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Originally published In Press as doi:10.1074/jbc.M601869200 on August 23, 2006

J. Biol. Chem., Vol. 281, Issue 42, 31430-31439, October 20, 2006
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Cross-species and Cross-compartmental Aminoacylation of Isoaccepting tRNAs by a Class II tRNA Synthetase*

Hsiao-Yun Huang{ddagger}, Yu Kuei{ddagger}, Hen-Yi Chao{ddagger}, Shun-Jia Chen{ddagger}, Lu-Shu Yeh§1, and Chien-Chia Wang{ddagger}2

From the {ddagger}Department of Life Science, National Central University, Jung-li, Taiwan 32001 and §Department of Life Science, Tzu-Chi University, Hua-lien, Taiwan 97041

Received for publication, February 27, 2006 , and in revised form, August 7, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
It was previously shown that ALA1, the only alanyl-tRNA synthetase gene in Saccharomyces cerevisiae, codes for two functionally exclusive protein isoforms through alternative initiation at two consecutive ACG codons and an in-frame downstream AUG. We reported here the cloning and characterization of a homologous gene from Candida albicans. Functional assays show that this gene can substitute for both the cytoplasmic and mitochondrial functions of ALA1 in S. cerevisiae and codes for two distinct protein isoforms through alternative initiation from two in-frame AUG triplets 8-codons apart. Unexpectedly, although the short form acts exclusively in cytoplasm, the longer form provides function in both compartments. Similar observations are made in fractionation assays. Thus, the alanyl-tRNA synthetase gene of C. albicans has evolved an unusual pattern of translation initiation and protein partitioning and codes for protein isoforms that can aminoacylate isoaccepting tRNAs from a different species and from across cellular compartments.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Typically there are 20 aminoacyl-tRNA synthetases in prokaryotes, one for each amino acid (1–4). These enzymes each catalyze the formation of an aminoacyl-tRNA by attaching a particular amino acid to the 3'-end of its cognate tRNA, with accompanying hydrolysis of ATP to AMP and pyrophosphate. The activated amino acid, i.e. aminoacyl-tRNA, is then transferred to ribosome for protein synthesis. In eukaryotes protein synthesis occurs not only in the cytoplasm but also in organelles, such as mitochondria and chloroplasts (5). Compartmentalization of the protein synthesis machinery within the cytoplasm and organelles of eukaryotes leads to isoaccepting tRNA species that are distinguished by nucleotide sequence, subcellular location, and enzyme specificity. Thus, eukaryotes such as yeast commonly have two genes that encode distinct sets of proteins for each aminoacylation activity, one localized to the cytoplasm and the other to the mitochondria. Each set aminoacylates the isoaccepting tRNAs within its respective cell compartment. Except for some algae (6), all aminoacyl-tRNA synthetases are encoded by nuclear genes regardless of the cell compartments to which they are confined.

In contrast to most known eukaryotic tRNA synthetases, two Saccharomyces cerevisiae genes, HTS1 (the gene encoding his-tidyl-tRNA synthetase) (7) and VAS1 (the gene encoding valyl-tRNA synthetase (ValRS)) (8), specify both the mitochondrial and cytosolic forms through alternative initiation from two inframe AUG codons. Each of these genes encodes mRNAs with distinct 5'-ends. Some of these mRNAs have their 5'-ends located upstream of the first AUG codon, whereas others have their 5'-ends located between the first and second AUG codons. The mitochondrial form of the enzyme is translated from the first AUG on the "long" messages, whereas the cytosolic form is translated from the second AUG on the "short" messages. As a consequence, the mitochondrial enzymes have the same polypeptide sequences as their cytosolic counterparts, except for a short amino-terminal mitochondrial targeting sequence. The transit peptide is subsequently cleaved away upon import into the mitochondria. Because the two isoforms are targeted to different subcellular compartments, they cannot substitute for each other in vivo (7–8). A similar scenario has been observed for the genes that encode the mitochondrial and cytoplasmic forms of Arabidopsis thaliana alanyl-tRNA synthetase (AlaRS),3 threonyl-tRNA synthetase, and ValRS (9).

Recently it was shown that ALA1, the only gene coding for AlaRS in S. cerevisiae, also encodes distinct protein isoforms (10–11). Although the cytoplasmic form is initiated from a canonical AUG triplet, its mitochondrial counterpart is initiated from two successive in-frame ACG triplets that are located 23 codons upstream of the AUG initiator, i.e. ACG(–25)/ACG(–24). These two forms function exclusively in their respective compartments and, thus, cannot substitute for each other under normal conditions. A similar scenario has been observed in GRS1 (12), the only active yeast gene coding for glycyl-tRNA synthetase. Because to date examples of native non-AUG initiation are still rare in low eukaryotes (10–12), we wondered whether a similar mechanism of translation initiation has been conserved in the AlaRS genes of other yeasts during evolution. In addition, we wondered whether the AlaRS gene of a closely related yeast species, such as Candida albicans, also provides function in both compartments and whether it can surmount the species barrier and charge the tRNAs of S. cerevisiae. It is our hope that results obtained from this study could provide further insight into the bifunctional nature of a particular nuclear gene and the diversity of mechanisms by which protein isoforms can be partitioned between two distinct compartments. In the work described here we presented experimental evidence that an ALA1 homologue of C. albicans (designated here as CaALA1) can rescue both the cytoplasmic and mitochondrial defects of a S. cerevisiae ala1– strain. Similar to the ALA1 gene in S. cerevisiae, two protein isoforms with distinct amino termini are alternatively generated from this gene; however, no non-canonical initiators are involved in this case. Instead, these isoforms are initiated from two in-frame AUG triplets. Even more unexpectedly, whereas the short form that is initiated from the second AUG is confined in the cytoplasm, the longer form that is initiated from the first AUG is dual-targeted and, thus, bifunctional. The implications of these observations will be further discussed in the context of co-evolution of tRNAs and their cognate tRNA synthetases.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Construction of Plasmids—Cloning of CaALA1 from C. albicans followed standard protocols (13). The wild-type CaALA1 sequence (base pairs –300~+2910 relative to ATG1) was amplified by PCR and cloned into pRS315 (a low copy number yeast vector) or pRS425 (a high copy number yeast vector). A short sequence coding for a His6 tag or FLAG was subsequently inserted in-frame into the 3'-end of the CaALA1 open reading frame. Various point mutations, such as ATG1/ATA2 to TCT/AGA and ATG9 to GCG, were introduced into the wild-type clone following standard protocols (the number 1 in ATG1 refers to the codon position in the open reading frame). To clone CaALA1 in pADH (a high copy number yeast vector with an ADH promoter), a segment of CaALA1 DNA containing base pairs –40~+2910 relative to ATG1 was amplified by PCR as an EagI/XhoI fragment and cloned into appropriate sites of this vector.

