Translation elongation factor 2 is encoded by a single essential gene in Candida albicans

Gene 229 (1999) 183–191

Translation elongation factor 2 is encoded by a single essential gene in Candida albicans Alfonso Mendoza, Marı´a J. Serramı´a, Laura Capa, Jose´ F. Garcı´a-Bustos * Research Department, Glaxo Wellcome, S.A., Severo Ochoa 2, E-28760 Tres Cantos, Spain Received 5 August 1998; received in revised form 28 November 1998; accepted 21 December 1998; Received by B. Dujon

Abstract Translation elongation factor 2 (eEF2) is a large protein of more than 800 amino acids which establishes complex interactions with the ribosome in order to catalyze the conformational changes needed for translation elongation. Unlike other yeasts, the pathogenic fungus Candida albicans was found to have a single gene encoding this factor per haploid genome, located on chromosome 2. Expression of this locus is essential for vegetative growth, as evidenced by placing it under the control of a repressible promoter. This C. albicans gene, named EFT2, was cloned and sequenced (EMBL accession number Y09664). Genomic and cDNA sequence analysis identified common transcription initiation and termination signals and an 842 amino acid open reading frame (ORF ), which is interrupted by a single intron. Despite some genetic differences, CaEFT2 was capable of complementing a Saccharomyces cerevisiae Deft1 Deft2 null mutant, which lacks endogenous eEF2, indicating that CaEFT2 can be expressed from its own promoter and its intron can be correctly spliced in S. cerevisiae. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Fungi; Promoter; Protein synthesis; Terminator; Yeast

1. Introduction The protein synthesis machinery in living cells is made up of molecules thought to have evolved at the origin of cellular life, being highly conserved in all extant life forms. Translation elongation factor 2 (eEF2) is part of this core machinery and its sequence has been analyzed to test phylogenetic hypotheses about the deepest branches of the ‘‘tree of life’’ (Cammarano et al., 1992). eEF2 and its eubacterial homolog, EF-G, act sequentially after eEF1a/EF-Tu to catalyze translocation of the ribosome during protein synthesis, to allow the peptidyl-tRNA to move from the A site to the P site, liberating the former site to accept a new mRNA triplet and its cognate ternary complex (eEF1-aminoacyltRNA-GTP). Translocation is greatly accelerated by * Corresponding author. Tel.: +34-91-8070606; fax: +34-91-8070550; e-mail: [email protected]. Abbreviations: CaEFT2, Candida albicans EFT2 gene; CAI, codon adaptation index; 5-FOA, 5-fluororotic acid; ORF, open reading frame; PFG, pulsed field gel electrophoresis; rRNA, ribosomal RNA; snc, supernumerary chromosome.

GTP hydrolysis. The GTP binding site is on all the soluble elongation factors, but its hydrolysis requires interaction with the ‘‘GTPase center’’ of the ribosome (Nygard and Nilsson, 1989). EF-Tu and EF-G (and presumably also eEF1a and eEF2) have overlapping binding sites on the ribosome (Moazed et al., 1988), possibly thanks to the fact that the shape of the larger EF-G resembles that of the smaller EF-Tu plus a tRNA molecule, a case of a protein structure mimicking RNA structure (Nyborg et al., 1996). The molecular details of how eEF2/EF-G helps the ribosome shift between different conformational states are unknown at present. Protein synthesis has been a very useful target in the search for antibacterial compounds, and it can be equally useful in the search for novel antifungals. In fact, eEF2 has recently been shown to be a target for a new family of antifungal compounds (Capa et al., 1998; Dominguez and Martin, 1998; Justice et al., 1998). As part of a program to investigate the protein-synthesis mechanisms of pathogenic fungi, we have cloned, sequenced and analyzed CaEFT2, the single gene encoding eEF2 in Candida albicans. The other known fungal eEF2 proteins, such as those from Saccharomyces cerevisiae and Schizosaccharomyces pombe, are encoded by two genes

0378-1119/99/$ – see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S0 3 7 8 -1 1 1 9 ( 9 9 ) 0 0 02 4 - 4

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bearing different promoters (Perentesis et al., 1992; Mita et al., 1997). C. albicans can apparently live normally with just one type of promoter for this gene, although being a diploid organism it has two copies per cell. Its chromosomal location, splicing sites and structure of the promoter and terminator regions are presented, and the differences with homologous genes from other yeasts are discussed. It is also shown that CaEFT2 is an essential gene.

