Two different genes from Schwanniomyces occidentalis determine ribosomal resistance to cycloheximide

Eur. J. Biochem. 213, 849-857 (1993) 0 FEBS 1993

I b o different genes from Schwanniomyces occidentalis determine ribosomal resistance to cycloheximide Lourdes Del POZO, Dolores ABARCA, Janet HOENICKA and Antonio JIMENEZ Centro de Biologia Molecular (U.A.M./C.S.I.C.), Universidad Aut6noma de Madrid, Spain (Received November 24, 1992) - EJB 92 1686/1

Two genes (SCRI and SCR2) encoding natural cycloheximide resistance in the budding yeast Schwanniomyces occidentalis have been cloned by expression in Saccharomyces cerevisiae. Both genes determine resistance to the inhibitory action of cycloheximide on the ribosome. SCRl and SCR2 are present as single copies in Schwanniomyces occidentalis, where they map on chromosomes I1 and V, respectively. The nucleotide sequence of SCR2 contains an open reading frame of 321 nucleotides which is interrupted by an intron of 452 nucleotides. It encodes a polypeptide of 106 amino acids of molecular mass 12.25 kDa and PI 11.19. The deduced amino acid sequence shows a high degree of similarity to the LA1 protein of the 60s ribosomal subunit from several eukaryotic organisms. The intron and the 5' non-coding region of SCR2 possess conserved elements which are typical of yeast ribosomal protein genes. A single amino acid change determines the resistance or sensitive phenotype to cycloheximide of the 80s ribosome since replacement of Gln56 in LA1 from Schwanniomyces with Pro, by site-directed mutagenesis, confers cycloheximide sensitivity. SCR2 may serve as a practical yeast cloning marker if integrated in a multicopy plasmid.

Schwanniomyces occidentalis [l] is a budding yeast which has attracted the attention of researchers because it secrets a variety of high-molecular-mass proteins. These include a-amylase and a glucoamylase which also shares a starch debranching activity. Therefore it solubilizes starch by complete hydrolysis to glucose [2]. Because of the potential industrial importance of this yeast, it is desirable to characterize in detail its genetic constitution and its spectrum of sensitivity or resistance to antibiotics. Indeed Sw. occidentalis is highly resistant in vivo to inhibition by the glutarimide antibiotic cycloheximide (cyh) [3], which blocks polypeptide chain elongation by eukaryotic ribosomes (for reviews, see [4, 51). This resistance is also shared by certain other yeast species including Kluyveromyces lactis and K. fragilis [6, 71, Candida maltosa and C. tropicalis [8, 91. In Saccharomyces cerevisiae, mutations at five different loci induce resistance to cyh [lo, 111. Of these, only cyh2 has been subjected to detailed biochemical and genetic studies. The presence of this gene confers resistance to cyh levels of 50-70 pg/ml. The gene encodes an altered ribosomal protein L29 which is presumed to reduce the affinity of the 60s ribosomal subunit for cyh [12, 131. The cyh2 gene has been Correspondence to A. Jimhez, Centro de Biologia Molecular, Universidad Autbnoma, Canto Blanco, E-28049 Madrid, Spain Abbreviation. CHEF, contour-clamped homogeneous electric field ; cyh, cycloheximide ; CyhR, cyh-resistance/resistant; CyhS, cyh-sensitivity/sensitive;NLS, nuclear localization sequence; YNB, yeast nitrogen base without amino acids; MIC, minimal inhibitory concentration; UAS, upstream activator site; RPG, ribosomal protein gene. Note. The novel nucleotide sequence data published here have been deposited with the EMBL sequence data bank and are available under the accession number X70807. The novel amino acid sequence data have also been deposited with the EMBL sequence data bank.