Cloning of CaALA1-VAS1c constructs followed a strategy described earlier (12). Basically, various CaALA1 sequences (–370~+102 bp, –370~+126 bp, or –370~+159 bp) were PCR-amplified as EagI-SpeI fragments and fused in-frame to the EagI/SpeI sites 5' to VAS1c (the open reading frame coding for the cytoplasmic form of ValRS) cloned in a low copy number vector pRS315, resulting in various CaALA1-VAS1c constructs in which the ATG initiator for the cytoplasmic form of ValRS had been mutated.

Mapping the 5'-Ends of CaALA1 Transcripts—Identification of the 5'-ends of CaALA1 transcripts was carried out with 5'-RACE (rapid amplification of cDNA ends; Invitrogen). Briefly, total RNA isolated from C. albicans was first treated with alkaline phosphatase to remove the 5'-phosphate group from truncated mRNA and non-mRNA and then with tobacco acid pyrophosphatase to remove the 5'-cap from intact full-length mRNA. An RNA oligonucleotide was subsequently fused to the 5'-end of the decapped mRNA with RNA ligase. The 5'-end modified mRNA was transcribed with SuperScript III reverse transcriptase into first strand cDNAs using an "antisense" CaALA1-specific primer that was annealed to a region 630-bp downstream of ATG1. The reaction mixture was treated with RNase H, and the first strand cDNA products were then amplified via PCR using Pfu DNA polymerase with a primer (provided by the manufacturer) annealed to the 5'-end of the cDNA and a nested CaALA1-specific primer annealed 600 bp downstream of ATG1. After PCR-driven amplification, the double-stranded cDNA products were cloned and sequenced.

Sequencing of the Mitochondrial Form of CaAlaRS—Determination of the amino terminus of the processed mitochondrial form of CaAlaRS was carried out by the Edman degradation method. First, mitochondria were isolated from transformants carrying the wild-type (pIVY97) and ATG9 mutant (pIVY118) constructs (14), and the His6-tagged proteins expressed were purified by nickel-nitrilotriacetic acid column chromatography. After SDS-polyacrylamide gel electrophoresis, the proteins were transferred to a nitrocellulose membrane and stained with Amido Black, and the protein band of the correct size was removed and sequenced.

Complementation Assays for the Cytoplasmic Function of ALA1—The yeast ALA1 knock-out strain TRY11 was as described (15). This strain is maintained by a plasmid encoding AlaRS and the URA3 marker. Complementation assays for the cytoplasmic function of plasmid-borne ALA1 and derivatives were carried out by introducing a test plasmid into TRY11 and determining the ability of transformants to grow in the presence of 5-fluoroorotic acid (5-FOA). The cultures were incubated at 30 °C for 3–5 days or until colonies appeared (photos for the complementation assays were taken at day 3 after incubation). The transformants evicted the maintenance plasmid with the URA3 marker in the presence of 5-FOA. Thus, only an enzyme with the cytoplasmic AlaRS activity encoded by the second plasmid (with the LEU2 marker) could rescue the growth defect.

Complementation Assays for the Mitochondrial Function of ALA1—Complementation assays for the mitochondrial function of plasmid-borne ALA1 and derivatives were carried out by introducing a test plasmid (carrying a LEU2 marker) and a second maintenance plasmid (carrying a HIS3 marker) into TRY11 and selecting on a plate containing 5-FOA. The second maintenance plasmid used in this assay contained ALA1(I(–1)stop), which expresses a functional cytoplasmic AlaRS but is defective in mitochondrial AlaRS activity. In the presence of 5-FOA, the first maintenance plasmid (containing a URA3 marker) was evicted from the co-transformants, whereas the second maintenance plasmid was retained. Thus, all co-transformants survived 5-FOA selections due to the presence of the cytoplasmic AlaRS derived from the second maintenance plasmid. The co-transformants were further tested on YPG plates for their mitochondrial phenotypes at 30 °C, with results documented at day 3 after plating. Because a yeast cell cannot survive on glycerol without functional mitochondria, the co-transformants do not grow on YPG plates unless a functional mitochondrial AlaRS is present. Complementation assays for various VAS1 constructs and their derivatives were conducted essentially the same way as those for ALA1 constructs, except that a VAS1 knock-out strain, CW1, was used as the test strain (16).


