# The Yeast VAS1 Gene Encodes Both Mitochondrial and Cytoplasmic Valyl-tRNA Synthetases*

(Received for publication, June 8, 1987)

Bruno Chatton, Philippe Walter, Jean-Pierre Ebel, François Lacroute, and Franco Fasiolo‡

From the Institut de Biologie Moleculaire et Cellulaire, Laboratoire de Biochimie, 15, rue René Descartes,
67084 Strasbourg Cedex, France

S1 mapping on the VAS1 structural gene indicates the existence of two classes of transcripts initiating at distinct in-frame translation start codons. The longer class of VAS1 transcripts initiates upstream of both ATG codons located 138 base pairs away and the shorter class downstream of the first ATG. A mutation that destroys the first AUG on the long message results in respiratory deficiency but does not affect viability. Mutation of the ATG at position 139 leads to lethality because the initiating methionine codon of the essential cytoplasmic valyl-tRNA synthetase has been destroyed. N-terminal protein sequence data further confirm translation initiation at ATG-139 for the cytoplasmic valyl-tRNA synthetase. From these results, we conclude that the VAS1 single gene encodes both mitochondrial and cytoplasmic valyl-tRNA synthetases. The presequence of the mitochondrial valyl-tRNA synthetase shows amino acid composition but not the amphiphilic character of imported mitochondrial proteins. From mutagenesis of the ATG-139 we conclude that the presequence specifically targets the cytoplasmically synthesized mitochondrial valyl-tRNA synthetase to the mitochondrial outer membrane and prevents binding of the enzyme core to cytoplasmic tRNA<sup>Val</sup>.

The existence of protein biosynthesis in yeast mitochondria is well established. A specific set of mitochondrial aminoacyl-tRNA synthetases is coded for by the nuclear genome as evidenced by the presence of normal levels of these enzymes in rho<sup>-</sup> mutants devoided of mitochondrial DNA (1). Some of these enzymes are specific for mitochondrial tRNAs and will not aminoacylate tRNA from the cytoplasm of the same organism (2). Based on these criteria the mitochondrial tRNA synthetases for phenylalanine, leucine, and methionine have been shown to be different from their cytoplasmic counterparts (1). These results suggest a specific gene for the synthesis of both cytoplasmic and mitochondrial aminoacyl-tRNA synthetase. Genetic confirmation was given by a mutation in the MES1 gene (coding for cytoplasmic methionyl-tRNA synthetase) which left the mitochondrial methionyl-tRNA synthetase intact (3). Recent work on the tryptophanyl- and threonyl-tRNA synthetases showed that distinct genes encode the cytoplasmic and mitochondrial forms (4-6).

However, the two forms of valyl-tRNA synthetase are indistinguishable by their chromatographic and kinetic properties. They have similar molecular weights and do not discriminate between the yeast tRNA<sup>Val</sup> species from cytoplasm and mitochondria (7), although the structures of the two tRNAs are different (8, 9). Successful aminoacylation of the heterologous tRNA<sup>Val</sup> from *Escherichia coli* showed that identical $K_m$ values were obtained with the two isoenzymes suggesting identity of the two enzyme forms. This observation is compatible with the existence of a single gene coding for the valyl-tRNA synthetase isoenzyme pair. We have cloned the gene coding for cytoplasmic valyl-tRNA synthetase (VAS1) using an immunological screening procedure (10). The VAS1 structural gene encodes 1104 amino acid residues and contains two closely positioned ATG codons in the 5'-end of the gene sequence. We show here by site-directed mutagenesis that these two ATG codons encode, respectively, the initiating methionine for the mitochondrial and cytoplasmic forms. N-terminal protein sequence data of the cytoplasmic valyl-tRNA synthetase further confirm translation initiation at the downstream methionine codon. These data indicate that the VAS1 transcriptional organization resembles the *Saccharomyces cerevisiae SUC2* gene encoding both secreted and cytoplasmic invertase (11, 12). A recent report presented genetic evidence that the *HTS1* gene also follows the *SUC2* model for the production of the two forms of histidyl-tRNA synthetase (13).