cloned, sequenced [ 141 and subsequently used as a dominant marker, at low cyh concentrations, for yeast cloning vectors [lo]. Of the other loci the cyh3 gene expresses, by means of a permeability barrier, a pleiotropic phenotype of multiple resistance to a large variety of antibiotics [15]. The RIM-C gene from C. maltosa also determines cyh resistance [8, 91; this gene has now been sequenced and shown to encode ribosomal protein L41 [16]. More recently [17], a further gene has been identified in C. maltosa that, when expressed in S. cerevisiae, causes resistance in vivo to 5-10 pg/ml of cyh. The product of this gene remains to be identified. In the present work we have cloned and partially characterized two new cyh resistance determinants (SCRI and SCR2) from Sw. occidentalis and have expressed them in S. cerevisiae. Drug resistance levels have been determined both in vivo and in vitro using concentrations of cyh that reflect the natural resistance levels (up to 1 mg/ml of drug) shown by Sw. occidentalis. We show that the essentially complete resistance of Sw. occidentalis to cyh is controlled by a ribosomal protein that is equivalent to LA1 in S. cerevisiae. This protein in Sw. occidentalis is encoded by the SCR2 gene which has been located on chromosome V as a single copy. The single-copy SCRl gene gives low-level resistance to cyh (100 pg/ml) in vivo and in vitro and is located on chromosome 11. MATERIALS AND METHODS

Strains, plasmids and culture conditions Sw. occidentalis (formerly Sw. alluvius and Sw. castellii [l]) was obtained as Sw. castellii ATCC 26077 from Dr. M. A. Delgado (Cruz del Camp0 S. A., Sevilla, Spain). S. cerevisiae IM1-8b (a, leu 2-3, 2-112, his4) was described else-

850 where [18, 191. The multicopy YEpl3 (CV13) and pAAR6 and single-copy YCp50 S. cerevisiaelEscherichia coli shuttle vectors were described elsewhere [20-221. E. coli plasmids were KS-bluescript (Stratagene) and pUC7 [23]. Yeasts were grown on YEPD medium (1% yeast extract, 2% peptone, 2% glucose) or YNB minimal medium (0.7% YNB from Difco, 2% glucose, supplemented, if required, with 40 p g / d each histidine and leucine). L medium [24] was used for culturing E. coli DH-5. If required, ampicillin was added at 100 pg/ml. Minimal inhibitory concentration (MIC) for cyh was determined by growing yeasts on agar plates containing minimal medium lacking, if relevant, the prototropy provided by the transforming plasmid. For Sw.occidentalis and S. cerevisiae IM1-Sb, MIC for cyh were >lo00 and 0.5 pg/ml, respectively.

rose) supplemented with cyh (2 pglml final concentration). Incubation was continued at 30°C for 3-4 days. A control in the absence of both leucine and cyh indicated that about lo4 transformants/pg plasmid DNA were obtained. Cyh was obtained from Calbiochem-Behring Corp., (La Jolla, California).

Transcription studies Total yeast RNAs were prepared as described [27]. The transcription initiation site from SCR2 was determined by the S1 nuclease protection method [29]. To perform Northern analysis, total yeast RNAs were developed by denaturing gel electrophoresis and then transferred to a nylon sheet which was hybridized to a 32P-labelledprobe as described elsewhere 1251.

DNA manipulations Recombinant DNA procedures were as described elsewhere [25]. DNA sequencing was performed by the dideoxy chain-terminating method [26]. Transformation of yeast spheroplasts was achieved as described [27]. Hybridization at high stringency was in the presence of SO% formamide, 0.45 M NaCl, 0.045 M sodium citrate, 1X Denhart solution, SO mM Hepes, 100 pglml salmon DNA, pH 7.4 at 45°C for 8 h. Conditions for hybridizations at low stringency were: 0.30 M NaCl, 0.03 M sodium citrate, 5 XDenhart and 100 pg/ml salmon DNA at 60°C for 12 h [25].