Figure 1
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  		FIGURE 1.
Determining the transcription initiation sites of CaALA1. A, the 5' sequences of CaALA1, extending from 141-bp upstream to 102-bp downstream of ATG1. The first two ATG triplets, ATG1 and ATG9, are boxed, and the upstream non-ATG triplets that differ from ATG by a nucleotide are underlined. Labeled on top of the nucleotide sequence is the transcription initiation site at bp –24 relative to ATG1. The amino acid residues deduced from the CaALA1 open reading frame (between Met-1 and Leu-34) are shaded. The cleavage site for mitochondrial matrix processing peptidase is marked with {blacktriangleup}. B, the cDNA product of 5'-RACE. Lane 1, DNA markers; lane 2, 5'-RACE product. The migrating positions of the DNA markers on a 2% agarose gel and the 5'-RACE product are labeled on the left and right, respectively. C, comparison of the sequence identities among AlaRS proteins of different origins. Ca, C. albicans; Sc, S. cerevisiae; Sp, S. pombe; Ec, E. coli. D, alignment of the amino-terminal sequences of AlaRS proteins of different origins. Amino acid residues conserved among these sequences are shaded.

 
Western Blot—The protein expression patterns of various CaALA1 constructs were determined by a chemiluminescence-based Western blot analysis following standard protocols. The CaALA1 constructs were first transformed into INVSc1 (Novagen), and the resultant transformants were subsequently grown in a selection medium lacking leucine. The total, cytoplasmic, and mitochondrial fractions were prepared from each of the transformants according to the protocols described by Daum et al. (14). 45 µg of the protein extracts were loaded onto a gel (size, 8 x 10 cm) containing 8% polyacrylamide and electrophoresed at 130 volts for 1~2 h. The resolved proteins were transferred onto a nitrocellulose membrane using a semidry blotting device. The membrane was hybridized with a horseradish peroxidase-conjugated anti-His6 tag (Invitrogen) or anti-FLAG antibody (Sigma) and then exposed to x-ray film after the addition of the appropriate substrates.

Growth Curve Assay—Growth curve assays for the plasmid-borne mitochondrial AlaRS activities were carried out in YPG broth. TRY11 was first co-transformed with a second maintenance plasmid (carrying a HIS3 marker) and a test plasmid (carrying a LEU2 marker), and the resultant co-transformants were plated on a FOA plate. After FOA selections, one colony of the survivors was picked and inoculated into 3 ml of SD broth lacking histidine and leucine and grown to stationary phase. The cells were washed three times with YPG broth, and appropriate amounts were transferred to a flask containing 10 ml of YPG broth to a final cell density of A600 = 0.1. The cell culture was shaken in a 30 °C incubator, and the cell density of the culture was checked every 4 h for a period of 48 h.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Cross-species Complementation of an S. cerevisiae ala1– Strain by a Homologue of C. albicans—Unlike most yeast tRNA synthetases that have two distinct nuclear genes (one coding for the cytoplasmic enzyme and the other for its mitochondrial counterpart), CaALA1 appears to be the only ALA1 homologue in the yeast C. albicans. We wondered whether this gene actually provides AlaRS function in vivo and whether it could code for both cytoplasmic and mitochondrial activities, as with the case of ALA1 in S. cerevisiae. To further our understanding on this gene, we first scanned the 5'-terminal nucleotide sequences of this gene for potential translation start codons that might be involved in the synthesis of protein isoforms. As shown in Fig. 1A, there are two nearby in-frame ATG codons, i.e. ATG1 and ATG9, close to the 5'-end of its open reading frame. In addition, four potential non-ATG initiators, i.e. non-ATG codons that differ from ATG by a single nucleotide, are present in the sequence between ATG1 and TGA(-47), the closest termination codon. We wondered which of these triplets is the authentic start sites for CaALA1. Before proceeding, the transcription profiles of this gene in vivo were elucidated using 5'-RACE. Fig. 1B showed that a single transcript, with its 5'-end mapped to nucleotide position –24 relative to ATG1, was amplified by RT-PCR using total RNA extracts of C. albicans as the templates (Fig. 1B). This transcript was, thus, considered to be the template for protein translation. In addition, sequences of the cDNAs (~600 bp determined) obtained from RT-PCR were identical to those of the genomic DNA (data not shown), suggesting that the possibilities of alternative splicing at the 5'-end of CaALA1 mRNAs and translation initiation from an AUG codon spliced from afar could be ruled out. Comparison of the protein sequences among AlaRSs of different origins showed that CaAlaRS (deduced from its putative open reading frame starting at ATG1) shares a significantly higher sequence identity to those from S. cerevisiae (cytoplasmic form) (~68%) and Schizosaccharomyces pombe (~61%) than to the Escherichia coli enzyme (~39%) (Fig. 1C). Most intriguingly, this protein appears to have an amino-terminal 15-residue appendage that is absent from the other yeast cytoplasmic enzymes compared (Fig. 1D). It should be noted that the cytoplasmic form of AlaRS of S. cerevisiae starts from the second Met on the sequence shown in Fig. 1D. We surmised that if this protein does have mitochondrial function, this appendage could serve as at least part of its mitochondrial targeting signal.


Figure 2
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  		FIGURE 2.
Cross-species complementation of a S. cerevisiae ala1– strain by CaALA1. TRY11 was transformed with the wild-type CaALA1 gene cloned in various vectors and then tested for its growth phenotypes. Complementation of the cytoplasmic and mitochondrial defects of the ala1– strain was shown by its ability to lose the maintenance plasmid and grow on a FOA and a YPG plate, respectively. A, summary of the ALA1 constructs and their complementation activities. B, complementation assays for the cytoplasmic function on a 5-FOA plate. C, complementation assays for the mitochondrial function on a YPG plate. In B and C, the numbers 1–5 denote constructs shown in A. Cyt, cytoplasmic; Mit, mitochondrial.

