced by measuring translational accuracy with mitochondrial ribosomes and comparing the error rate to that of degradation. However, currently in vitro translation systems with mitochondrial ribosomes have not been established, and this will thus remain an open question.


   FOOTNOTES
 
* This work was supported by Grant MCB-0344002 from the National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Microbiology, The Ohio State University, 484 W. 12th Ave., Columbus, OH 43210. Tel.: 614-292-2120; Fax: 614-292-8120; E-mail: ibba.1{at}osu.edu.

2 The abbreviations used are: aa, aminoacyl; RS, tRNA synthetase. Back


   ACKNOWLEDGMENTS
 
We thank S. Ataide, C. Hausmann, and J. Levengood for critical reading of the manuscript, S. Walker and K. Fredrick for modifications to the CCAse labeling procedure, and F. Fasiolo, D. Tirrell, O. Uhlenbeck, and R. Zimmermann for gifts of strains and plasmids.



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 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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