involved in mRNA splicing - Europe PMC

The EMBO Journal vol.7 no.2 pp.473-483, 1988

The NAM2 proteins from S.cerevisiae and S.douglasii are mitochondrial leucyl-tRNA synthetases, and are involved in mRNA splicing

Christopher J.Herbert, Michel Labouesse, Genevieve Dujardin and Piotr P.Slonimski Centre de Genetique Moleculaire du CNRS, Laboratoire propre associe a l'Universite Pierre et Marie Curie, 91 190 Gif-sur-Yvette, France

Communicated by P.Slonimski

We have cloned and sequenced the NAM2 gene of Saccharomyces douglasii, which is a homologue of the NAM2 gene of Saccharomyces cerevisiae. The wild-type S.douglasii gene possesses the suppressor functions of the mutant S.cerevisiae NAM2-1 allele, being able to cure a nmtochondrial bl4 maturase deficiency. By sequence comparisons and direct measurements we have demonstrated that the NAM2 genes encode mitochondrial leucyl tRNA synthetases (EC 6.1.1.4.). Using a derivative of the NAM2 gene, where the expression of the gene is under the control of the UAS GAL]O, we have shown that the processing of the pre-mRNA from the two mosaic genes oxi3 and cob-box is impaired when transcription of the gene is repressed. These results lead us to conclude that the mitochondrial leucyl tRNA synthetase is involved in protein synthesis and mRNA splicing. Sequence comparisons show that the mitochondrial and Escherchia coli leucyl tRNA synthetases are highly homologous; however, significant features which may be important for the splicing functions of the mitochondrial enzymes are absent from the bacterial enzyme. Key words: mitochondrial leucyl tRNA synthetase/mitochondrial mRNA splicing/nuclear suppressor/zinc fingers/yeast

Introduction Although the basic mechanism of RNA splicing is understood, with all the reactions proceeding by a series of transesterifications (for a review see Cech and Bass, 1986), our knowledge of the extrinsic factors involved, such as proteins and other RNAs, varies considerably from one system to another. RNA processing in fungal mitochondria is of particular interest as it requires proteins synthesized in two cellular compartments: mRNA maturases which are encoded by certain mitochondrial introns, translated in mitochondria and necessary for the excision of specific introns (Lazowska et al., 1980 and Banroques et al., 1986, for a review see Tabak and Grivell, 1986), and nuclear encoded proteins, synthesized in the cytoplasm and then imported into the mitochondria. These can be specific for individual introns (McGraw and Tzagoloff, 1983) or can be involved in the metabolism of several introns (Akins and Lambowitz, 1987). To try and discover nuclear genes whose products might be involved in mitochondrial RNA splicing in Saccharomyces cerevisiae, we have isolated nuclear suppressors of mitochondrial splicing deficient mutants. In this way several genes have been identified (Dujardin et al., 1980, Groudinsky et ©IRL Press Limited, Oxford, England

al., 1981). One of these genes NAM2 was identified by the ability of dominant alleles (NAM2-J . . NAM2-7) to suppress mutations that inactivate the maturase encoded by the fourth intron of the mitochondrial cytochrome b gene (bl4 maturase). This maturase is necessary for the excision of the intron b14 (De la Salle et al., 1982) and the fourth intron of the mitochondrial gene encoding subunit 1 of cytochrome oxidase (al4) (Labouesse et al., 1984). Experiments with double mutants have shown that the suppressor activity of the NAM2-1 allele is dependent on the ORF of the intron al4 (Dujardin et al., 1983). The suppressor activity of the NAM2-1 allele is subject to a gene dosage effect being more efficient when cloned on a multicopy vector. This suppressor phenotype is not due to the acquisition of a new function, as the wild-type NAM2 gene has a weak suppressor activity when cloned on a multicopy plasmid. Rather it is the result of the enhancement of an activity already present in the wildtype gene (Labouesse et al., 1987). In S. cerevisiae null nam2 alleles have been constructed by creating deletions and/or insertions in the gene. These were not lethal for the cell, but lead to the formation of 100 % cytoplasmic rho- petites, showing that the NAM2 gene is essential for the maintenance of an intact mitochondrial genome (Labouesse et al., 1985). When the deduced protein sequence was analysed two domains could be identified; one was a nucleotide binding site, and the other was a nucleic acid binding site. Both of these domains are found in some aminoacyl tRNA synthetases, but we were not able to decide whether the NAM2 protein was a synthetase or just resembled a synthetase (Labouesse et al., 1987). We also noted that in mitochondria mRNA splicing and translation occur in the same compartment and we hypothesized that the NAM2 gene may be involved in both processes. Recently Akins and Lambowitz (1987) have shown that the cytl8 gene of Neurospora crassa encodes the mitochondrial tyrosyl tRNA synthetase and that this protein is involved in the splicing of group I mitochondrial introns. Also they were able to separate chromatographically two types of tyrosyl tRNA synthetase activities, one of which was associated with a

splicing activity. In this paper we describe several approaches we have used in order to broaden our understanding of the function of the NAM2 gene. The first approach was to isolate and characterize a new NAM2 gene from a different yeast species, the NAM2D gene of S.douglasii, (for the sake of clarity we shall henceforth call the S. cerevisiae gene NAM2C, and the S.douglasii gene NAM2D). We show that this wild-type S. douglasii gene possesses the suppressor functions of the mutated S.cerevisiae NAM2-1 allele. In a second approach sequence comparisons, direct tRNA charging assays in mitochondrial extracts and inhibition by specific antibodies, allowed us to show that the NAM2 proteins are mitochondrial leucyl tRNA synthetases. Finally the use of a conditional allele of the S. cerevisiae NAM2C gene allowed us to study the effect of a depletion of the NAM2C protein

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C.J.Herbert et al.

could encode a protein of 894 amino acids (mol. wt 102 kd). This long ORF is exactly the same size as the long ORF found in the S. cerevisiae NAM2C gene (Labouesse et al., 1987). When the deduced protein sequences of the S. douglasii and S.cerevisiae ORFs were compared they were found to be very similar, with only 76 replacements in 894 amino acids, 35 of which could be considered as conservative changes. The replacements are scattered throughout the protein but are more frequent in the region of 280-450. These results indicate that S.douglasii contains a gene which we shall call the NAM2D gene, which is the homologue of the S. cerevisiae NAM2C gene (the comparison of the protein sequences is shown in Figure 4).

on translation and mitochondrial mRNA splicing. Recently Hartlein and Madern have established the sequence of the Escherichia coli leucyl tRNA synthetase (Leu RS). The mitochondrial and bacterial enzymes display considerable homology; however, significant features are absent from the E. coli enzyme and we believe that these may be important for the splicing functions of the mitochondrial enzymes.