2. Materials and methods 2.1. Strains and growth conditions C. albicans 1006, 1001, SC5314 and WO-1 strains were used to isolate genomic DNA. C. albicans RM1000 (Dura3/Dura3 Dhis1/Dhis1) (Negredo et al., 1997) was used as host for recombinant DNA. C. albicans AM3 (EFT2/pPCK1::EFT2::HIS1) and C. albicans AM4 ( pPCK1::EFT2::HIS1/Deft2::URA3) are RM1000 derivatives obtained in this work. S. cerevisiae strain MMM11-b (MATa ura3-52 leu2-3,112 his3-D200 trp1-D901 lys2-81 suc2-D9 Deft1::HIS3 Deft2::HIS3) was used for complementation studies. This strain was derived from SEY6210 by substituting HIS3 for a 380 bp internal fragment of EFT1 and EFT2. The double disruptant is inviable and the strain was kept alive by transformation with ScEFT2 cloned in YEp352 (2 mm URA3 ampr). Escherichia coli strain DH5a [F−, endA1, hsdR17, supE44, thi-1, recA1, gyrA96, relA1, DlacU169(W80lacZDM15)] was used for transformation and preparation of plasmid DNA. Yeast strains were grown routinely in YPD-rich medium or SD minimal medium with the necessary additions for auxotrophic strains. B medium (SD medium with 2% succinate as carbon source) was used to derepress the PCK1 promoter and was made according to (Stoldt et al., 1997). Ura− revertants were selected on SD plates containing 1 mg/ml of 5-fluoroorotic acid. E. coli was grown in LB medium supplemented with 100 mg/ml of ampicillin for plasmid selection. 2.2. Genetic analysis To test complementation in S. cerevisiae, strain MMM11-b was transformed with the CaEFT2 ORF under the control of its own promoter, cloned as a 4.4 kb EcoRI–SphI fragment in the multicopy vector YEplac112 (2 mm TRP1 ampr). To investigate whether CaEFT2 is essential for viability of C. albicans, one copy of the CaEFT2 locus was replaced by a construct carrying the CaEFT2 ORF under control of the PCK1 promoter, plus a HIS1 marker for selection of transformants. The CaPCK1 promoter was cloned in pCA01, a pUC21 derivative

(Leuker et al., 1997) (provided by J.F. Ernst). A 3.5 kb fragment containing the ORF and terminator regions of CaEFT2 was amplified by PCR and cloned into pCA01 downstream of the CaPCK1 promoter, generating plasmid pAM1. A 420 bp fragment corresponding to the −861 to −441 region of CaEFT2 (relative to the ATG codon) was added 5∞ of the PCK1 promoter, and a HIS1 marker was inserted at position +3177, leaving 350 bp at the 3∞ end for homologous recombination with the +3178 to +3528 region of the CaEFT2 locus. This whole 6.8 kb construct was linearized and transformed into C. albicans RM1000, selecting for histidine prototrophy in SD medium. His+ colonies were screened for the correct integration by PCR (see below). The second chromosomal copy was disrupted by the ‘‘URA blaster’’ technique (Fonzi and Irwin, 1993). Two kilobases of the CaEFT2 ORF, encoding amino acids 100–775 of the factor, were replaced by a 4 kb SmaI fragment containing the CaURA3 gene flanked by hisG repeats. The deletion construct was transformed into a His+ strain carrying a pPCK1::EFT2 allele obtained as described above. His+, Ura+ prototrophs were selected in B-medium (succinate as carbon source) to derepress the PCK1 promoter and allow transcription of EFT2. Several identical isolates were obtained in which one EFT2 allele was linked to a repressible promoter and the second allele was disrupted by URA3. All correct integrations of DNA were confirmed by PCR (Fig. 1). Genomic DNA from wild-type and transformant strains was amplified using primers hybridizing outside and inside the region targeted for disruption. The resulting structures at the CaEFT2 loci and the expected PCR fragments are shown in Fig. 1. The wildtype loci are expected to generate a 1.5 kb PCR fragment ( Fig. 1B, lane 3). Insertion of the PCK1 promoter increases the size to 2.5 kb for one of them (Fig. 1B, lane 2). When the second allele is disrupted with URA3, the wild-type fragment present in the heterozygous strain disappears ( Fig. 1B, lane 1). To show the presence of the URA3 marker in the double transformant, a new amplification was done using a primer which hybridizes with the marker and the same outside primer used before. A new specific fragment of 2.8 kb is amplified only when genomic DNA from the double transformant is used as template (Fig. 1C, lane 1). DNA probes for library screening were prepared using an Amersham Multiprime DNA labeling Kit using [a-32P)dCTP (Amersham). Probes for Southern detection and chromosomal localization were labeled using a non-radioactive ECL Labeling and Detection System (Amersham). The C. albicans ordered array library contains genomic DNA from C. albicans ATCC 10261 in the YCp50 vector (URA3 CEN4 ampr tetr) and has 6000 clones with inserts of 9–12 kb on average. Duplicate filters containing the library were hybridized overnight at 42°C in 5×SSC, 5×Denhard’s solution,