## MATERIALS AND METHODS

*Mapping of VAS1 mRNA Termini*—S1 nuclease protection experiments were performed essentially as described by Berk and Sharp (14). The DNA probe (the 607-base pair EcoRI-MstII fragment shown on the map in Fig. 2) was dephosphorylated with calf intestinal phosphatase, 5'-labeled at its MstII end with T4 polynucleotide kinase, and the strands were separated as described by Maxam and Gilbert (15). The isolated single strands (5000 cpm Cerenkov) were hybridized to 15 µg of poly(A<sup>+</sup>) mRNA prepared from wild type yeast cells. The hybridization mixture was diluted 10-fold in S1 buffer containing various amounts of S1 nuclease and digested for 2 h at 25 °C. S1-resistant fragments were separated on a 6% sequencing gel and visualized by autoradiography.

*In Vitro Mutagenesis*—In vitro mutagenesis experiments were performed according to Zoller and Smith (16). Template DNA was a 1.6-kb<sup>1</sup> EcoRI fragment from the VAS1 region (see the map in Fig. 2B) cloned in phage M13mp19. The synthetic oligonucleotide V1 (AGATGGTATCGATAAGTGGT) was used to generate a ClaI site at the ATG encoding Met-1 of the VAS1 region. Oligonucleotide V2 (GTTCTTTAAAACGATCGATCTTGAT) was used to generate a PvuI site at the ATG encoding Met-47 of the VAS1 region. The mutated M13 recombinants were screened with the synthetic oligonucleotides at 50 °C. The presence of the ClaI and PvuI sites in the mutated M13 recombinants was confirmed by DNA sequence analysis.

<footnote>* This work was supported by grants from the Centre National de la Recherche Scientifique. 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.
‡ To whom correspondence should be sent.</footnote>

<footnote n="1">¹ The abbreviations used are: kb, kilobase(s); FAB-MS, fast atom bombardment mass spectra; CID, collision-induced decomposition.</footnote>
FIG. 1. Map of the pFF-V1 and pFF-V2 plasmids. The numbers indicate the coordinates of the restriction sites on pUC19, URA3, and *vas1* sequences. E, EcoRI; C, ClaI; P, PvuI; B, BglII.

<figure><img src="image_1.png" /></figure>

*Transformation of S. cerevisiae*—Yeast cells were transformed according to the method of Hinnen *et al.* (17).

*Plasmids*—The recombinant plasmids pFF-V1 and pFF-V2 (Fig. 1) were constructed by inserting the mutated *vas1* sequences into the EcoRI site of the polylinker region of the integrative vector pFL34. The latter was constructed by the insertion of the *URA3* HindIII fragment with BglII linkers into the AluI-629 site of pUC19. Before its integration in pUC19, the *URA3* gene was subjected to the following modifications: (i) the *PstI* site was removed using the 3' to 5' exonuclease activity of T4 DNA polymerase; (ii) the *SmaI* and *XmaI* sites were removed after cleavage with *XmaI* and filling by DNA polymerase I (large fragment) followed by blunt-end ligation. The *HincII* site and the two *AccI* sites of *URA3* were untouched. The *vas1* EcoRI fragment was cloned in opposite orientation in plasmids pFF-V1 and pFF-V2.

*Verification of the Protein Sequence*—Thirty-five nanomoles of S-carbamidomethylated valyl-tRNA synthetase were digested with tosylphenylalanyl chloromethyl ketone-treated trypsin at an enzyme to substrate ratio of 1:50 (w/w). A portion of the digest corresponding to 2 nmol was separated by reverse-phase high performance liquid chromatography into 27 fractions using a 1%/min linear gradient of the high strength eluant (low strength eluant, 0.05% trifluoroacetic acid/H₂O; high strength eluant, 0.035% trifluoroacetic acid/CH₃CN). Fast atom bombardment mass spectra (FAB-MS) and collision-induced decomposition (CID) spectra were determined with a JEOL HX110/HX110 tandem mass spectrometer.

RESULTS

*VAS1 Transcriptional Organization*—The transcription starts of the *VAS1* gene were analyzed by S1 nuclease protection (Fig. 2, A and B). The analysis showed the existence of two classes of messages: a long one containing two ATGs located at positions +1 and +139, and a shorter one containing only the downstream ATG at +139. These two ATGs encode Met-1 and Met-47, respectively, in the *VAS1* structural gene. There is size heterogeneity in both classes of transcripts, but major sites can be identified at position -26 for the longer species and at positions +30, +34, and +61 for the shorter species. Densitometry scanning of the respective bands established that the shorter class of message is four times more abundant than the longer one. The existence of two sets of messages differing in size but having the same coding sequences was consistent with the biosynthesis of two forms of valyl-tRNA synthetase from a single gene. According to the *SUC2* model mentioned above, the DNA region between the two ATGs of the *VAS1* gene is expected to encode the presence of the mitochondrial isoenzyme.