Results Hybridization studies and cloning of the S.douglasii NAM2D gene In order to see if a gene structurally similar to the NAM2C gene of S. cerevisiae exists in other yeasts Southern blots were performed with genomic DNA from S. cerevisiae, S. douglasii and Shizosaccharomyces pombe, using an S. cerevisiae NAM2C gene probe. S.pombe and S.douglasii were chosen as they represent yeasts both distantly (S.pombe) and closely (S.douglasii) related to S. cerevisiae. The results of this experiment (Figure 1) show that S.pombe does not contain sequences that hybridize to the S. cerevisiae NAM2C gene under the conditions used; however, S.douglasii does contain sequences that hybridize to the S. cerevisiae NAM2C gene. Figure 1 also shows that some restriction site polymorphism exists between the S. cerevisiae and S. douglasii sequences so they cannot be identical. In order to further study the S. douglasii gene we decided to clone it. A bank of S.douglasii nuclear DNA was made in the lambda phage EMBL4 and recombinant phages containing the S.douglasii NAM2 sequences were identified by hybridization (see Materials and methods).

Nature of the NAM2 proteins Similarities between NAM2 and other proteins especially aminoacyl tRNA synthetases. In a previous study (Labouesse et al., 1987) we showed that the NAM2C protein of S. cerevisiae has an N-terminal sequence typical of proteins that are transported into the mitochondrial matrix. The deduced N-terminal sequence of the NAM2D protein also shows the same features, being enriched for arg, leu and ser residues, with few asp, glu, val and ile residues (10 versus 2 in the first 25 residues) which according to Roise et al. (1986) are characteristic of mitochondrial import signals. Two regions of similarity with other proteins, which could correspond to functional domains were also identified. The first was a putative nucleotide binding site similar to those found in aminoacyl tRNA synthetases and dehydrogenases. This consists of two blocks, residues 53-66 and residues 153-169; only one change occurs in these sequences in the S.douglasii protein, an alanine (residue 61) is replaced by a valine. The second was a putative nucleic acid binding site, residues 349 -369, 462475 and residues 503 -516; there are no changes in the critical residues in these regions between NAM2D and NAM2C. The nucleic acid binding site is based on the motifs Cys-X2-Cys or CysX2/3-His, which are able to form 'Zinc fingers' (Miller et al., 1985 and Berg, 1986) and are found in many proteins, such as retroviral proteins, regulatory proteins and mRNA maturases.

Sequence of the S.douglasii NAM2D protein If the S. douglasii NAM2 hybridizable sequences are part of a gene and encode a protein, the comparison of the S. cerevisiae and S. douglasii restriction maps, indicates that this gene should be contained within a 4-kb SacI-SphI fragment (Figure 1). This fragment was isolated from XSdA, and its sequence was determined as described in the Materials and methods. Analysis of the sequence shows a single long ORF which

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Fig. 1. Structure of the S.douglasii NAM2D gene. The left hand panel shows a Southern blot analysis of genomic DNA from S.cerevisiae (C), S.douglasii (D), and Spombe (P). Hybridization was carried out at 42°C, in the presence of 25% formamide, the probe was pGMC031 which contains the NAM2C gene cloned in pBR322 (Labouesse et al., 1985). Molecular size markers (in kb) are indicated on the left. The right hand panel shows a restriction map of the S.douglasii NAM2D region above the S.cerevisiae NAM2C region. The position of the NAM2C ORF is marked by an open bar and the two NAM2C transcripts are shown by arrows. The data for S.cerevisiae was derived from Labouesse et al. (1985, 1987), and the data for S.douglasii was derived from Southern blot and sequence analysis.

474

Mitochondrial leucyl tRNA synthetase and mRNA splicing

Further comparisons have revealed another region of similarity. Affinity labelling studies and sequence comparisons allowed Hountondji and collaborators to identify the amino acid sequence KMSKS and an invariant lysine residue, as part of the binding site for the 3' end of the tRNA in aminoacyl tRNA synthetases (Hountondji et al., 1985, 1986a,b). Both the NAM2D and NAM2C proteins contain this sequence at residues 646-650. In view of the fact that two of these regions of similarity occur in some aminoacyl tRNA synthetases, we decided to investigate more closely the possible relationship between tRNA synthetases and the NAM2 proteins. Aminoacyl tRNA synthetases all catalyse the same overall reaction, but apart from the regions described above little similarity has been detected at the primary sequence level for synthetases that charge different amino acids (Hountondji et al., 1986b). Much more homology is seen between synthetases that charge the same amino acid, in particular the two yeast mitochondrial synthetases sequenced to date show considerable homology with their counterparts from E. coli (Pape et al., 1985 and Myers et al., 1985). Thus if the NAM2C and NAM2D proteins are mitochondrial aminoacyl tRNA synthetases, we would expect to find considerable homology with other synthetases that charge the same amino acid. Until now all the comparisons were made by visual alignments and no statistical analysis of the similarity has been performed. A Aminoacyl tRNA synthetases charging the same amino acid from different organisms, or different cellular compartements have very similar sequences.