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common. The C. albicans chromosome blot was a gift from F. Navarro-Garcia. Both strands of a 4.4 kb EcoRI–SphI fragment containing the EFT2 ORF, plus 861 bp of promoter region and 802 bp 3∞ of the coding region, were partially sequenced by subcloning. The full 4.4 kb fragment was sequenced by J. Arin˜o at the Autonomous University of Barcelona, using an Applied Biosystems 373 automatic DNA sequencer. All nucleotide positions were determined at least three times in total and at least once on each strand. C. albicans total RNA was isolated with an acid phenol/guanidinium hydrochloride solution, ULTRASPEC-II RNA Isolation System (Biotecx), and cDNA synthesis from 1 mg of total RNA was carried out with the CapFinder cDNA Library Construction Kit (Clontech). Oligonucleotide primers were obtained from MedProbe. A standard Perkin-Elmer GeneAmp PCR Reagent Kit was used to generate DNA fragments by amplification. 2.3. Sequence analysis DNA and protein sequence analysis was carried out using the EGCG extensions to the Wisconsin Package (Genetics Computer Group) (Rice, 1996) and data present in the EMBL, TFSITES, PROSITE and the ENTREZ system of databases (National Center for Biotechnology Information, Bethesda, MD)

3. Results Fig. 1. Genetic structures resulting after each disruption event at the CaEFT2 locus. (A) Schematic representation of the different genetic elements drawn to scale. DNA sequences used to transform C. albicans are represented as solid lines, flanking chromosomal sequences as dashed lines. The 3∞ annealing positions of PCR primers used to check the integrations are represented as half-arrows; the length of the primers is not drawn to scale. (B) PCR amplification using primers 1 and 2 of genomic DNA from C. albicans strains AM4 (pPCK1:: EFT2::HIS3/Deft2::URA3) ( lane 1); AM3 (pPCK1::EFT2:: HIS3/EFT2) ( lane 2) and RM1000 (EFT2 / EFT2) ( lane 3). (C ) PCR amplification using primers 1 and 3 of genomic DNA from C. albicans strains AM4 ( pPCK1::EFT2::HIS3/Deft2::URA3) ( lane 1); AM3 (pPCK1::EFT2::HIS3 /EFT2) ( lane 2) and RM1000 (EFT2/EFT2) ( lane 3). Numbers to the right of (B) and (C ) indicate the migration of size markers (in bp).

0.3% SDS, 100 mg/ml denatured herring sperm DNA and 20% of N,N-dimethyl formamide. Excess probe was removed by washing twice in 1×SSC, 0.1% SDS for 15 min and twice with 0.1×SSC, 0.1% SDS, for 15 min at room temperature. Four positive clones were selected and re-hybridized in the same conditions with amino and carboxy-terminal probes from ScEFT2. Restriction analysis of the three clones that hybridized with both ends of the S. cerevisiae ORF had a 3 kb segment in