 
Because the genetic system has not been well developed for C. albicans, we wondered whether we could test the biological functions of CaALA1 in a closely related and well developed yeast system such as S. cerevisiae. To this end, CaALA1 cloned in various vectors was transformed into a S. cerevisiae ala1– yeast strain, TRY11, and tested for its complementing activity. As shown in Fig. 2, the wild-type CaALA1 gene cloned in pRS315 (a low copy number vector), pRS425 (a high copy number vector), or pADH (a high copy number vector with a constitutive ADH promoter) efficiently rescued both the cytoplasmic and mitochondrial defects of TRY11, i.e. transformants carrying the plasmid-borne CaALA1 gene formed colonies after 2~3 days of growth on FOA (Fig. 2B) and YPG (Fig. 2C) plates, respectively. These results indicated that the homologous gene from C. albicans could overcome the species barrier and encodes both cytoplasmic and mitochondrial AlaRS activities.

Generation of Two Functionally Overlapping Protein Isoforms through Alternative Translation Initiation—The question arose as to how many protein isoforms are generated from CaALA1 and whether the upstream non-ATG triplets (relative to ATG1) are involved in the translation initiation. To shed light on this matter, various CaALA1 constructs were cloned using pRS425 as the vector and tested for their complementation activities. First, the codon at position –1 was mutated to a stop codon TAA to block all the possible translational events initiated upstream of ATG1 and then tested for its effect on the cytoplasmic and mitochondrial functions of this gene. Fig. 3 shows that the newly introduced stop codon affected neither the mitochondrial nor cytoplasmic function of this gene (see pIVY102), suggesting that the upstream potential non-ATG initiators are not involved in the synthesis of the alanine enzyme(s). We next aimed at the two nearby ATG triplets, i.e. ATG1 and ATG9, for their possible participation in translation. Mutation of ATG1 (see pIVY100 or pSAM35) or insertion of two nucleotides between ATG1 and ATG9 (causing ATG1 to be out-of-frame with respect to the rest of the open reading frame) (see pSAM25) specifically impaired the mitochondrial function of this gene (Fig. 3, B and C), suggesting that ATG1 was the sole initiator responsible for the translation of the mitochondrial form and the cytoplasmic form was initiated elsewhere, possibly from ATG9. Further mutation of ATG9 in pIVY100 (resulting in pIVY104) abolished the remaining cytoplasmic activity, indicating that the cytoplasmic function of pIVY100 was indeed provided by protein product initiated at ATG9. However, much to our surprise, mutation of ATG9 alone did not impair the cytoplasmic function as expected (see pSAM33); instead, the ATG9 mutant still retained both activities. These results suggested that the long form that is initiated from ATG1 provided both the cytoplasmic and mitochondrial functions, whereas the shorter form that is initiated from ATG9 provided only the cytoplasmic activity. Given that the out-of-frame mutant (see pSAM25) still retained the cytoplasmic function, it is likely that the second ATG triplet can be recognized by scanning ribosomes as a remedial translation start site even in the presence of the first ATG triplet. Therefore, the cytoplasmic function of this gene is probably contributed by both isoforms under normal conditions. It is noteworthy that mutation of ATG1 to GCG (resulting in pSAM35) also created an out-of-frame ATG triplet (between the nucleotides –2 and +1) and had a phenotype similar to that of pSAM25. To provide a more quantitative data on the mitochondrial complementation activity of these mutants, a growth curve assay was subsequently carried out in YPG broth. Consistent with the observations made from the complementation assay (Fig. 3C), transformants carrying pSAM33 (with ATG9 inactivated) or pSAM20 (with the wild-type gene) grew well in YPG broth, whereas growth of those carrying pIVY100 (with ATG1 inactivated), pSAM25 (with an out-of-frame mutation between the two ATG codons), or pIVY104 (with both ATG triplets inactivated) was severely impaired (Fig. 3D). Analysis of the relative levels of specific mRNAs generated from each of these constructs indicated that similar levels of CaALA1 transcripts were generated from or maintained in the transformants carrying the wild-type or mutant CaALA1 constructs as determined by a semiquantitative RT-PCR experiment (Fig. 3E). This observation suggested that these mutations had little effect on the stability of the specific mRNAs generated from the constructs and, therefore, most likely modulated only the translation initiation activity of the individual initiators.


Figure 3
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  		FIGURE 3.
Identifying the translation initiation sites of CaALA1. Various mutations were individually introduced into CaALA1 cloned in pRS425 and tested for their effect on the complementing activities. A, summary of the CaALA1 constructs and their complementation functions. TRY11 was transformed with the wild-type and mutant CaALA1 constructs and then tested for its growth phenotypes. Nucleotide sequences shown include nucleotides –3~+27, i.e. codons –1~+9, relative to ATG1. Codons that have been mutated are shaded. B, complementation assays for the cytoplasmic function on a 5-FOA plate. C, complementation assays for the mitochondrial function on a YPG plate. D, growth curves of the TRY11 transformants containing various plasmid-borne CaALA1 constructs in YPG broth. E, RT-PCR. The relative amounts of specific CaALA1 mRNA generated from some of the constructs shown in A were determined by RT-PCR. As an internal control, the relative amounts of actin-specific mRNA in each sample were also determined. In B--E, the numbers 1–8 denote constructs shown in A. Cyt, cytoplasmic; Mit, mitochondrial.

 


Figure 4
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  		FIGURE 4.
Complementation by CaALA1 constructs cloned in a low copy number vector. Various mutations were individually introduced into CaALA1 cloned in pRS315 and tested for their effect on the complementing activities. A, summary of the CaALA1 constructs and their complementation functions. B, complementation assays for the cytoplasmic function on a 5-FOA plate. C, complementation assays for the mitochondrial function on a YPG plate. In B and C the numbers 1–6 denote constructs shown in A. Cyt, cytoplasmic; Mit, mitochondrial.