*Verification of the Initiating Methionines of the Two Forms of Valyl-tRNA Synthetase*—To verify the *in vivo* functioning of the assumed initiation methionine codon for the mitochondrial and cytoplasmic valyl-tRNA synthetase genes, the corresponding ATGs were mutated by *in vitro* oligonucleotide mutagenesis and the altered DNA sequences introduced in place of the wild type sequence on the chromosomal DNA by the two-step replacement technique of Scherer and Davis (18). We used the *URA3* gene as a selectable marker for the integration step because it is easy to select in a second step

looping out events for the resulting *Ura*⁻ cells using 5-fluoroorotic acid (19).

*Mutagenesis of the Putative Initiating Met-1 Codon for the Mitochondrial Valyl-tRNA Synthetase*—The mutated *vas1* sequence integrated at the chromosomal locus was a 1.6-kb EcoRI fragment containing approximately 1.2 kb of coding sequence and 0.4 kb of 5'-upstream sequence (see the map in Fig. 2). This fragment was introduced into the pFL34 integrative vector (see "Materials and Methods") after alteration of the Met-1 codon. The mutagenesis experiment was designed in order to create a new *ClaI* site. The recombinant plasmid (pFF-VI), opened at the *MstII* site, was used to transform a *ura3* triple mutant diploid strain (*ura3-251,329,373*) to prototrophy. One of the numerous *URA3*⁺ transformants was induced to sporulate, and tetrads were dissected. The germination was poor; only one complete tetrad and four triads were recovered. These were tested on minimal medium supplemented with: (i) glucose, (ii) uracil plus glucose, (iii) uracil plus glycerol and ethanol. The *URA3*⁺ character segregated 2:2 in the unique tetrad, and 2-1 or 1-2 in the triads; moreover, all the *URA3*⁺ haploids were respiratory deficient (*Pet*⁻) and all the *URA3*⁻ haploids were respiratory competent. Thus the integration of the recombinant plasmid with an altered Met-1 codon results in a recessive *Pet*⁻ mutation (inability to grow on a nonfermentable carbon source like glycerol or ethanol but able to grow on glucose). One of the *Pet*⁻:URA3⁺ spores was grown for about 25 generations on complete medium. Derivatives of *Pet*⁻ haploids having undergone a second crossing-over with loss of plasmid and the *URA3*⁺ marker were selected for 5-fluoroorotic resistance. The majority of the *URA3*⁻ strains were indeed respiratory-deficient. However, in view of the strong induction of cytoplasmic respiratory deficiency by 5-fluoropyrimidines, we verified that the *Pet*⁻ phenotype was indeed due to a chromosomal mutation. For this purpose we crossed a *Pet*⁻:URA3⁻ haploid with a wild type haploid and induced meiosis. Tetrad analysis showed unambiguously that the *Pet*⁻ mutation segregated 2:2 and was independent from the segregation of the *URA3*⁻ character, demonstrating that the respiratory deficiency was a consequence of a chromosomal mutation.

To obtain direct evidence of the genetic events in the transformed *Pet*⁻:URA3⁺ haploid and the cured *Pet*⁻ haploid, we made Southern blots of their respective chromosomal DNAs. The result of *ClaI* digestion of DNA from *Pet*⁻:URA3⁺ haploid is shown in Fig. 3, lane 1. Four fragments of 1.9, 3.5, 5.9, and 8.3 kb were visualized. The sizes of these fragments were predicted by the map in Fig. 3 showing the integration at the chromosomal locus of two copies of the pFF-V1 plasmid. The transforming plasmid was shown in the dimeric state in order to illustrate the result of *ClaI* digestion in the *Pet*⁻:URA3⁺ haploid, although it is not clear whether dimerization occurs before or after the integration event. Partial digestion was assumed to account for the observation of an 8.3-kb *ClaI* fragment in the *URA3*⁺ transformant.
Mitochondrial and Cytoplasmic tRNA Synthetase from the VAS1 Gene