Sequences MetRS E. coli ThrRS E. coli ThrRS E. coli ThrRS S. cerevisiae cyto TrpRS E. coli TyrRS E. coli TyrRS E. coli

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Sequences to lleRS E. coli to GluRS E. coli All other comparisons (n=76)

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Z value 34 20 20 generations. Panels C and D: Extracts from HM200/1 grown on 2% galactose minimal medium (25 ug) were pre-incubated overnight at 4°C in 50 10 mM Tris pH 7.5 with 25 ,ul of control serum or antiserum raised against the ,Bgalactosidase/NAM2C fusion protein prior to the charging assays.

D.Madern communicated to us the sequence of the LeuRS of E. coli (Hartlein and Madern, in press). When this was compared with the NAM2 sequences the homology was overwhelming, with a Z value of 141. This is the highest value we found in all of our comparisons (cf. Figure 2). The three proteins display 34% identical amino acids and 20% conservative replacements in the 839 residues that can be aligned unambiguously (Figure 4). The homology is higher in the first 220 residues (46% identical residues and 15% conservative replacements), medium in the central part, residues 221-660 (34% identical residues and 20% conservative replacements), and lower at the end, residues 661-879 (17% identical residues and 20% conservative replacements).

Role of the NAM2 proteins in mitochondrial mRNA splicing Suppressor activity of the S.douglasii NAM2D gene in S.cerevisiae. Cytoduction experiments by Claisse et al. (1987) indicate that a b14 maturase mutation is alleviated by the nucleus of S.douglasii. As this activity resembles the suppressor activity of the NAM2-1 allele we decided to see if the NAM2D gene was able to suppress b14 maturase mutations in S.cerevisiae. To test the suppressor activity of the different NAM2 alleles, an S. cerevisiae strain which has an inactive b14 maturase (CKG18) was transformed with the S.cerevisiae NAM2-1 suppressor allele, or the S.douglasii

476

NAM2D gene, on centromeric and multicopy plasmids and the ability of the transformants to grow on glycerol was determined. The results in Figure 5 show that the S.douglasii NAM2D gene is able to suppress an S. cerevisiae b14 maturase mutation, and that the suppressor activity is subject to a gene dosage effect, being more effective when present on a multicopy plasmid. In a previous study we have shown that the suppressor mutations in three S.cerevisiae alleles (NAM2-1, NAM2-6 and NAM2-7) are located within a central 1-kb BamHI fragment, and that the same codon (residue 240) is affected in all three cases (Labouesse et al., 1987). In the wild type non-suppressor NAM2C protein this residue is a glycine. When compared with the wild type NAM2C protein, the sequence of the NAM2D protein shows 34 amino acid changes in the region corresponding to the -kd BamHI fragment. However, at position 240 there is a glycine as in the wild type S. cerevisiae protein. To determine if this region of the S. douglasii gene is able to confer a suppressor activity we constructed a series of hybrid genes, taking advantage of the fact that the BamHI sites of the NAM2C and NAM2D genes are conserved in both position and frame. An S. cerevisiae strain which has an inactivated b14 maturase (CKG18) was transformed by the plasmids YEpGMC050 (which contains the wild type NAM2C gene), YEpGMC053 and YEpGMC054, where the BamHI fragment from NAM2-1 and NAM2D respectively replaces the equivalent

Mitochondrial leucyl tRNA synthetase and mRNA splicing 50

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Fig. 4. Alignment of the two yeast mitochondrial and the E. coli leucyl tRNA synthetases. The alignment was made using the Dayhoff PAM matrix, LRSSd respresents the S.douglasii NAM2D sequence, LRSSc the wild-type S.cerevisiae NAM2C sequence (Labouesse et al., 1987), and LRSEc the E.coli sequence (Hartlein and Madem, in press). LRS is the consensus sequence, upper case letters show identical residues and lower case letters show conservative changes (Dayhoff, 1978). The N-terminal residues 1-23 of the mitochondrial leucyl tRNA synthetases display the characteristic features of a mitochondrial import signal and differ greatly from the bacterial sequence. There are only three differences between S.douglasii and S.cerevisiae in this region, (position 3: pro to ser, position 13: gln to lys, and position 18: arg to pro). The first amino acids aligned are residues 24 in the two yeast sequences and residue 14 in the E.coli. The nucleotide sequence of LRSSd is available in the EMBL data bank.

fragment in the wild type gene. Transformants were tested for growth on glycerol (Figure 6), and transformants harbouring YEpGMC054 were able to grow at least as well, if not better than those harbouring YEpGMC053. This shows that the 1-kb BamHI fragment of the NAM2D gene contains sufficient information to confer a suppressor activity when it replaces the corresponding 1-kb BamHI fragment from the S. cerevisiae NAM2C, even though it has a glycine residue at position 240. Effect of reducing the quantity of NAM2 protein on RNA splicing. Disruption of the NAM2C gene of S. cerevisiae leads to the loss of the mitochondrial genome, preventing the analysis of the function of the gene. To circumvent this problem we have used a conditional allele of the gene in the plasmid YCpGMC068 where the coding sequence of the NAM2C gene is under the control of the UAS GALIO (see Materials and methods). The phenotype conferred by this plasmid was assayed in a strain carrying a disrupted nam2C chromosomal gene. When these strains are grown in a galactose medium, almost all the cells are able to form a colony when plated on glycerol. When grown in a glucose medium the cells gradually lose the ability to form a colony on glycerol plates. This is consistent with the idea that a factor

essential for growth on glycerol is being diluted out and becoming limiting. The proportion of rho- petites produced by these strains was very low (5 % compared to 1 % in the control strain) and remained stable during growth on galactose and glucose (as a control the plasmid YCpGMC068 was transformed into a strain with a wild type nam2C gene). When the RNA from these strains was examined with probes for various mitochondrial genes (Figure 7) we see that the amount of mRNA for cytochrome oxidase subunit III remains almost constant (panel D), (the 21S rRNA and the mRNA for ATPase subunit 9 and cytochrome oxidase subunit LI, were also examined and found not to be significantly affected; data not shown). However, the mRNA from the two mosaic genes cob-box and oxi3 disappears (panels A and B), instead high mol. wt RNA precursors containing unexcised IVSs accumulate. The stable circular excised form of the group II intron all also disappears (panel C). These effects are seen between 10-19 generations in a glucose medium, when innoculated from a galactose medium. We believe that this lag is due to the enormous over-expression of the NAM2C protein from this construction when grown on galactose (at least a 200-fold increase, data not shown) which must be diluted before an effect can be seen. In a similar type of study Patterson and Guthrie 477