 
Because the observations made above were a result of AlaRS proteins produced from a high copy number plasmid, we were afraid that they might not accurately reflect protein activity at physiological levels. Therefore, wild-type and mutant CaALA1 genes were cloned into a low copy number shuttle vector (Fig. 4A) and tested for their cytoplasmic and mitochondrial functions. As shown in Fig. 4, B and C, these constructs had complementing activities similar to the corresponding constructs cloned in a high copy number vector.

Partition Pattern of ATG1- and ATG9-initiated CaAlaRS Isoforms—To investigate whether the CaALA1 constructs contain similar activities when highly expressed from a constitutive ADH promoter, some of the representative constructs shown in Fig. 3A were subcloned into pADH and tested for their complementation functions. As shown in Fig. 5, A–C, these constructs contained similar complementation functions to those cloned in pRS425, except for the ATG1 mutant, which contained only cytoplasmic function when cloned in pRS425 but contained both cytoplasmic and mitochondrial functions when cloned in pADH (compare pIVY100 and pIVY98). One likely possibility leading to this outcome is that the ATG9-initiated form contains a cryptic mitochondrial targeting signal that normally does not play a role in mitochondrial localization but can be recruited to function when the protein is highly expressed.


Figure 5
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  		FIGURE 5.
Localization of CaAlaRS isoforms expressed from a high copy number vector. Various mutations were individually introduced into CaALA1 cloned in pADH and tested for their effect on the complementing activities. A, summary of the CaALA1 constructs and their complementation functions. Nucleotide sequences shown include nucleotides –3~+27, i.e. codons –1~+9, relative to ATG1. Codons that have been mutated are shaded. B, complementation assays for the cytoplasmic function on a 5-FOA plate. C, complementation assays for the mitochondrial function on a YPG plate. Cyt, cytoplasmic; Mit, mitochondrial. D, fractionation and Western blots. The total, cytoplasmic, and mitochondrial fractions of the transformants harboring various CaALA1 constructs were isolated and analyzed by Western blots using anti-His6 tag antibody (upper three panels). As internal controls, the cytoplasmic and mitochondrial fractions extracted from these transformants were probed with an antibody mixture containing both anti-phosphoglycerate kinase (PGK) and anti-Hsp60 (lower two panels). The rightmost lane in the lower two panels shows the hybridization patterns of the total extracts of transformants containing pIVY97 (shown as Cont.). Indicated on the right are the relative migrating positions of CaAlaRS, Hsp60, and phosphoglycerate kinase (PGK), respectively. E, relative levels of CaAlaRS isoforms. The total fractions of the transformants were serially diluted and analyzed by Western blots. In B–E, the numbers 1–5 denote constructs shown in A.

 
To directly look at the protein expression levels and elucidate the correlations between complementation functions and partition patterns of the isoforms within the cell, the total, mitochondrial, and cytoplasmic fractions were isolated from each of the transformants harboring various CaALA1 constructs. As shown in Fig. 5D, the proteins expressed from the wild-type CaALA1 construct were partitioned between cytoplasm and mitochondria (lane 1, pIVY97), but mutations that inactivated both of the ATG initiators completely abolished the synthesis of the isoforms (lane 4, pIVY149). Interestingly, when the first ATG initiator was inactivated (lane 2, pIVY98), the protein levels in the total fraction remained almost unchanged, whereas the protein band in the mitochondrial fraction drastically decreased (compare lanes 1 and 2), suggesting that the mitochondrial proteins came largely from initiation at ATG1. In addition, it came as a surprise to us at first to find that the protein levels in the cytoplasmic fraction of this mutant appreciably increased. We surmised that the basis underlying this unexpected observation could probably be attributed to the fact that ATG9 served only as a remedial initiation site in the wild-type construct (lane 1) but became the first available ATG initiator in the ATG1 mutant (lane 2), leading to the higher expression of the "cytoplasmic" form. To assess the initiating activity of the remedial initiation site, i.e. ATG9, more accurately, ATG1 was left unaltered, and two extra nucleotides were inserted into the sequence between the two ATG initiators, causing the first ATG to be out-of-frame with respect to the rest of the open reading frame (lane 5, pIVY152). Under such conditions the protein band in the total fraction only slightly decreased due to loss of the ATG1-initiated protein, whereas the protein level in the cytoplasmic fraction was almost unchanged (compare lanes 1 and 5). But most significant of all, no protein was seen in the mitochondrial fraction of this out-of-frame mutant (lane 5), suggesting that the ATG9-initiated protein form was exclusively confined to the cytoplasm when ATG9 serves only as a remedial initiation site (compare lanes 1 and 5) and could be forced into mitochondria, possibly due to the presence of a cryptic mitochondrial targeting signal, when ATG9 serves as the first available initiator, resulting in higher expression of the short form (compare lanes 2 and 5). By contrast, when the second ATG initiator was inactivated (lane 3; pIVY118), the levels of the proteins in the total or cytoplasmic extracts drastically decreased, suggesting that the cytoplasmic proteins came largely from initiation at ATG9 in the wild-type construct (compare lanes 1 and 3), and the ATG1-initiated protein form can be partitioned in both compartments (lane 3), with the major portion targeted to the mitochondria. It is noteworthy that the protein level in the mitochondrial fraction of this mutant appreciably increased as compared with the wild-type construct (compare lanes 1 and 3). We surmised that perhaps this unexpected increase was due to alterations of the potential MPP cleavage site, which might affect its processing and in turn its distribution. As a control, the mitochondrial and cytoplasmic fractions were also probed with a mixture of antiphosphoglycerate kinase (a cytoplasmic marker protein) and anti-Hsp60 (a mitochondrial marker protein) to check for cross-contamination. As shown in Fig. 5D, no serious cross-contamination was seen in these preparations (lower two panels). To further quantify the initiating activity of ATG1 and ATG9, the relative protein levels in the total fractions of pIVY152 (an out-of-frame mutant) and pIVY118 (an ATG9 mutant) were compared. Fig. 5E showed that the initiating activity of ATG9 is around 4-fold as high as that of ATG1 under the conditions used. As a control, the relative protein levels generated from pIVY97 (wild type) were also shown. This result suggested that the long and short forms account for ~20 and ~80% of the total proteins generated from CaALA1.