<figure><img src="image_2.png" /><figcaption>FIG. 2. Determination of the 5'-ends of VAS1 transcripts. A, S1 mapping. The 5'-labeled single-stranded MstII-EcoRI fragment with coordinates -377 to +230 was hybridized to poly(A+) mRNA and digested by S1 nuclease. The S1-protected strands are displayed along with Maxam-Gilbert sequencing reactions (G and T+C) of the coding strand. *Lane 1* shows a control reaction without mRNA. *Lanes 2 and 3* show digestions of RNA-DNA hybrids by 2000 and 6000 units of S1 nuclease. *Lanes 4 and 5, G and T+C* sequencing reactions. The numbering is relative to A of the first initiating ATG codon of the *VAS1* structural gene. The arrows on the left-hand side indicate the positions of the major transcription sites. *B*, map of the *VAS1* gene and location of the 5'-ends on the *VAS1* upstream region. The letters above the line indicate some of the restriction sites: *E*, *EcoRI*; *M*, *MstII*; *H*, *HincII*. The map shows the *EcoRI* fragment used in the mutagenesis and gene replacement experiments. The box below the line shows the extent of the *VAS1* coding region. The solid portion of the box corresponds to the region that encodes the leader peptide of the mitochondrial form. The *EcoRI-MstII* fragment was used to map the 5'-ends. The figure shows the sequence of the mRNA identical strand of the *EcoRI-MstII* fragment. Twenty percent of the transcripts map in the -37 to -26 region. The most abundant *VAS1* transcripts (80%) map in the + 29 to +60 coding region. The relative abundance of the two classes of messages is indicated by the heights of the arrows. *V1* and *V2* correspond to the sequences of the oligonucleotides used to mutagenize the initiating Met-1 and Met-47 codons.</figcaption></figure>
<figure><img src="image_3.png" /></figure>

FIG. 3. Replacement of chromosomal VAS1 segment with an altered ATG at the Met-1 codon. The scheme shows the sequence of events giving rise to integration of the altered sequence into a diploid Ura⁻ stain. The top line represents the chromosomal VAS1 region with some of the restriction sites: C, ClaI; E, EcoRI; S, StuI. Numbers under the line represent the distances in kb from the ClaI site. The box indicates the extent of the labeled probe used in the Southern analysis. The integrative plasmid pFF-V1 is represented as a dimer; the thick line represents plasmid vector sequences and the thin line the 1.6-kb EcoRI fragment from the VAS1 region in which the altered sequence gives rise to a ClaI site. The arrow indicates the position of the mutated ATG in the VAS1 gene. The diploid Ura⁻ recipient was transformed with the linearized integrative plasmid. The cross indicates the site of integration at the opened MstII site. The bottom line represents the linear resolution of the integrated plasmid dimer. The numbers in brackets refer to the size in kb of the respective ClaI fragments predicted by DNA-DNA hybridization using the DNA probe shown on the map. Ura³⁺ transformants were induced to sporulate and the tetrads dissected. DNA from Pet⁻:Ura³⁺ haploid was extracted, digested by ClaI, run on an agarose gel, and subjected to Southern hybridization (shown on the right, lane 1). The sizes of the ClaI fragments determined experimentally are indicated and correspond to the sizes predicted by the map. The Southern blot also shows: lane 2, StuI-ClaI digest of the Pet⁻:Ura³⁺ haploid as confirmation of the integration at the VAS1 locus; lane 3, ClaI digest of DNA from the Pet⁻:Ura³⁻ haploid. Two fragments of 2.4 and 6.4 kb were obtained. They resulted from plasmid loss by a second crossover upstream to the mutated ATG and prove gene replacement at the VAS1 locus. Lanes 4 and 6, chromosomal DNA extracted from the Ura³⁻ diploid and digested by ClaI (lane 4) and StuI (lane 6); lane 5, HindIII digest from λ DNA. The numbers on the right indicate the size in kb of the marker DNA.

Looping-out in the Pet⁻:Ura³⁺ strain resulted in the loss of the plasmid vector with concomitant replacement of the sequences surrounding the ATG at Met-1 by the altered *vas1* sequences. This could be verified by *ClaI* digestion of DNA from Pet⁻:Ura³⁻ haploids. Two fragments of 2.4 and 6.4 kb were obtained (Fig. 3, *lane* 3). The sizes of these fragments were predicted by the map in Fig. 3 and proved gene replacement at the *VAS1* locus.