C.J.Herbert et al. urowwlh on glycerol of a b14 maturase mutant in the eresence :lf di-fferernt NAM2 allelles

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Fig. 6. Suppression of a b14 maturase mutation by a hybrid S.douglasii-S.cerevisiae NAM2 gene. The left hand panel shows construction and the YEpGMC053 and YEpGMC054 where the 1-kb BamHI fragment of the wild-type NAM2C gene is replaced by the 1-kb BamHI fragment from NAM2-1 and NAM2D respectively. The right hand panel shows the growth on glycerol of an S.cerevisiae strain with an inactive b14 maturase (CKG18) which has been transformed by these plasmids. After 10 days YEpGMC051 confers no growth, YEpGMC050 confers very slight growth, while YEpGMC053 and YEpGMC054 confer strong growth. structure of

(1987) have constructed gene fusions between GAL] and SNR7, they also found a considerable lag before the effect of glucose repression was seen. In summary when the concentration of the NAM2C protein is lowered in the cell, the excision of several IVSs of the mosaic genes cob-box and oxi3 is considerably impaired, while the mRNAs of continuous genes, and rRNA are not significantly affected.

Discussion Here we have shown that the NAM2C gene of S. cerevisiae 478

(previously localized to chromosome XII, Dujardin et al., 1983) encodes the mitochondrial leucyl tRNA synthetase. Direct aminoacyl tRNA synthetase assays in mitochondrial extracts (Figure 3 panels A and B) showed that the leucine charging activity was almost absent in a strain with a disrupted nam2C gene and greatly increased in a strain where the gene is overexpressed, while other charging activities were not affected The residual charging activity seen in mitochondrial extracts of the disrupted strain (Figure 3, panel A), is probably due to the presence of a small amount of contaminating cytoplasmic synthetase, which must be

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-.

1C 19

Block 1 sdlrs .......... sclrs .......... eclrs .......... scvrs .......... ecirs .......... consensus ........

sdlrs sclrs eclrs scvrs ecirs cons.

.NH2-55 .NH2-55 .NH2-41 .NH2-189

.......... PYPSGvLHIG HLRvYVISDS .......... PYPSGALHIG HLRvYVISDS ... .......... PYPSGrLHHG HVRnYtIGDv .......... PnvTGALHIG ....... . .NIH2-57 ..... PYAnGSIHIG HsvnkILXD1 .......... .......... H. ....I.D. ...

...

HaltiaIqDS

PypsG.lHiG

Block 2 AnFD VdREVTTCDP AnFD WdREITTCDP FGLPAE ..-29...... fgYD VSRELATCtP aGIATQ .. -47-.. AsYD WSREafTlsP hiGLPiE .. -42-.. vlgD WSHpylTEDf yD W.re ..T. .p Glpae..

LnRFyKqRGY IVIhPMGWDA FGLPAE .. -29-.. LnRFyKqKGY I aRYqHiGlck LiRYnRNKGk IvKskgLsGY 1.ry.k. .G.

NVIhPNGWDA FGLPAE .. -29-..

IVLqPIGWDA tVLflpGFDh DspyvpGWDc nvi.. GvD.

.......

4.. sdlrs

.4 mRNA

4

-

_*

r

r

m

scirs ecirs scvrs ecirs

ft.

cons.

sdlrs sclrs

C COXI intronic probe n for all introf Dcca _

ecirs

be

scvrs

ecirs cons.

NAM2 allele

WT

EYYKFtOwiF LRLFEIGLaY RKeAeIIWdP vDkTVLAIEO EYYKFtQviF LKLFEIGLaY RKeAeINWdP vDnTVLAIEO EYYRleQkfF teLYkkGLVY RKTSaVIWcP nDqTVLANIE EltKsvEeaF VRLhDEGVIY RaSklVIVWsv kInTaISNlE kteanliral gKIigNGhLh KgAkpVHWcv dcrSaLAEaE e. .k. .qv.f .kl ..nGl.y r .... vnV . t.lan.q

eclrs scvrs

ecirs mRNA-

Ve ...... rs

Block 3 OWFLgITKFA ..-44-.... LIVFTTRPET IFAvqYVAL. QWFLgITKFA ..-44-.... LIVFTTRPET 1FAvqYVAL. QWFIkInaYA ..-43-.... LtVYTTRPDT f*GcTYLAV. veFgvLTsFA ...-9-.... LIIaTTRPET eFGdTaVAV. ld.Va.fQav DqdalkAKFA ...-9-.... LVIVTTRrgl clPtAqslL. V... q.. q.t itfA .... LivfTTRpet ..g... val. Block 4

sdlrs

sclrs

VEnkdvksRT VEyydKTspS

gAlVEKKQLK gAiVEKKOLK dTkVERKEIp llsVpgyDeK

a _ted -.

dlsr.pted

GLU-GLU - xl -GLJ --~~~~~~~~~~ 0 10 *9 0 10_ 11

VDaQGRSWRS VDaQGRSWRS V.1DGccVRc

. . . . . .

-65-.. .A -65-... A -69-.. .G -37-.. .G -72-.. .G

cons.

....

PSAVMGcPGH DsRDFE

PSAVHGcPGH DNRDFE

Block 5 .. .. .. .. ..

-84-...... DVlI SRQRYVGTPI -84-...... DVI SRORYWGTPI -71-...... DvgV SRORYWGAPI

TGAVNAvPGH DORDYE TGAVkitPAH DQnDY. -116-....DWcI SRQlVVGhrc TGAVhTaPGH gpdDYv -116-.... DVcI SRORtVGvPM

g tgAV.g.PgH d ..Dye

. .....