3.1. Cloning and sequence analysis of CaEFT2 An arrayed genomic library of C. albicans in YCp50 was probed with a 518 bp DNA fragment corresponding to amino acids 274–445 of S. cerevisiae eEF2. Four positive clones were detected and all had a segment of approx. 3 kb in common by restriction mapping. Subclones were made and their ends were sequenced. Sequences obtained were found to be highly homologous to the ORF in S .cerevisiae EFT1 and EFT2 genes. A 4.4 kb EcoRI–SphI fragment was completely sequenced on both strands by primer walking ( EMBL accession number Y09664) and found to contain a reading frame of 842 amino acids. At the DNA level, the coding region is 85% identical to S. cerevisiae, while at the protein level 89% and 93% of the residues are found to be identical and homologous, respectively, to those in S. cerevisiae eEF2 by the BestFit program. We have accordingly named the gene EFT2, for elongation factor 2. A search for transcription factor binding motifs in the promoter region indicated the presence of putative CCAAT boxes in the −453 to −417 region, relative to the initial ATG codon. These are common enhancer

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binding sites in eukaryotes and are used in S. cerevisiae to activate transcription in the presence of non-fermentable carbon sources (see McNabb et al., 1995 and references therein). Their function, if any, in the predominantly respiratory C. albicans remains to be investigated. Putative TATA boxes are located at positions −124 and −99. Transcription starts within a TC(G/A)A site (see below), located 67 bp downstream of the last A in the proximal TATA. This topology is also seen in numerous S. cerevisiae genes. There is a 63 bp-long polypyrimidine tract directly upstream from the transcription initiation site. This sequence is absent from the S. cerevisiae genes encoding eEF2, but shorter versions are common in the promoters of highly expressed genes in C. albicans and other fungi [e.g., those encoding C. albicans actin (Losberger and Ernst, 1989) and eEF1a from Aureobasidium pullulans ( Thornewell et al., 1995) or Mucor racemosus (Sundstrom et al., 1990)]. The region 3∞ of the stop codon (position 2726) contains sequences proposed to represent consensus sites for transcription termination and polyadenylation. There are yeast terminator sequences fitting the consensus TAG..TAGT..(AT rich)..TTT from position 2754 to 2772, and two sequences resembling the TTTTTATA motif at 2790 and 2812 (inverted). Polyadenylation signals typical of higher eukaryotes are also present, including the AATAAA motif (at 2818) and the T/Grich region (2840–2860). 3.2. eEF2 is encoded by a single gene located on chromosome 2 of C. albicans There are two genes in S. cerevisiae encoding identical versions of the eEF2 protein under control of different promoters and both are expressed, albeit at different rates ( Veldman et al., 1994). Genetic relatedness between S. cerevisiae and C. albicans made it probable that C. albicans would also have two genes for this elongation factor. Hybridization experiments (Southern blot) with total genomic DNA from C. albicans strain 1006 suggested, however, that there is only one kind of eEF2 gene in this fungus. Genomic DNA was digested with 7 restriction enzymes, transferred to a nylon membrane and probed with the 1.3 kb HindIII fragment of CaEFT2, which represents the 3∞ region of the ORF. One hybridizing band was obtained with enzymes which did not cut inside the probe and two with those which did, although an additional unexplained faint band is also visible in the EcoRI digest ( Fig. 2). We interpreted these results to mean that there was probably a single eEF2-encoding gene per haploid genome in C. albicans. This interpretation was also supported by the finding that expression of the identified CaEFT2 locus is essential for cell viability (see below). A chromosomal blot of C. albicans strains 1001,

Fig. 2. Copy number of CaEFT2 determined by Southern blot. C. albicans chromosomal DNA was digested with the restriction enzymes shown at the top. Fragments were separated by gel electrophoresis and transferred to a membrane, which was probed with the HindIII fragment of EFT2 indicated in the scheme below the photograph. There are no restriction sites inside the probe for BamHI, EcoRI, HindIII or SalI. Electrophoretic migration of size markers in this experiment is indicated to the right of the blot (in kb). A, AflIII; H, HindIII; P, PinA1; S, SphI.

SC5314 and WO-1 was probed with the same HindIII fragment of CaEFT2 mentioned above. As shown in Fig. 3, the probe hybridized to chromosome 2 from all three strains, plus chromosome 2* of strain 1001, indicating that CaEFT2 is located on the 2U fragment of the SfiI map of C. albicans (Navarro-Garcia et al., 1995). 3.3. C. albicans EFT2 has an intron Conceptual translation of CaEFT2 showed a reading frame which, although highly homologous throughout S. cerevisiae eEF2, had no initial methionine. It was therefore likely that CaEFT2 had an intron which, when processed, would restore a Met residue to the first position of the protein. A computer search for consensus splice sites in C. albicans genes, theoretically or experimentally determined, revealed that they all had 5∞ and