We next checked the protein partition patterns for CaAlaRS isoforms expressed from a low copy number plasmid. As shown in Fig. 6, the partitioning patterns obtained were very similar to those shown in Fig. 5. However, the protein levels expressed from ATG1 under native conditions were only about 4% relative to those initiated from ATG9 (Fig. 6B). Because the relative amount of protein produced from ATG1 under native conditions is much lower than that produced under conditions of overexpression, the faint band seen in lane 6 (and possibly a portion of the protein seen in lane 4) is likely a result of slight contamination from the cytoplasmic fraction. Such contamination becomes significant under native conditions but is too slight to be seen under conditions of overexpression.


Figure 6
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  		FIGURE 6.
Localization of CaAlaRS isoforms expressed from a low copy number vector. Transformants harboring various CaALA1 constructs cloned in pRS315 were subjected to fractionation, and the relative levels of cytoplasmic and mitochondrial CaALA1 proteins were probed by Western blot. A, fractionation and Western blots. The total, cytoplasmic, and mitochondrial fractions of the transformants harboring various CaALA1 constructs were isolated and analyzed by Western blots using anti-FLAG antibody (upper panel). As internal controls, the total, cytoplasmic, and mitochondrial fractions extracted from these transformants were probed with an antibody mixture containing both anti-phosphoglycerate kinase (PGK) and anti-Hsp60 (lower panel). Indicated on the right are the relative migrating positions of CaAlaRS, Hsp60, and phosphoglycerate kinase, respectively. Lanes 1, 4, and 7, wild-type; lanes 2, 5, and 8, ATG9 mutant; lanes 3, 6, and 9, out-of-frame mutant. B, relative levels of CaAlaRS isoforms. The total fractions of the transformants were serially diluted and analyzed by Western blots. In B the numbers 2 and 3 (circled) denote constructs shown in A. Cyt, cytoplasmic; Mit, mitochondrial.

 
To determine whether the ATG1-initiated protein form is indeed processed in mitochondria, we subsequently sequenced the His6-tagged CaAlaRS proteins purified from the mitochondrial fraction of transformants harboring pIVY97 (wild-type) and pIVY118 (Met-9 mutation to Ala-9). The results showed that the purified proteins have an amino-terminal sequence of MSSNTTI and ASSNTTI, respectively, suggesting that the ATG1-initiated protein can be targeted to mitochondria and processed by matrix-processing peptidase between residues eight and nine (Fig. 1A). To rule out the possibility that proteins overexpressed from the ADH promoter may overload the processing system and lead to aberrant cleavage, the His6-tagged CaAlaRS protein was also purified from the mitochondrial fraction of transformants harboring a ATG9 mutant construct cloned in a low copy number vector and sequenced. As it turned out, the processed mitochondrial form contained an expected amino-terminal sequence of ASSNTTI.

Demonstration of Two Distinct Protein Isoforms Initiated from ATG1 and ATG9—Because the ATG1- and ATG9-initiated protein isoforms are very similar in size (~106 kDa), it is impractical to directly distinguish them by Western blot. Furthermore, the long protein form may be processed in the mitochondria to a size similar to the short form (Fig. 5). To ascertain whether two distinct proteins are independently initiated from these two nearby initiators, we next fused the 5'-terminal sequence of CaALA1 (base pairs –40~+30) to a smaller gene, lexA (thereby both avoiding mitochondrial localization and increasing relative size difference of the resulting fusions) and tested for its protein expression profile. These fusions were expressed under the control of an ADH promoter and separated on an 18% SDS-PAGE (size, 16 x 20 cm). As shown in Fig. 7, a minor upper band and a major lower band were simultaneously generated from pIVY155 (containing both ATG1 and ATG9), whereas mutation of ATG1 or introduction of an out-of-frame mutation between ATG1 and ATG9 impaired the production of the upper band (see pIVY156 and pIVY159), suggesting that the upper band is initiated from ATG1 and the lower band shown in pIVY156 and pIVY159 is initiated from ATG9. Consistently, further mutation of ATG9 in pIVY156, resulting in pIVY161, abolished both protein bands (lane 4). However, when only ATG9 was mutated (pIVY160, lane 3), two distinct protein bands of similar amounts could be seen, suggesting that the ATG1-initiated protein was partially processed to a molecule with size similar to the ATG9-initiated protein. Therefore, the lower band shown in pIVY155 (lane 1) is likely composed of proteins initiated from ATG1 (processed) and ATG9. It appears that mutation of ATG9 to GCG (Met-9 to Ala-9) in pIVY160 reduces the processing rate of the protein by matrix processing peptidase (compare lanes 1 and 3 and Fig. 5D, lanes 1 and 3). Thus, ATG9 functions as a remedial translation initiator even in the presence of ATG1 (pIVY159) and is about 4 times as efficient as ATG1 (compare lanes 3 and 5), similar to results obtained in Fig. 5. Similar results were observed when these fusions were expressed from the native CaALA1 promoter (data not shown).


Figure 7
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  		FIGURE 7.
Demonstration of two distinct protein isoforms initiated from ATG1 and ATG9. A, summary of the CaALA1-lexA* constructs. A wild-type or mutant CaALA1 sequence (bp –40~+30) was fused in-frame to the 5'-end of lexA* (where its ATG initiator has been mutated), resulting in various CaALA1-lexA* fusions. The ATG initiators and their mutants (shaded) were labeled on top of the sequences. The open and striped boxes represent CaALA1 and lexA sequences, respectively. B, Western blot. The constructs were transformed into INVSc1, and their protein expression was analyzed by Western blot using anti-LexA antibody. In B, the numbers 1–5 correspond to constructs shown in A.