....

iD.1 SRQryVG.pl

Block 6

sdlrs sclrs eclrs scvrs

Fig. 7. Effect of a depletion of the NAM2C protein on mitochondrial transcripts. The strain HM200 has its chromosomal NAM2C gene deleted and harbours a plasmid (YCpGMC068) where the transcription of the NAM2C gene is under the control of the UAS GALIO (see Materials and methods). The control strain harbours the same plasmid but its chromosomal NAM2C gene is wild type (+), both the strains are rho+ and were grown first on galactose (left lanes), then transferred to glucose and samples were taken after 10 and 19 generations. Total RNA was extracted and comparable amounts were analysed by Northern blotting with various probes. Panel A, 350bp Hinfl fragment internal to the fourth exon of the gene for COXI. Panel B, 2200 bp BamHI fragment of the intron-free cytb gene (Labouesse and Slonimski, 1983). Panel C, 850 bp HindII-MboI fragment of the first intron of the COXI gene. Panel D, 450 bp HindlIl fragment of the COXIJI gene.

encoded by a separate gene as a deletion of the NAM2C gene is not lethal. Finally, the leucyl tRNA synthetase activity was specifically inhibited by antiserum raised against a ,B-galactosidase/NAM2C chimeric protein (Figure 3 panels C and D). When the S.cerevisiae and S.douglasii NAM2 sequences were compared to the E.coli LeuRS, they were found to be highly homologous, with the highest resemblance coefficient found amongst aminoacyl tRNA synthetases (cf. Figure 2). This reinforces previous observations that mitochondrial synthetases closely resemble their homologues in E. coli (Myers and Tzagoloff, 1985; Pape et al., 1985). When the sequences of the different leucyl tRNA synthetases were compared with those of the yeast cytoplasmic VaiRS and the E. coli IleRS an interesting situation was revealed. Several motifs, or regions of homology could be identified. Two of these regions (blocks 1 and 6, Figure 8) have been described before (Webster et al., 1984; Hountondji et al., 1986b), but the others (blocks 2-5) have not previously been detected. Thus these synthetases show a greater level of similarity with each other than is normally found between synthetases charging different amino acids (cf. Figure 2). In view of the fact that they all charge large aliphatic amino acids, it is tempting to speculate that they form a subset or family of synthetases that have evolved from a common

ecirs cons.

PII..-185-....KHSKSKh NGaDPNEcIl RhGAD..-228-COOH PII..-185-....KMSKSKy NGaDPNEcIl RhGPD..-228-COOH PnV..-183-....KMSKSKn IGIDPQvHVe RyGAD..-220-COOH PVy..-122-....KNSKSlg NvIDPlDVIt giklD..-380-COOH

SLf..-124-....KNSKSig ITVsPQDVMn KIGAD..-317-COOH pi ........ S KSKS.. Ng.dPqd.l. r.gaD ..... ...

...

Fig. 8. Common sequence motifs between different aminoacyl tRNA synthetases charging large aliphatic amino acids. Block 1 was discovered by Webster et al. (1984), and block 5 by Hountondji et al. (1986). Blocks 2-5 have not been described previously. The NAM2D sequence of S.douglasii (Sdlrs) was taken from Figure 4, the NAM2C sequence of S.cerevisiae (Sclrs) was taken from Labouesse et al. (1987), leucyl tRNA synthetase of Ecoli (Eclrs) from Hartlein and Madern (in press), valyl tRNA synthetase of S.cerevisiae (Scvrs) from Jordana et al. (1987), and isoleucyl tRNA synthetase of Ecoli (Ecirs) was taken from Webster et al. (1984). The consensus sequence shows residues belonging to the same group (Dayhoff, 1978) when present in four cases out of five (lower case) and when all five are identical (upper case).

ancestor. At a recent Cold Spring Harbor meeting (August 1987) the isolation and sequence of the wild-type gene corresponding to the pet- complementation group G59 was reported (Repetto, Tzagoloff, Akai and Kurkulos, personal communication). This gene was shown to encode the mitochondrial LeuRS. The deduced protein sequence is identical to the previously published NAM2C sequence (Labouesse et al., 1987). We have demonstrated that S.douglasii contains a gene, the NAM2D gene, which is the homologue of, and closely

resembles the S. cerevisiae NAM2C gene (the two genes are also functionally interchangeable in both S.cerevisiae and S. douglasii, Herbert et al., in preparation). We have shown that the wild-type NAM2D gene of S. douglasii acts like the suppressor alleles of the NAM2C gene and in S. cerevisiae is able to suppress mutations that inactivate the b14 maturase (Figure 5). By constructing hybrid genes where the internal BamHI fragments have been exchanged between the NAM2C and NAM2D genes, we have shown that the information sufficient for the suppression of splicing defects is contained within this fragment in NAM2D (Figure 6). This, and previous results (Labouesse et al., 1987) suggest that in the NAM2C gene there are very limited possibilities for single amino acid changes to enhance the suppressor activity; while 479

C.J.Herbert et al.