*Mutagenesis of the Putative Initiating Met-47 Codon of the Cytoplasmic Valyl-tRNA Synthetase*—The introduction at the chromosomal locus of the mutated ATG encoding Met-47 was realized with the integrative plasmid pFF-V2 containing a newly created *PvuI* site in the *VAS1* region (Fig. 4). This plasmid, opened at the *MstII* site, was used to transform the Ura³⁻ diploid, and one of the Ura³⁺ transformants was analyzed genetically. The results obtained from dissection of 10 tetrads showed a 2:2 segregation for a lethal phenotype. All viable spores were Ura³⁻. This shows that the integration event caused nonviability of the Ura³⁺ spores because the initiating methionine of the cytoplasmic valyl-tRNA synthetase has most likely been destroyed. The demonstration that the integration had occurred at the *VAS1* locus was shown by

<figure><img src="image_4.png" /></figure>

FIG. 4. Replacement of the chromosomal VAS1 segment with an altered ATG at Met-47 codon. Ura³⁻ diploid was transformed with pFF-V2 plasmid. The scheme shows the steps of the integration event. The legend is similar to Fig. 3. Restriction sites on the VAS1 chromosome and pFF-V2 plasmid are: B, BglII; E, EcoRI; P, PvuI. The numbers refer to the sizes in kb of the respective fragments. Those predicted from DNA-DNA hybridization using the DNA probe shown on the map are indicated in brackets. DNA from the resulting Ura³⁺ transformant was digested by PvuI-BglII and subjected to Southern analysis (lane 7). The sizes of the fragments obtained (0.62, 1.4, and 1.9 kb) are indicated. They fit the size of the fragments predicted by the map. The Southern analysis also shows: *lanes 2–4*, genomic DNA from Ura⁻ diploid digested by *StuI*, BglII, and *StuI-PvuI*, respectively; *lane 6*, DNA from Ura³⁺ diploid digested by *BamHI*; *lanes 1 and 5*, marker DNA fragments whose sizes in kb are indicated on the right part of the figure.

Southern analysis. DNA isolated from the Ura³⁺ diploid was subjected to BglII-PvuI digestion and probed with a fragment covering the major part of the 1.6-kb VAS1 EcoRI fragment. Three fragments of 0.62, 1.4, and 1.9 kb were visualized (Fig. 4, lane 7). The sizes of these BglII-PvuI fragments were predicted by the map in Fig. 4, indicating integration of plasmid pFF-V2 at the VAS1 locus. This study demonstrates that the region of the VAS1 gene encoding the cytoplasmic valyl-tRNA synthetase is essential for cell viability.

*FAB-MS Protein Sequence Data*—The purified yeast cytoplasmic valyl-tRNA synthetase was not amenable to Edman degradation due to a blocked N terminus. Therefore, FAB-MS experiments were necessary to identify the N-terminal sequence of the cytoplasmic enzyme. A tryptic digest of the protein was fractionated by reverse-phase high performance liquid chromatography, and the molecular weights of the peptides in each fraction were determined by FAB-MS. A total of 79 peptides were characterized by their molecular weights. These peptides corroborated 80% of the DNA sequence (Fig. 5) and established Met-47 as the initiation site for the cytoplasmic enzyme since no proteolytic peptides from the amino acid region between Met-1 and Met-47 were detected. One signal at *m/z* 1351.6 did not correspond to the protonated molecular ion, (M+H)⁺, of any tryptic peptide predicted from the cytoplasmic enzyme. The CID spectrum of this ion was recorded in order to determine its sequence (20, 21). Fragmentation along the peptide backbone indicated the sequence, Ac-Ser-Asp-Leu-Asp-Leu-Pro-Val-Asp-Lys, which corresponds to the sequence 48–59.<fnref n="2" /> From this we conclude that translation of the cytoplasmic enzyme begins at Met-47 which is removed post-translationally before acetylation of the newly formed N-terminal serine.