Site-directed mutagenesis Conversion of a CAA (Glu) to CCA (Pro) in the coding sequence of SCR2 gene was performed essentially as described [28]. Mutagenesis took place on a 1.4-kb Hue111 DNA fragment which contained the SCR2 coding sequence. This fragment was inserted in the Hind11 site of pUC7. The resulting plasmid (pSCU1) was digested with PvuII and a 1.7-kb fragment carrying SCR2 was isolated, then amplified by the polymerase chain reaction using oligonucleotides 5’-AACCAAACCAATTTTCC-3’and 3’-TTGGTTTGGTTAAAAGG-5‘, to generate the mutation, and the universal and reverse primers for amplification. For hybridization of the oligonucleotides, a temperature of 42°C was used. The reaction products were separated by electrophoresis on lowmelting-point agarose, the 1.S-kb fragment isolated and digested with HindIII to obtain a 210-bp fragment containing the base change. The original 210-bp HindIII fragment was removed from pSCUl by digestion with HindIII and the altered fragment substituted. Six out of seven of the resulting plasmids carried the expected base substitution as shown by DNA sequencing. From one of these plasmids, the 1.4-kb BamHI fragment containing the mutated SCR2 gene was isolated and inserted in the BamHI site of YEpl3. A plasmid (pSCE3m) containing this insert in the correct orientation was isolated. Isolation of cyh-resistance determinants A YEpl3-based gene library of total DNA from Sw. occidentalis was used to transform S. cerevisiae spheroplasts and transformants were recovered on plates with 30 ml regeneration medium (YNB supplemented with 1M sorbitol, 0.4% YEPD, 40 pg/ml each histidine and leucine and 3% agar) for 24 h at 30°C. The plates were overlaid with 3 ml soft regeneration medium (containing only 0.6% aga-

Pulsed-field gel electrophoresis Infact chromosomal DNA from yeasts was separated by the CHEF technique [lo, 301. For samples containing Sw. occidentalis DNA, the electrophoresis conditions were: 0.8% agarose gel in 0.089 M Tris/borate, 0.002 M EDTA, pH 8.0; pulse times of 150, 180, 200, 250 and 300 s, 24 h each at 150, 150, 120, 100 and 100 V, respectively.

Preparation of ribosomes and supernatant fractions for polyphenylalanine synthesis Yeast cells were grown on minimal media in the absence of cyh. In yeast transformants, plasmid maintenance was achieved by growing prototrophically. High-salt-washed 80s ribosomes and partially purified yeast supernatant (S100) fractions were prepared as described [31]. Preparation of 60s and 40s ribosomal subunits was carried out as described [311. Poly(U)-directed polyphenylalanine synthesis in yeast cell-free extracts was as described [31]. Reactions took place for 20 min at 30°C.

Computer-based sequence analyses Nucleotide and amino acid sequences were analyzed with the aid of the Wisconsin programs [32] on a Micro Vax R215F station in conjunction with sequence data from GenBank, EMBL, NBRF-protein and SwissProt.

RESULTS Cloning of two different CyhRdeterminants Several cyh-resistant (CyhR) S. cerevisiae transformants were obtained from a Sw.occidentalis gene library raised in YEpl3 and plasmid DNA from these clones was used to transform E. coli. Independently isolated transformants carried plasmids which, according to the restriction map of their inserts, conform to one of two different groups (Fig. 1). Plasmids pSC3 and pSC5 had a common DNA insert of 8.5 kb. Plasmids pSC1, 2, 4, 6 and 7 had a common DNA insert of 3.2 kb. S. cerevisiae was transformed with one plasmid of each group and transformants selected in the presence of 2 pg/ml cyh. The levels of CyhRin the resulting clones were then examined. It was apparent that plasmids of the first group induced levels of CyhR (2-100 pg/ml) much lower than those of Sw.occidentalis (>lo00 pg/ml). In contrast, plasmids from the second group conferred levels of CyhR