changing several residues in this region, as in NAM2D, may have an effect equivalent to the one produced by a single amino acid replacement at position 240. At this point, we should address the question of why and how are mitochondrial leucyl tRNA synthetases able to suppress a splicing deficiency caused by an inactive b14 maturases? Other studies have shown that both the NAM2C and the NAM2D suppressor alleles need the ORF of the intron al4 for their suppressor activity (Dujardin et al., 1983 and Herbert et al., in preparation). The al4 ORF is 70% homologous to the b14 ORF, but in a wild-type strain it has no known function and the b14 maturase is responsible for the excision of the intron a14 (Labouesse et al., 1984). However, the mitochondrial mutation mim2 which causes a single amino acid change in the a14 ORF (glu- lys), activates the latent a14 maturase which is then competent for the excision of the introns al4 and b14 (Dujardin et al., 1982). A priori two types of hypothesis can be advanced to explain the suppression of the RNA splicing deficiency. In the first it is due to a translational effect, where the mutated synthetase of S. cerevisiae (or the wild-type synthetase of S. douglasii) activates the latent al4 maturase. A possible formulation of this hypothesis would be that mischarging produces sufficient specific translation errors, mimicking the mim2 mutation, i.e. replacement of glutamic acid residue by a lysine, to render the normally inactive al4 protein active. We have amply discussed this hypothesis previously (Labouesse et al., 1987). If the NAM2 genes encoded tRNA synthetases charging lysine or glutamate this hypothesis could have been retained. We have shown that they encode leucyl aminoacyl tRNA synthetases. Therefore it becomes unlikely, since it is implausible that a leucyl tRNA synthetase would charge a lysine on a glutamic tRNA. In the second hypothesis the NAM2 proteins would have a second role, as well as being LeuRS they would also be involved more directly in mitochondrial mRNA splicing. In this case the suppressor mutations may change the affinity for another part of the splicing apparatus such as an intron RNA or a protein, which may be intron encoded. It should be stressed that the NAM2D protein is also able to recognise and manipulate the S. cerevisiae b14 and a14 intron RNAs, although the mitochondrial genome of S. douglasii does not contain introns identical to b14 and al4 (Kotylak et al., 1985; Claisse et al., 1987 and Lazowska et al., in preparation). However, as the secondary structure of introns is highly conserved and certain motifs within maturases are also conserved (Michel et al., 1982), it is possible that in S. douglasii the NAM2D protein is involved in the splicing of introns other than b14 and a14. Thus the NAM2 proteins may have a more general role in mRNA splicing than has previously been considered. This is consistent with the results obtained with the galactose controlled conditional allele showing that the NAM2C gene is involved in the excision and metabolism of several introns (Figure 7). These data can be interpreted in two ways; either the effects on mRNA splicing seen in Figure 7 are caused by a reduction in protein synthesis which leads to a maturase deficiency or they are the result of a more direct effect on mRNA splicing. Several lines of evidence lead us to support the second hypothesis. If the level of the LeuRS is reduced, protein synthesis will be impaired and consequently the translation of the intron encoded mRNA maturases will be diminished.

480

However there is considerable evidence to show that the amount of maturase required for intron excision is very low. Using antibodies raised against the b14 maturase Jacq et al. (1984) have shown that this maturase can only be detected in mutant strains that overproduce the maturase. Dujardin et al. (1984) were able to show that paromomycin preferentially suppresses nonsense mutations in mRNA maturases and Weiss-Brummer et al. (1984) have demonstrated that frame-shift mutations in the upstream exons of the cob-box gene are sufficiently leaky to allow the processing of the oxi3 transcript, which implies that mRNA maturases are synthesized. All these data support the idea that only a small amount of maturase is needed for splicing. In the experiments with the conditional allele there are several reasons for believing that the glucose repressed cells are capable of sustaining a sufficient level of protein synthesis to produce mRNA maturases. First, protein synthesis is necessary for the maintenance of an intact mitochondrial genome (Myers et al., 1985), the inactivation of the nam2C gene, like the inactivation of other mitochondrial tRNA synthetases causes the formation of 100% rho-petites (Labouesse et al., 1985) and growth in the presence of mitochondrial protein synthesis inhibitors such as chloramphenicol also induces rho-petite formation (Williamson et al., 1971). Thus it is important that the glucose repressed cells do not produce a high level of rho-petites (5% compared to 1 % in the wild type), indicating that protein synthesis is not abolished. Second direct measurement of the leucyl tRNA synthetase activity in the glucose repressed cells shows that they contain 50% of the wild-type enzyme level (Figure 3, panel A), while the TyrRS activity is not affected (Figure 3, panel B). It is highly improbable that a reduction of 50% in the activity of one aminoacyl tRNA synthetase will decrease protein synthesis to a level which will not allow maturase formation. We feel that there is sufficient evidence to postulate that the mitochondrial leucyl tRNA synthetases are involved in both protein synthesis and mRNA splicing. We also conclude that the involvement in mRNA splicing is distinct from the translation of maturases. Since the suppression of splicing defects requires conjoinedly the NAM2 gene product and the product of the a14 ORF, it is possible that it is due to the activation, by an unknown mechanism, of the 'dormant' al4 reading frame, although a translational activation appears

unlikely. Comparisons between the two mitochondrial and the bacterial leucyl tRNA synthetases (Figure 4) reveal a remarkable feature: the first two have a characteristic structure which could form a typical 'Zinc-finger' (Miller et al., 1985 and Berg, 1986). 334\

1

N

346

G

-GC 331

E HI l

350

co484

446

P S

N D

HC 443

COGl

487

On the contrary, the E. coli enzyme cannot form any such structures because the essential Cys and His residues are replaced by amino acids that cannot act as metal ligands (positions from Figure 4 are: Val at 331, Phe or Ala at 346. Tyr at 350, Leu at 443, Gly at 446, Val at 484 and Gly or

Mitochondrial leucyl tRNA synthetase and mRNA splicing

Asn at 487). Alternative finger like structures can be formed by residues around positions 354 and 645 -657. Here again the crucial Cys and His residues are replaced by Ile, Met, Tyr and Phe in the bacterial enzyme. In general the positions of the cysteine residues diverge the most between the mitochondrial and bacterial enzymes. A total of 20 positions are occupied by cysteine residues in Figure 4. However only one of these is common to the mitochondrial and bacterial enzymes, although the overall conservation between the three sequences is 34%. These replacements are even more striking because the regions surrounding them are well conserved between the mitochondrial and bacterial enzymes. It is tempting to speculate that these structures are involved in the mRNA splicing displayed by the mitochondrial enzymes and presumably absent from the bacterial. Although the 'fingerlike' domains are mainly known to interact with DNA or 5S RNA (Miller et al., 1985), intronic RNAs are highly structured (Michel et al., 1982) and contain many double stranded regions which could be recognized by such domains. This hypothesis is amenable to experimental testing by site-directed mutagenesis of the putative finger forming residues. At present we are in the process of isolating different alleles of the NAM2 gene in an attempt to separate the synthetase and splicing functions. It is interesting to note that there is no sequence similarity between the two aminoacyl tRNA synthetases that are involved in mitochondrial RNA splicing (the tyrosyl tRNA synthetase in N. crassa (Akins and Lambowitz, 1987) and the leucyl tRNA synthetase in yeast, this study). Thus we can ask whether they fulfil equivalent functions in Neurospora and yeast; or, whether they act at distinct steps in the splicing process and the role of other tRNA synthetases remains to be discovered. Whatever the answer to the coevolution of two types of molecules, tRNA and intron RNA on the one hand, and the protein that is involved in recognizing them on the other, is an interesting problem.