 


Figure 8
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  		FIGURE 8.
Mapping the mitochondrial targeting sequence of CaAlaRS. The vas1– strain, CW1, was transformed with various CaALA1-VAS1c constructs and then tested for its growth phenotypes. A, summary of the CaALA1-VAS1c constructs and their complementation activities. Various CaALA1 fragments (–370~+102 bp, –370~+126 bp, or –370~+159 bp) were fused in-frame to the 5'-end of VAS1c, resulting in pIVY117, pIVY105, and pSAM37, respectively. The ATG initiators were labeled on top of the sequences. The open and solid boxes represent CaALA1 and VAS1c sequences, respectively. B, complementation assays for the cytoplasmic function on a 5-FOA plate. C, complementation assays for the mitochondrial function on a YPG plate. Cyt, cytoplasmic; Mit, mitochondrial. D, RT-PCR. The relative amounts of specific CaALA1-VAS1c mRNA generated from the constructs were determined by RT-PCR. As an internal control, the relative amounts of actin-specific mRNA in each sample were also determined. In B–D, the numbers 1–3 denote constructs shown in A.

 
Mapping the Mitochondrial Targeting Signal of CaAlaRS—As shown in Fig. 1D, the protein form initiated from ATG1 appears to have an amino-terminal 15-residue appendage that is absent from the other yeast cytoplasmic AlaRSs compared; therefore, we wondered whether this leader peptide actually participates in protein localization into mitochondria. To map the mitochondrial targeting sequence of CaAlaRS, the cytoplasmic form of ValRS (designated here as ValRSc) was chosen as the reporter protein, which is by itself confined exclusively in the cytoplasm even when overexpressed (15). Three CaALA1 fragments with distinct 3'-ends were independently amplified by PCR and fused in-frame to the 5'-end of VAS1c (coding for ValRSc), resulting in various CaALA1-VAS1c constructs (Fig. 8A). These constructs were transformed into CW1, a vas1– strain, and tested for their ability to rescue the growth defects of the knock-out strain on FOA and YPG plates, respectively. Contrary to our anticipation, fusion of the peptide containing residues 1–34 of CaAlaRS to ValRSc (resulting in pIVY117) did not confer a mitochondrial phenotype to the fusion, i.e. transformants harboring pIVY117 could not grow on the YPG plate (Fig. 8, B–C). The passenger protein could be successfully targeted to mitochondria only when the CaAlaRS portion was extended to residue 42 or 53 (see pIVY105 and pSAM37, respectively). This result suggested that the mitochondrial targeting sequence of CaAlaRS extends from residue 1 to 42. To check if these fusions express similar levels of mRNAs, the relative levels of specific CaALA1-VAS1c mRNAs generated from each construct were determined by a semiquantitative RT-PCR experiment using a set of primers, with the forward primer annealed to base pairs +1~+25 of CaALA1 and the reverse primer to base pairs +400~+425 of VAS1. Fig. 8D showed that similar levels of the cDNA products were generated from these fusions, suggesting that lack of the mitochondrial function in pIVY117 was not caused by different levels of RNA expression (or degradation) but, rather, by the absence of an efficient mitochondrial transit signal.


Figure 9
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  		FIGURE 9.
Testing the efficiency of the mitochondrial targeting signal of CaAlaRS. The vas1– strain, CW1, was transformed with various CaALA1-VAS1c constructs and then tested for its growth phenotypes. A, summary of the CaALA1-VAS1c constructs and their complementation activities. A wild-type or mutant CaALA1 sequence (–370~+126 bp) was fused in-frame to the 5'-end of VAS1c, resulting in various CaALA1-VAS1c constructs. The ATG initiators and their mutants (shaded) were labeled on top of the sequences. The open and solid boxes represent CaALA1 and VAS1c sequences, respectively. B, complementation assays for the cytoplasmic function on a 5-FOA plate. C, complementation assays for the mitochondrial function on a YPG plate. In B and C, the numbers 1–4 denote constructs shown in A. Cyt, cytoplasmic; Mit, mitochondrial.

 
Given that the long form of CaAlaRS can be dual-targeted, we wondered whether this unique feature could be attributed to a poor mitochondrial targeting signal such that only a portion of the preprotein is targeted to the mitochondria and whether this feature could be passed on to the CaAlaRS-ValRSc fusion proteins. As shown in Fig. 9, mutation of ATG1 to TCT (resulting in pIVY106) selectively impaired the mitochondrial function of the CaALA1-VAS1c fusion (compare pIVY105 and pIVY106), whereas mutation of ATG9 to GCG (resulting in pIVY107) specifically eliminated the cytoplasmic function of the fusion (compare pIVY105 and pIVY107). Additionally, double mutations that inactivated both of the initiators led to a construct (see pIVY108) that was defective in both functions. This observation suggested that the ATG1- and ATG9-initiated fusions were targeted exclusively to mitochondria and cytoplasm, respectively. Thus, the ATG1-initiated signal peptide (containing residues 1–42) is in effect a strong mitochondrial targeting signal under the conditions used.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present work we discovered that CaALA1, the only alanyl-tRNA synthetase gene in C. albicans, could overcome the species barrier and substitute for both the cytoplasmic and mitochondrial functions of ALA1 in S. cerevisiae (Figs. 2 and 3). As with the case in S. cerevisiae (10), these functions are provided by two distinct protein isoforms that are synthesized through alternative initiation from two nearby in-frame start codons (Fig. 3). However, several characteristic features regarding the mechanism of translation initiation and partition of the protein isoforms appear to be idiosyncratic to the gene and are worthy of further attention. First, although two distinct protein forms are generated from this gene, the short form appears to be redundant and dispensable for the cytoplasmic function (Fig. 3). Second, different from ALA1 of S. cerevisiae, no non-canonical initiators are involved in the translation of a minor, mitochondrial form in this case; instead, both of the isoforms of CaAlaRS are initiated from AUG triplets (Fig. 3). Last but not least, whereas the bifunctional phenotype of ScALA1 is contributed by two functionally exclusive protein isoforms; the long form of CaAlaRS per se is a dual-targeted (and, thus, bifunctional) protein (Fig. 3). To our knowledge, this appears to be the first example in yeast, wherein a naturally occurring form of a tRNA synthetase can play roles in both compartments.