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similar to those of Sw. occidentalis (Fig. 1). These findings suggested the presence of at least two different genes for CyhRin Sw.occidentalis. The gene determining low levels of CyhRwas named SCRl and that conferring high levels SCR2. By subcloning experiments, plasmids pSC2.1, containing SCRI,and pSC3.13i and 2.22, containing SCRZ,were obtained (Fig. l), permitting the localization of both genes on discrete DNA fragments. We note that different levels of CyhR could be determined by plasmids of the first group (low-level resistance) according to the orientation of their DNA inserts; this may be due to promoter function and/or upstream activator sites (UAS) activity of the DNA flanking the BamHI site of the tetracycline-resistance gene of YEpl3 133, 341. To examine the efficiency of transformation by direct selection for CyhR, S. cerevisiae was transformed with pSC3.13i and pSC2.22. In both cases regeneration of spheroplasts took place in the presence of either 2, 5 and 10 pg cyNml. For both plasmids, recovered transformants were of the order 104, 3 X lo3 and 5 X lo2, respectively. Biochemical analysis of CyhR

Taking into account that Cyh" in C.maltosa [8]and Kluy-

CYH CONC [MI

Fig. 2. The effect of cyh on poly(U)-directed polyphenylalanine synthesis by different yeast cell-free systems. Reactions were performed as described [31], except that samples were incubated for 20 min. All reactions contained 100000X g supernatants from S. cerevisiue (YEpl3) and ribosomes from (0)S. cerevisiae (YEpl3) (A) S. cerevisiae (pSC7 [SCRI]) (0) S. cerevisiue (pSC3.13i [SCRZ]) (A)Sw. occidentulis. Control values were (0)9.4 mol ['4C]phenylalanine incorporatedlmol ribosomes, (A) 9.1 mol/mol, (0)10.3 mol/mol and (A)9.6 moYmo1.

veromyces sp. [6] is expressed at the ribosomal level, the

effect of cyh was studied on poly(U)-directed polyphenylalanine synthesis in cell-free extracts of the different yeast strains used in this study. Cell-free systems using homologous ribosome and supernatant fractions from Sw. occidentalis or S. cerevisiae (containing SCR2)gave high level resistance to cyh that reflected in vivo resistance levels (up to

1 mg/ml of drug). Ribosomes and supernatant fractions from S. cerevisiae (containing SCRl) were resistant, as was the strain in vivo, to cyh concentrations up to 100 pg/ml. Combinations of ribosomal and supernatant fractions indicated that resistance was located on the 80s ribosomes (Fig. 2); all reaction mixtures contained the 100000 X g fraction from

852

Fig. 3. Chromosomal mapping of cyh-resistance genes. Chromosomal DNA from Sw. occidentulis was developed by CHEF and transferred to nylon sheets, which were hybridized at high stringency to either the 0.21-kb Hind111 fragment from SCRZ (right) or to the 3.2-kb DNA insert from pSC2.1 containing SCRl (left). S. cerevisiae chromosomal DNA served as markers. Roman numberals indicate chromosomes. Numbers in brackets indicate the size (in kb) of the chromosomes.

S. cerevisiae (YEpl3). Resistance was attributed, as expected, to the 60s ribosomal subunit (data not shown). Because in all these experiments yeast cells were grown in minimal media lacking cyh (see Materials and Methods), it may be concluded that SCRl and SCR2 are expressed constitutively in S. cerevisiae. Chromosomal location of SCRl and SCR2 The DNA inserts of plasmids pSC3 and pSC7 (Fig. 1) showed no cross-hybridization, even at low-stringency conditions (data not shown). In another experiment, genomic DNA from both Sw. occidentalis and S. cerevisiae, digested with several restriction endonucleases, was probed under low-stringency conditions with a 0.21-kb Hind111 DNA fragment internal to the SCR2 gene (see below). The results suggested that a single SCR2 gene was present in Sw. occidentalis, whereas two genes homologous to SCR2 were present in S. cerevisiae. This was confirmed by showing that the same probe hybridized to intact genomic DNA from S. cerevisiae fractionated by the CHEF technique, indicating similar sequences in chromosome V or VIII and in chromosome XIV (data not shown). Chromosomal DNA from Sw. occidentulis was also analysed by the CHEF technique. A minimum of seven different chromosomes were clearly detected (Fig. 3). Southern blot hybridizations showed that SCRl and SCR2 were located in chromosome I1 and V, respectively (Fig. 3). Nucleotide sequence of SCR2 The SCR2 gene sequence was determined as indicated under Materials and Methods. The resulting sequence (2283