Materials and methods Construction of plasmids Unless otherwise stated the plasmids used have been described in Labouesse et al. (1985 and 1987). YEpGMC104 is a multicopy plasmid containing the NAM2D gene and was constructed by adding SphI linkers to the 7.2-kb EcoRI -SphI fragment of XSdA, and cloning it in the SphI site of YEpl3. YCpGMC109 was made by cloning the 4. 1-kb SacI -SphI fragment of XSdA which contains the NAM2D gene in the polylinker of M13mp18 and reisolating it as an SphI-EcoRI fragment which was cloned in YCpGMC 102. The plasmid pGMCl 11 was made by adding BglII linkers to a 4.5-kb ApaI fragment containing the NAM2C gene and cloning it in the BamHI site of pUC 19. YCpGMC068 was made by inserting the 1.7-kb HindIII-BamHI fragment of pLGSD5-ATG which contains the URA3 gene, the UAS GAL1O and the promoter/leader region of CYC1 (Guarente et al.,

1981), between the Sall-BamHI sites of pFL39 (an ARS, CEN, TRPI yeast vector based on pUC19 and donated by Dr F.Lacroute). The wild type NAM2C gene was cloned between the KpnI-EcoRI sites of this plasmid to give YCpGMC068. The KpnI site in front of the NAM2C gene had been introduced by site directed mutagenesis using the oligonucleotide 5'GCATTTTTCCTCGGTACCCACACGTC complementary to positions +4 to -22 relative to the AUG. The plasmid pGMC043 used to disrupt the NAM2C gene was made by cloning the internal 2.3 kb PstI fragment of the NAM2C gene in pBR322; the 1-kb BamHI fragment internal to the PstI fragment was then replaced by a 2.8-kb BamHI fragment containing the LEU2 gene.

Strains, phages and media S.douglasii 2B12D was donated by Dr D.Hawthorne, and issued from a cross between S.douglasii 4898-2B (MALI, his4, trp5x, metl3x, ade2, adeS, 7, leulx, 'ys], are4) and S.douglasii 4873-12D (trpSy, metl3y, adel,

leuly), S.douglasii 2B 12D/50 is the rhoo derivative of this strain. Spombe was donated by Dr M.Claisse. The strains of S.cerevisiae used as listed

in Table I. The strain HM199/50 was obtained by transforming the strain CW04 to leucine prototrophy with the 4. 1-kb PstI fragment of pGMC043; leucine prototrophs were glycerol negative, rho- petite, and had an inactive nam2C gene with the LEU2 gene inserted within it. A rhoo derivative was isolated from one transformant using the standard methods. HM200/ 1 was constructed in two steps; first the plasmid YCpGMC068 was introduced into CW04 by transformation to give the strain CW04/YCpGMC068. In the second step the NAM2C gene was disrupted by transformation with the 4. 1-kb PstI fragment of pGMC043 as described above, these transformants were selected on galactose minimal medium without leucine. Such transformants could have been disrupted in the chromosomal or the plasmid NAM2C gene; out of 12 transformants selected on galactose, 4 were disrupted on the chromosome and 8 on the plasmid. HM200/1 is a transformant that was selected on galactose and that has its chromosomal nam2C gene disrupted; the properties of this strain are described in the results section. Ecoli JM101 (A (lac proAB), thi, supE, F' traD36, proAB, lacIQ, lacAM15) and Q358 (hsdR-; hsdM-, supE, 480 ) were used for the propagation of M13 and lambda phages, respectively. For standard cloning experiments E.coli JA221 (recA-, hsdR-, M+, leuB6, trpE5, lacY) was used. M13 vectors mpl8 and mpl9 were obtained from Pharmacia, the lambda vector EMBL4 (Frischauf et al., 1983) was obtained from Drs F.Caron and B.Guiard. Galactose minimal medium GO is galactose 2% and 0.67% yeast nitrogen base, other media and genetic methods used for yeast were described in Dujardin et al. (1980) and for Ecoli in Maniatis et al. (1982).

Preparation of nucleic acids Yeast nuclear DNA was prepared according to Cryer et al. (1975), with minor modifications. Small scale preparations of phage lambda DNA were performed according to Maniatis et al. (1982), and M 13 DNA was isolated using the method of Messing (1983). Total yeast RNA was prepared as described by Carlson and Botstein (1982), except that the breaking buffer was 0.1 M NaCI, 50 mM Tris (pH 7.5), 10 mM EDTA and 5% SDS without diethyloxydiformate.

Transfer of nucleic acids and hybridization DNA, RNA and lambda plaques were transferred to nitrocellulose filters as described in Maniatis et al. (1982). Filters were washed and hybridized according to Thomas (1980). For hybridization under nonstringent conditions, the pre-hybridization and hybridization were carried out at 42°C in the presence of 25% formamide, and the final wash was performed at 55°C. Radioactive probes were prepared by nick-translation in the presence of [a-32P]dATP.