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of the PCR product and comparison with the genomic sequence revealed that the predicted splicing event had indeed taken place, established the sequences of the 5∞ and 3∞ intron junctions and located the main transcription start site at position −25 relative to the first codon ( Fig. 4). 3.4. Analysis of the protein sequence encoded by CaEFT2

Fig. 3. Chromosomal location of CaEFT2. PFGE separation of chromosomes from three different C. albicans strains ( left panel ) and hybridization of a DNA probe from EFT2 (see Sections 2.2 and 3.2) with a chromosomal blot from the same gel (right panel ). Strain designations are written on top. Chromosome numbering follows Chu et al. (1993) and Navarro-Garcia et al. (1995).

3∞ splice sequences like those described in S. cerevisiae, including the presence of the universally conserved branch point sequence TACTAAC within 55 bp of the 3∞ splice site ( Table 1) (Langford et al., 1984). All these sequence elements were present in CaEFT2. The splice sequences defined a putative 200 bp intron separating a single methionine from what would be the second amino acid by comparison with S. cerevisiae eEF2. To test for the splicing event, cDNA was prepared from C. albicans and the 5∞ end of EFT2 was amplified using the capfinder commercial oligonucleotide (Clontech) plus a reverse primer specific for the CaEFT2 ORF. Sequencing

The gene product of CaEFT2 is highly homologous to all eukaryotic eEF2 proteins, homology being maximal with the other fungal factors in the databases (from S. cerevisiae and Sch. pombe, 93% and 88% homology, respectively) and minimal when compared with that from the microsporidian protozoan Glugea plecoglossi, although still at the 56% homology level as calculated by the BestFit program. The encoded protein has the conserved residues involved in GTP binding present in domains G1–G4 of G-proteins (Bourne et al., 1991). A search in the PROSITE database revealed the nucleotide-binding (‘‘Ploop’’, position 26) and the ‘‘GTP-binding elongation factor’’ (position 58) motifs. Interestingly Thr57, a phosphorylation site in mammalian eEF2 (Redpath et al., 1993) is missing in C. albicans eEF2, which has a Met residue at that position. S. cerevisiae eEF2 has been proposed to be phosphorylated and hence regulated in the same manner as the mammalian factor (Donovan and Bodley, 1991), but obviously this cannot be the case in C. albicans. At the carboxyl terminal half of the protein, absolute sequence conservation is observed in all eEF2 proteins at the His residue that is modified to diphthamide ( H699) and flanking positions. Positions 797–830 fit,

Table 1 Intron sequences in C. albicans translated genes Protein

5∞ splice sequence

n1

Branch sequence

n2

3∞ splice sequence

Acc. numbera

Actin aTubulin bTubulin intron A bTubulin intron B Ribosomal protein L41 Ribosomal protein L39 Elongation factor 1 b Cytosine deaminase Calmodulin Meiosis specific homolog intron A Meiosis specific homolog intron B IMP dehydrogenase Peptide transporter ras-related protein Consensus Elongation factor 2

GTATGT GTATGT GTATGT GTATGT GTATGT GTATGT GTAAGT GTATGT GTATGT GTATGT GTATGT GTATGT GTATGT GTATGT GTATGT GTATGT

628 391 180 132 333 391 331 43 193 54 38 223 43 51

TACTAAC TACTAAC TACTAAC TACTAAC TACTAAC TACTAAC TA . TAAC TACTAAC TACTAAC TACTAAC TATTAAC TACTAAC TACTAAC TACTAAC TACTAAC TACTAAC

9 18 10 16 18 18 19 12 13 11 5 15 12 13

TAG TAG TAG TAG TAG TAG TAG CAG AAG TAG CAG TAG TAG CAG ( T/C )AG TAG

X16377 U38534 M19398 M19398 D67040 U37010 X96517 U55194 M61128 U39808 U39808 U85049 U09781 U46158

a Acc. number, Accession number in EMBL/GenBank/DDBJ.