<footnote n="2">² Biemann K., and Scoble, H. A. (1987) *Science* **237**, 992–998.</footnote>
<figure><img src="image_5.png" /><figcaption>FIG. 5. Gene-derived amino acid sequence of the cytoplasmic valyl-tRNA synthetase and FAB-MS verification. Peptides identified by FAB-MS from a fractionated tryptic hydrolysate of cytoplasmic valyl-tRNA synthetase are underlined. The N-terminal serine occupies position 48 in the polypeptide sequence deduced from the VAS1 coding frame (see Fig. 2B). The N-terminal dodecapeptide was identified by CID experiments. For clarity, peptides arising from cleavage at each lysine or arginine of a partial peptide were not underlined.</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>FIG. 6. Wheel diagram representation of a portion of the N-terminal region of the cytoplasmic valyl-tRNA synthetase. The numbering includes the initiating methionine.</figcaption></figure>

DISCUSSION

Our data show that a single VAS1 gene encodes both the cytoplasmic and mitochondrial valyl-tRNA synthetases. The polypeptide portions of these two forms are synthesized from two distinct mRNAs differing in length but having the same coding frame. The longer messages contain two AUGs and code for the mitochondrial enzyme; the shorter ones contain only the second AUG at position +139 and code for the cytoplasmic enzyme. Derivation from a single structural gene implies structural and kinetic similarities. Indeed, the molecular weights of the two proteins estimated by gel filtration are similar (M, 120,000 ± 5,000), and the measured K<sub>m</sub> values for yeast mitochondrial and cytoplasmic tRNA<sup>Val</sup> are identical for both valyl-tRNA synthetase forms, but different for the tRNA species (7).

The cytoplasmic valyl-tRNA synthetase polypeptide chain has several interesting structural features (10). Approximately 170 amino acid residues from the N-terminal region build a strong α-helix, followed by a nucleotide binding domain responsible for aminoacyl-adenylate catalysis. This domain, comprised of alternating α/β structures, is separated from the C-terminal helical region by the functional Lys-Met-Ser-Lys-Ser region which probably binds the 3'-terminal end of tRNA<sup>Val</sup>. In addition to these structural features the mitochondrial valyl-tRNA synthetase has a presequence of 47 amino acids composed of only basic, hydrophobic, and hydroxylated residues. Characteristically, mitochondrial presequences lack a specific sequence for the targeting function (22). However, a statistical analysis of more than 20 mitochondrial targeting sequences indicates that they have the potential to form an amphiphilic helix with one highly charged and one hydrophobic face (23). Secondary structure predictions were made for the valyl-tRNA synthetase presequence. It turns out that the probability for the polypeptide chain to form a β-sheet is highest. This observation confirms the view that an amphiphilic helix is not an absolute structural requirement for transport into mitochondria. Allison and Schatz (24) genetically engineered nonamphiphilic mitochondrial presequences and showed that they target subunit IV of cytochrome c as well as amphiphilic structures. In contrast, the N-terminal region of the cytoplasmic valyl-tRNA synthetase shows a strong resemblance to an amphiphilic helix (Fig. 6). The folded helix has a cluster of 9 lysines exposed toward the solvent and a constellation of hydrophobic amino acids on

the opposite side, an unusual finding in cytosolic N-terminal sequences. Among the 132 N-terminal cytosolic sequences analyzed by von Heijne (23), only the so-called agnoprotein from SV40 meets the criteria for an amphiphilic helix. This protein is a small basic molecule that may have a role in DNA-protein interactions (25). What role might this helix play in the yeast cytoplasmic valyl-tRNA synthetase? Any functional significance related to catalysis can be excluded since the homologous *E. coli* valyl-tRNA synthetase lacks this structural domain. Sequence comparison has shown that the yeast valyl-tRNA synthetase extends about 170 amino acid residues beyond the N terminus of the *E. coli* enzyme.<fnref n="3" /> Chain extension at the N terminus seems to be a general feature of yeast aminoacyl-tRNA synthetases compared to their prokaryotic homologues. This is the case for the yeast and *E. coli* methionyl-tRNA synthetases and the threonyl-tRNA synthetase pairs (cited in Ref. 26), although the additional domain shows a much less pronounced amphiphilic character. Both synthetase pairs nonetheless acquired the property to bind to polyanionic carriers like the other yeast and mammalian aminoacyl-tRNA synthetases (27, 28). Considering that the evolutionary acquisition of polyanionic binding is of functional importance *in vivo*, Cirakoglu and Waller (29) proposed that this acquisition promotes the compartmentalization of these enzymes within the cytoplasm through electrostatic interactions with negatively charged cellular components, presumably ribosomes. We propose an alternative explanation which takes into account the amphiphilic character of the N-terminal helix of valyl-tRNA synthetase. It has been shown that *E. coli* alanyl-tRNA synthetase controls its synthesis by transcriptional autoregulation (30). This control is mediated by the intracellular level of the specific amino acid and explains derepression of the alanyl-tRNA synthetase under amino acid limitation. Regulation studies on yeast valyl-tRNA synthetase show that this enzyme is also derepressed by valine restriction (31). One plausible explanation is that the N-terminal helix of yeast valyl-tRNA synthetase (with a high density of basic residues on one side) has nuclear targeting activity allowing access of the enzyme to the promoter region of the VAS1 gene.