nucleotides), shown in Fig. 4, presents an open reading frame (OW) of 321 which is interrupted by a putative intron of 452 between 4-456, containing the consensus sequences of yeast ribosomal protein gene introns [35]: GTATGT and TTCTATATAG at the 5’ and 3’ ends, respectively. In addition, the sequence ATACTAACA appears 22 nucleotides preceding the intron 3‘ end. This sequence is highly conserved and essential for mRNA splicing [36]. The non-translated 5’ region of SCR2 contains one TATA box at -154 (Fig. 4); (most other yeast promoters are found between -30 and -120 [37]). One copy of a CAAT box is present, at -176 (Fig. 4). Between positions -363 and - 353 the sequence AACATGCATGC (Fig. 4) corresponds to the consensus AACATC(T/C)(G/A)t(A/G)CA found at around -300 in the yeast ribosomal protein genes (RPG). Between -300 and -288, the sequence ACCCAGCCATCGT is similar to the RPG box [ACCCATACATT(T/A)]which is located at about -300 in these genes. As in the case of SCR2, when both motifs are present the RPG box is located next to the 3‘ end of the other motif [38]. The 3’ non-coding region of SCR2 contains several motifs, indicated in Fig. 4, which have been proposed to play roles in transcription termination and polyadenylation from eukaryotic genes [39, 401.

The SCR2 gene encodes ribosomal protein L41 The 321-nucleotides O W of the SCRZ gene (Fig. 4) determines a putative protein (SWLA1) of 106 amino acids, molecular mass 12.25 kDa, and a PI of 11.19. Such a basic PI is typical of most ribosomal proteins, which contain a high

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proportion of basic amino acids. Since ribosome assembly takes place in the nucleus, most ribosomal proteins contain a nuclear localization sequence (NLS) motif for transport through the nuclear membrane [41, 421. SWL41 has the sequence PKTRK at positions 5-9, which could be a NLS motif. The codon usage of the SCR2 coding region was examined by the CODONPREFERENCE program [32], using as reference the nucleotide sequences of the Sw. occidentalis genes INV [43], EGl [44], AMY1 [45] and CYCl [46]. SCR2 has a codon usage which is typical of Schwanniomyces, with a bias to the use of A or T in the third position, which is in agreement with the low content (34.9%) of G + C in the genome of this yeast (data not shown). Comparison of the amino acid sequence of S W 1 with the data base sequences showed the presence of nine riboso-

mal proteins with a high similarity (91-62% identity) to SWL41. A protein sequence alignment including the ribosomal protein KLLAl from K. lactis, which promotes cyh-resistance in this yeast [47], is shown in Fig. 5. The highest similarities were found with LA1 ribosomal proteins from yeasts (S. cerevisiae, K. lactis, K. fragilis, C. maltosa and C. tropicalis). All the L41 proteins represented in Fig. 5 share a highly conserved central region. The similarities are less pronounced at the amino and carboxy termini.

Site-directed mutagenesis in vitro Comparison of the amino acid sequences of L41 proteins from CyhRand Cyh' organisms shows that all CyhRproteins differ from CyhS proteins at position 56: CyhRhave Gln56, CyhShave Pro56 (Fig. 5), suggesting that this may be at least

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