171

13

Y09664

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Fig. 4. Location of the transcription start site and the intron sequence in CaEFT2. DNA sequence around the amino terminus of the EFT2 ORF at the chromosomal locus and at the mRNA (as cDNA) is shown. A sequence corresponding to a typical transcription start site in S. cerevisiae is boxed. The first four residues of the conceptual translation of the eEF2 protein are also indicated.

with one mismatch, the PROSITE consensus for the helix–turn–helix motif of homeo-box proteins. This region could therefore be one that interacts with nucleic acids, rRNA in this case. Ribosomal protein L11 has recently been shown to bind RNA using a homeo-boxlike motif ( Xing et al., 1997). 3.5. Expression of the CaEFT2 gene is essential for cell viability If CaEFT2 encodes the only eEF2 function in C. albicans, its expression ought to be essential for cell viability. Since this yeast seems to be a permanent diploid, positive proof of essentiality can be obtained only by manipulating both homologous copies of the locus under study. One copy was simply disrupted with URA3 ( Fig. 1) (see Materials and Methods) and the other was placed under the control of the PCK1 promoter, which is repressed by glucose and derepressed by gluconeogenic carbon sources, such as succinate. Growth on sucrose allows intermediate levels of expression (Leuker et al., 1997). The ability of wild-type and transformed C. albicans strains to grow on different carbon sources was tested on solid media ( Fig. 5). Strains carrying at least one intact allele of EFT2 were able to grow in all three carbon sources tested. When eEF2 expression was controlled by the PCK1 promoter, only a medium containing succinate as carbon source supported cell growth, showing that CaEFT2 is an essential gene and also indicating that it needs to be expressed at relatively high levels, as partial derepression on sucrose is not enough to support the growth. PCK1 promoter activity in C. albicans growing on sucrose is reported to be 10% of the activity measured when growing on succinate (Leuker et al., 1997). This also confirms that there are no other genomic loci expressing eEF2 in C. albicans. 3.5.1. CaEFT2 can provide eEF2 function in S. cerevisiae C. albicans and S. cerevisiae seem to use the same codons for highly expressed proteins. Using S. cerevisiae codon adaptation index tables, CaEFT2 appears to be a very highly expressed gene (CAI=0.795), with a bias towards AT at the third codon position as expected

Fig. 5. Effect of repressing CaEFT2 expression on cell growth. C. albicans strains with the genotype indicated on the scheme were streaked on plates with the carbon source indicated and incubated at 30°C during 3 days.

from an organism with a 35% G+C content. There are none of the CTG codons which in C. albicans noncanonically encode Ser rather than Leu. Thus, CaETF2 should be efficiently translated in S.cerevisiae. To ascertain whether CaEFT2 does indeed encode a functional homolog of eEF2, we tested its ability to supply eEF2 function, from its own promoter, to an S. cerevisiae strain in which both genes encoding eEF2 had been disrupted (see Section 2.1). Because at least one of the two genes encoding eEF2 in S. cerevisiae is required for viability, haploid strains with deletions in either EFT1 or EFT2 were constructed and mated to each other. The resulting diploid was transformed with

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ScEFT2 cloned in the YEp352 vector (URA3). Haploid segregants with the double disruption were obtained and found to be incapable of growing in the presence of 5-FOA, indicating that their growth depended on the ScEFT2 gene carried by the URA3 plasmid. One of the segregants was used in a plasmid shuffle experiment to substitute CaEFT2 for its S. cerevisiae homolog. The Deft1 Deft2 double disruptant was transformed with the C. albicans gene cloned in YEplac112 (TRP1) and transformants with restored growth on 5-FOA plates were selected, to recover cells which had lost the URA3 plasmid. Viable Ura− colonies were obtained. PCR amplifications on two of the colonies showed that the EFT1 and EFT2 loci were still disrupted, indicating that CaEFT2 is expressed in S. cerevisiae to provide eEF2 function despite differences in promoter sequence and the intron.