Aminoacyl-tRNA synthetases and tRNAs are present in the cytoplasm in the micromolar range (32), a concentration 10 times greater than their binding constants. We calculated that this is true for yeast valyl-tRNA synthetase and its cognate tRNA from their known protein and nucleic acid

<footnote n="3">³ P. Walter and F. Fasiolo, unpublished results.</footnote>
contents. This implies that both macromolecules exist as
complexes and are not free in solution. The synthesis on
cytoplasmic ribosomes of the mitochondrial valyl-tRNA syn-
thetase, functionally interchangeable with the cytoplasmic
valyl-tRNA synthetase, raises the question of the partition of
the two isoenzymes. Possibly a pool of precursor forms of
mitochondrial valyl-tRNA synthetase could compete with the
cytoplasmic isoenzyme for tRNA<sup>Val</sup> and ensure the essential
functions of the cytoplasmic protein-synthesizing machinery.
However, since synthesis of mitochondrial valyl-tRNA syn-
thetase cannot prevent lethality when the initiating methio-
nine of cytoplasmic valyl-tRNA synthetase is mutagenized,
we know that there is no pool of precursor mitochondrial
valyl-tRNA synthetase that can compensate for the loss of
cytoplasmic valyl-tRNA synthetase. It is not reasonable to
assume that the precursor valyl-tRNA synthetase is unable
to bind to cytoplasmic tRNA<sup>Val</sup> because the C-terminal do-
main generally carries the major tRNA binding sites (33, 34).
Furthermore, the plasmid-born VAS1 gene encoding the mi-
tochondrial presequence is capable of complementing an E.
*coli* temperature-sensitive valyl-tRNA synthetase mutant.<fnref n="4" />
This indicates that the precursor mitochondrial valyl-tRNA
synthetase is able to aminoacylate *E. coli* tRNA<sup>Val</sup> and con-
sequently cytoplasmic yeast tRNA<sup>Val</sup> since both forms are
kinetically indistinguishable as stated above. Since import of
mitochondrial proteins occurs post-translationally (35, 36),
we, therefore, admit that binding of the precursor valyl-tRNA
synthetase to the mitochondrial surface is very specific and
prevents binding to cytoplasmic tRNA<sup>Val</sup>. Current data on
translocation of imported proteins having precursor polypep-
tides suggest that binding requires common receptors or sol-
uble components (37). To achieve specificity, the receptor
binding affinity has to be much greater than affinity of the
protein core to cytoplasmic tRNA<sup>Val</sup>. It turns out that speci-
ficity of the import of mitochondrial valyl-tRNA synthetase
is governed by the ratio of the association constants of two
distinct domains of the enzyme molecule: the K<sub>a</sub> of prese-
quence for the mitochondrial membrane receptor versus the
K<sub>a</sub> of the rest of the molecule for the tRNA<sup>Val</sup>. This idea could
be tested experimentally. Any mutation lowering the affinity
of the presequence for the receptor should overcome lethality
resulting from mutagenesis at the initiating Met-47 codon.

Acknowledgments—We are grateful to Drs. K. Biemann and H. A. Scoble for supplying us with the FAB-MS and CID experiments on cytoplasmic valyl-tRNA synthetase. We thank D. Kern for his gift of valyl-tRNA synthetase. We acknowledge the skillful technical assistance of G. Nussbaum.

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<footnote n="4">⁴ M.-P. Weisshaar and F. Fasiolo, unpublished results.</footnote>