Table I. List of S. cerevisiae strains used Name

Nuclear genotype

Mitochondrial genotype

Reference or origin

W303-lB/50

a

his3-11,15 leu2-3,112 ade2-1 trpl-l ura3-1 canl-100 NAM2C+ as W303-lB/50 as CW04 with plasmid YCpGMC068 as W303-lB/50 except nam2C::LEU2 as HM199/50 with plasmid YCpGMC068 a his3-11,15 leu2-3,112 can' NAM2C2

rhoo

Labouesse et al., 1987

rho+ mit+ rho+ mit+

Labouesse et al., 1987 This study This study This study Labouesse et al., 1985

CW04 CW04/YCpGMC068 HM 199/50 HM200/1 CKG18

rhoo rho+ mit+ box7-V328

The S. cerevisiae strains used in this study and their origin are listed; all the mitochondrial genomes are derived from 777-3A.

481

C.J.Herbert et al. Cloning the S.douglasii NAM2D gene Genomic DNA from S.douglasii 2B12D was partially digested with Sau3A; fragments of 15-20 kb were purified and ligated into BamHI cut lambda EMBL4. After in vitro packaging the libary was screened by plaque hybridization using pGMC003, which contains an internal BamHI fragment of the S.cerevisiae NAM2-1 gene cloned in pBR322 (Labouesse et al., 1985). Hiybridization was carried out under non-stringent conditions and two filters were made from each plate, 15 000 plaques were screened and two positive signals which coincided on the duplicate filters were obtained. The recombinant phages XSdA and XSdB corresponding to these signals were purified and the DNA isolated, restriction analysis showed that they both contained all the NAM2 homologous sequences.

Acknowledgements

DNA sequencing

References

DNA sequencing was performed using the chain termination method of Sanger in conjunction with the M13 phage system of Messing. A SacI-SphI fragment containing the NAM2D gene was cloned in mpl9, the single stranded DNA was linearized and a series of deletions were made using the exonuclease activity of T4 DNA polymerase (Dale et al., 1985). These clones allowed the sequence of the first strand to be determined. The sequence of the second strand was determined using clones constructed by the deletion procedure, and by cloning specific restriction fragments.

Preparation of mitochondrial extracts Mitochondria were extracted from exponentially growing cells and all operations were carried out at 4°C; the cells were broken in a cell disintegrator using glass beads, in a buffer that contained Tris-HCl 50 mM pH 7.5, EDTA 2 mM and sorbitol 0.7 M (TE+sorbitol 0.7 M). The debris was removed by two centrifugations at 2500 r.p.m. for 10 min and the mitochondria pelleted by centrifuging at 15000 r.p.m. for 15 min. The mitochondria were resuspended in the same buffer using a tight fitting dounce homogenizer and layered onto a sucrose step gradient (20, 36, 50% in TE), this was developed at 25 000 r.p.m. for 1 h in a Beckman SW41 rotor. The mitochondria were collected from the interface of the 36 and 50% steps, diluted with 4 vol of TE + 0.5 M sorbitol and pelleted by centrifuging at 15 000 r.p.m. for 15 min. They were then resuspended in Tris-HCI 10 mM pH 7.5, mercaptoethanol 1 mM and PMSF 1 mM at a protein concentration of 0.5-2.0 mg/ml, the suspension was lysed with 0.3% Triton. The lysate was centrifuged at 100 000 g for 1 hand the supernatant was dialysed overnight against Tris-HCI 10 mM pH 7.5 before use.

Aminoacyl tRNA synthetase assay

Aminoacyl tRNA synthetase assays were carried out at 30°C in a total volume of 200 A1 and contained 100 mM Tris-HCI, pH 8.3, 10 mM ATP, 25 mM MgCl2, 1 mM CTP, 10 ug/ml BSA, 80ytg/ml glutathione, 500 ytg Ecoli tRNA, 5 /ACi [3H] amino acid (leucine 160 Ci/mmol, tyrosine 48 Ci/mmol) and 25 tig of mitochondrial protein. E. coli tRNA was used as it was readily available and Schneller et al. (1976) have shown that the E.coli tRNA-leu is efficiently charged by the mitochondrial enzyme. The reactions were started by the addition of the enzyme, samples were taken at different times and precipitated in 5% TCA. The precipitates were collected on GFC filters, washed and dried, the filters were then counted to monitor the progress of the reaction.

Production of an antiserum raised against part of the NAM2C protein In order to isolate a part of the NAM2C protein to raise an antiserum, a fusion protein was constructed between the 5' section of LacZ and 3' section of the nam2C gene, using the plasmid pJB13.01 (pJB13.01 was constructed and donated by Dr J.Banroques). This plasmid allows the construction of fusions with the 5' section of LacZ (280 amino acids), which are under the control of a thermosensitive phage lambda repressor. The BamHI-EcoRI fragment of pGMCl 11 which contains the 3' portion of the nam2C gene (see Figure 1) was isolated and ligated into the BamHI site of pJBI3.01 after the addition of 12 bp BamHI linkers to create the correct reading frame. Plasmids containing the insert in the correct orientation were identified by restriction digestion. Induction of the gene fusion gave rise to a chimeric protein of the expected size (containing 363 amino acids of the C-terminal of the NAM2C protein). The fusion protein was purified from polyacrylamide gels and used to immunize rabbits.

Miscellaneous Restriction enzyme digestions, agarose gel electrophoresis, fragment purification, ligation, transformation and in vitro packaging were all performed as described in Maniatis et al. (1982). In vitro packaging extracts were provided by Drs F.Caron and B.Guiard. S. cerevisiae was transformed by the method of Ito et al. (1980).

482

This paper is dedicated to the memory of Marika Somlo. We would like to thank Drs Hartlein and Madern for communicating the sequence of the E.coli leucyl tRNA synthetase prior to publication, Drs D.C.Hawthorne

and M.Claisse for gifts of strains, and Drs L.Guarente, F.Lacroute and J.Banroques for their gifts of plasmids. We would also like to thank J.Renowicki and M.O.Mosse for their help with the computer analysis. This work was supported by grants from the INSERM, CNRS, ATP Biologie contre le Cancer and Moleculaire du Gene, la Ligue Nationale la Fondation pour la Recherche Medicale. C.J.H. was the recipient of a NATO Research Fellowship.

Francaise

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Received on October 28, 1987; revised on November 30, 1987

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