4. Discussion We report here the structure of C. albicans EFT2 gene, which encodes translation elongation factor 2 (eEF2) in this pathogenic yeast and represents the third published fungal eEF2 sequence. The encoded 842 amino acid protein is most closely related to the ORF in S. cerevisiae EFT1 and EFT2 genes (88% sequence identity overall ) and it can supply eEF2 function from its own promoter in the latter yeast. The similarity to Sch. pombe eEF2 is somewhat lower, 79% of amino acid positions being identical in this case. Unlike the situation in S. cerevisiae and Sch. pombe, eEF2 seems to be encoded by a single gene per haploid genome in C. albicans. The physiological consequences of this difference are unclear. The genes for elongation factors 1a and 2 in S. cerevisiae seem to lie in duplicated chromosomal fragments, explaining the origin of two genes for each factor in baker’s yeast ( Wolfe and Shields, 1997). Whether this also applies to Sch. pombe is unknown at the moment. Gene duplication in S. cerevisiae has been proposed to be instrumental in its adaptation to fermentative metabolism and C. albicans lacks that specialization, but so does Sch. pombe. It is also unlikely that C. albicans will have less need to regulate eEF2 expression than the other two yeasts, which could theoretically fine-tune eEF2 transcription having two different promoters available. A search for transcription factor binding sites on the published yeast EFT2 promoters did not offer obvious clues to explain the differences in gene number either. The implications of the number of elongation factor genes for the regulation of translation needs to be investigated, but the differences between C. albicans on one hand, and S. cerevisiae and Sch. pombe on the other, throw a note of caution on the use of gene duplications to estimate the relative timing of phyloge-

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netic divergence, and of the postulated genome duplication in S. cerevisiae ( Wolfe and Shields, 1997). CaEFT2 promoter structure is typically fungal, with TATA boxes located at a distance from the transcription initiation site that more or less fall within the ‘‘initiation window’’ of yeast promoters. Nothing is known about the existence of CCAAG-box binding factors in C. albicans, capable of binding to the CCAAG elements located upstream of the TATA boxes in CaEFT2. C. albicans may have homologs of the HAP gene products of S. cerevisiae, and if so it would be interesting to know their function. The polypyrimidine tract is absent or much shorter in ScEFT1, ScEFT2 and the Sch. pombe homologs, but it is present in highly expressed genes from many fungi. It does not span the transcription initiation site, so it is unlikely to represent the 5∞TOP element present in higher eukaryotic genes encoding components of the translation machinery. These elements confer mitogen regulation and make translation of the downstream ORF sensitive to the drug rapamycin ( Terada et al., 1994; Jefferies et al., 1997). It should be noted, however, that rapamycin also has a selective inhibitory effect on yeast translation (Barbet et al., 1996). When comparing different eEF2 protein sequences, it is worth noting the lack of Thr57 in C. albicans eEF2. Of the 17 eukaryotic eEF2 proteins found in a database search, only five lack that residue (those from C. albicans, Sch. pombe, Dictyostelium discoideum, Glugea plecoglossi and Trypanosoma cruzi). In all cases, a Met residue has been substituted for Thr, a remarkable parallelism likely to have some functional significance. Thr57 is a calmodulin-dependent protein kinase III (eEF2 kinase) phosphorylation site, which in higher eukaryotes is involved in translation regulation in response to growth factors (Nairn et al., 1987; Palfrey et al., 1987), a signaling pathway which is a target for cyclosporine and rapamycin (Gschwendt et al., 1988; Redpath et al., 1996). There is some evidence suggesting that in S. cerevisiae eEF2 may be regulated as in mammalian cells (Donovan and Bodley, 1991). Complementation by C. albicans eEF2, which lacks the critical Thr residue, shows that such regulation is not essential for vegetative growth, without excluding a more subtle role. CaEFT2 is an essential gene, additionally supporting the above conclusion that it is also unique in the C. albicans genome. We were able to obtain positive evidence of its being essential thanks to the recent availability of repressible C. albicans promoters (Brown et al., 1996; Srikantha et al., 1996; Leuker et al., 1997). As expected from the fact that eEF2 needs to be roughly equimolar with ribosomes, CaEFT2 seems to require expression at above a certain level to be able to sustain growth. Only when the PCK1 promoter is fully derepressed by growth on succinate can the pPCK1::EFT2

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allele supply enough eEF2 function to allow colony formation on agar plates (Fig. 5). eEF2 is an ancient and fascinating protein which seems to have evolved to mimic some RNA structures (Nissen et al., 1995). Studies in progress at different laboratories, including our own, will soon shed light on the three-dimensional structure of this protein and on the complex events which bring about ribosomal translocation.

Acknowledgements We are indebted to Maria-Stella de Tiani for making us a replica of the C. albicans arrayed genomic library, to Marı´a Go´mez for disrupting the ScEFT genes and to Federico Navarro-Garcia and Jesu´s Pla for generously providing the chromosomal blot and strain RM1000. We also thank J.P. Garcia Ballesta for critical reading of the manuscript.

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