Despite the fact that the mitochondrial targeting signal of CaAlaRS extends from amino acid residue 1 to 42 (Fig. 8), the cleavage site of matrix-processing peptidase was mapped between residue positions 8 and 9 (Fig. 1). As a result, the "processed" mitochondrial form has an amino terminus identical to that of the short form. Analysis of the CaAlaRS isoforms with the PSORTII program (17) showed a 65% likelihood of mitochondrial import for the long form but only a 26% likelihood for the short form. Furthermore, as with many classical mitochondrial targeting signals (18), this 42-residue peptide is rich in positively charged (17%) and hydroxylated residues (26%) but is almost devoid of acidic residues (2%). Strangely enough, although the long form could be distributed and, thus, was functional in both compartments, fusion of the full-length signal peptide to a cytoplasmic passenger did not confer a bifunctional phenotype to the fusion (Fig. 9). These results suggested that the mechanism of protein localization for CaAlaRS is different from what we have observed for a truncated version of the mitochondrial form of ValRS, where a mitochondrial preprotein can be made bifunctional simply by weakening its mitochondrial targeting signal (15). More experiments are currently under way to elucidate the mechanism that contributes to the dual-targeting nature of the long form. In this aspect the yeast FUM1 gene (coding for fumarase) represents an interesting example. A single species of primary translation product is generated from FUM1 and is responsible for both the cytoplasmic and mitochondrial fumarase activities in vivo (19). As it turns out all FUM1 gene products are first targeted to the mitochondrial matrix and then a significant fraction of the processed proteins arrives back in the cytoplasm (20). Thus, the mature forms of the cytoplasmic and mitochondrial fumarases have the same amino termini (21).

In mammalian cells the small ribosomal subunit often skips a weak translation initiator, such as a non-AUG codon or an AUG codon within a suboptimal sequence context, and continues scanning downstream on the message until it encounters an AUG triplet within a more favorable sequence context. This process is referred to as "leaky scanning" (22–23). Although this mechanism has been observed frequently in mammals (24–27), there are only a few known examples in yeast. Examples include MOD5 (coding for isopentenyl pyrophosphate:tRNA isopentenyl transferase) (28) and CCA1 (coding for ATP (CTP):tRNA nucleotidyltransferase) (29). In these two instances leaky scanning occurs probably because the first AUG codon is located too close to the 5'-end of the mRNA, making it less accessible to the initiating ribosome. In the case of CaALA1, because only one transcript with its 5'-end located at nucleotide position –24 relative to ATG1 is available (Fig. 1), it is, therefore, likely that recognition of the second AUG triplet as well as production of the short isoform is also mediated by leaky scanning. Evidence supporting this argument came from the observation that expression of the AUG9-initiated short form drastically increased when AUG1 was mutated (Fig. 5 and 7).

The specificity of an aminoacylation reaction is accomplished by direct recognition of the cognate tRNA by the specific synthetase. In some instances recognition depends mainly on the anticodon, whereas in others it depends more on the sequences/structures in the acceptor stem (right next to the amino acid coupling site) (30). For example, Drosophila melanogaster cytoplasmic tRNAAla has a G3-U70 base pair in the acceptor stem as its major identity determinant, whereas the GU pair has been translocated to position 2:71 in its mitochondrial isoacceptor. Consequently, D. melanogaster mitochondrial AlaRS can only aminoacylate its mitochondrial tRNAs but not its cytoplasmic equivalents (31). We should mention that the G3-U70 base pair is conserved in all known tRNAAla sequences from prokaryotes, Archaea, eukaryote cytoplasm, and chloroplasts. In this sense low eukaryotes such as S. cerevisiae and C. albicans appear to represent a divergent point in the course of coevolution of tRNAAla and its cognate synthetase, where a single nuclear AlaRS gene is capable of aminoacylating alanyl-tRNAs in both compartments of the same cell in these organisms (10). Furthermore, the C. albicans enzymes are capable of cross-species complementation, adding further emphasis to the importance of the G3-U70 element in recognition specificity.


   FOOTNOTES
 
* This work was supported in part by National Science Council (Taiwan) Grant NSC 94-2311-B-008-009 (to C.-C. W.) and by Institute of Nuclear Energy Research, Atomic Energy Council (Taiwan) Grant 94-2001-INER-EE-009 (to C.-C. W.). 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 may be addressed. Tel.: 886-3-856-5301 (ext. 7541); Fax: 886-3-857-2526; E-mail: susanyeh{at}mail.tcu.edu.tw. 2To whom correspondence may be addressed. Tel.: 886-3-426-0840; Fax: 886-3-422-8482; E-mail: dukewang{at}cc.ncu.edu.tw.

3 The abbreviations used are: AlaRS, alanyl-tRNA synthetase; ADH, alcohol dehydrogenase; 5-FOA, 5-fluoroorotic acid; ValRS, valyl-tRNA synthetase; YPG, yeast extract-peptone-glycerol; RACE, rapid amplification of cDNA ends; RT, reverse transcription. Back


   ACKNOWLEDGMENTS
 
We are grateful to Grace Lin of National Central University for critical reading of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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