Cloning and characterization of the gene encoding the ADP-ribosylation factor in Candida albicans

Gene, 110 (1992) 123-128 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/92/$05.00

123

GENE 06245

Cloning and characterization of the gene encoding the ADP-ribosylation factor in C a n d i d a albicans (GTP-binding proteins; Saccharomyces cerevisi~ie; human; sequence homology; fungi)

Kenneth T. Denieh, Peter J. Malloy and David Feldman Division of Endocrinology, Stanford University School of Medicine, Stanford, CA 94305 (U.S.A.) Received by J.A. Gorman: 21 May 1991 Revised/Accepted: 30 September/7 October 1991 Received at publishers: 30 October 1991

SUMMARY

We have cloned and sequenced the gene (ARF) encoding the ADP-ribosylation factor (ARF) of Candida albicans. The gene contains an open reading frame of 537 nucleotides (nt) that codes for a protein with an Mr of 20 259. The C. albicans ARF gene is 67-70~ identical at the nt level to other ARF sequences including those of humans; the deduced amino acid sequence of C. albicans ARF shows a 78-83~ identity and 89-92~ similarity to the other ARFs. Southern analysis of C. albicans genomic DNA suggested the presence of a second ARF gene. The presence of multiple ARF genes is a consistent finding among the other organisms previously shown to have ARFs.

INTRODUCTION

ADP-ribosylation factor (ARF) acts as a co-factor for the covalent modification of the stimulatory guanine ntbinding protein of adenylate cyclase (Gs) by cholera toxin (Kahn and Gilman, 1984; 1986). Ribosylation of Gs results in the irreversible activation of adenylate cyclase (Casey and Gilman, 1988; Kahn et al., 1983; Moss and Vaughan, 1988). The physiological role of ARF in the absence of

Correspondence to: Dr. D. Feldman, Division of Endocrinology, S005, Stanford University School of Medicine, Stanford, CA 94305 (U.S.A.) Tel. (415)723-6054; Fax (415)725-7085. Abbreviations: A, absorbance; aa, amino acid(s); ARF, ADP-ribosylation factor; ARF, gene encoding ARF; bp, base pair(s); C., Candida; CaCBP, C. aibicans corticosteroid-binding protein: Gs, see INTRODUCTION: kb, kilobase(s) or 1000 bp; nt, nucleotide(s); oligo, oligodeoxyribonucleotide: ORF, open reading frame: RFLP, restriction fragment length polymorphism; S., Saccharomyces; SDS, sodium dodecyl sulfate: SSC, standard saline citrate: UV, ultraviolet; Xaa, any aa.

cholera toxin remains to be defined. However, this 20-kDa protein (Kahn et al., 1988), itself a GTP-binding protein (Enomoto and Gill, 1979; Schleifer et al., 1982), has been found in every eukaryotic cell examined to date, including human (Freissmuth et al., 1989; Kahn et al., 1988), bovine (Tsai et al., 1987; 1988), and rabbit (Kahn and Gilman, 1984), as well as slime mold and yeast (Freissmuth et al., 1989; Kahn et al., 1988). Its abundance is particularly high in the brain (1% of cell protein) (Sewell and Kahn, 1988) suggesting a fundamental and critical role in cellular physiology. Recently, it has been postulated that ARF may play a pivotal role in the secretory pathway. ARF has been found to be associated with the Golgi complex and may be involved in secretion in yeast (Stearns et al., 1990a,b). Our laboratory has been investigating the function of steroid-binding proteins present in various pathogenic fungi (Feldman, 1988) including C. albicans (Loose and Feldman, 1982). During the course of our attempts to clone the gene encoding the CaCBP using mixed oligos, we isolated and sequenced a gene which was very similar to the mammalian and yeast ARF genes. In this report we describe the putative C. albicans ARF nt sequence, the predicted aa sequence and a comparison to other cloned ARF genes.

124 EXPERIMENTAL AND DISCUSSION

(a) Isolation of the gene for ADP-ribosylation factor A 64-fold degenerate 29-mer oligo probe synthesized on the basis of the microsequence analysis of peptides isolated from purified C. albicans CBP was used to screen a ~.EMBL-3 C. albicans genomic D N A library as described in the legend to Fig. 1. Five hybridizing clones were isolated and shown by restriction mapping and Southern analysis to contain overlapping sequences. A 6.6-kb EcoRI fragment which was common to each clone and hybridized to the oligo probe was mapped for restriction sites and subcloned. Further analysis of restriction enzyme digests localized the hybridizing sequence to a 1.8-kb BglII-EcoRI fragment which was subsequently sequenced. Fig. 1 shows a restriction map of this region as well as the sequencing strategy employed. The nt sequence of the 1.8-kb fragment is depicted in Fig. 2. Analysis of the sequence data revealed an ORF of 537 nt, which, when translated, encodes a protein of 179 aa with a predicted M r of 20 259. The C. albicans

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Fig. !. Restriction map and sequencing strategy for clones pCa2 and pCa3. DNA isolated from C. albic'ans strain stn-! was used to construct a genomic library. The DNA was partially digested with Sau3Al and fractionated by sucrose density gradient centrifugation. Size-selected (820 kb) DNA was then ligated to the BamHl arms of ~.EMBL-3, in vitro packaged, and used to transfect E. co/t LE392. The unamplified library contained 106 recombinants with less than 1% parentals, Methods. The library was plated and approx, 30 000 plaques were screened as described by Benton and Davis (1977), Duplicate filters were prehybridized for 12 h in 6 x SSC (1 x = 0,15 M NaCI/0,015 M Na~.citrate pH 8)/2 x Denhardt solution ( I x =0.02% bovine serum albumin/0,02% Ficoll/0.02% polyvinylpyrolidone)/0.5% SDS/0,05% Na'pyrophosphate/denatured

salmon sperm DNA (100/~g/ml). The labeled oligo probes were then

added and allowed to hybridize for 16 h at 42°C. Positive hybridizing plaques were purified and the DNA was isolated as described by Tsonis and Manes (1988). The DNA was subcloned into the phagemid vector Bluescript II (Stratagene, San Diego, CA), digested with restriction endonucleases, blotted to nitrocellulose (Maniatis et al., 1982) and hybridized to the mixed oligo probe. A 6.6-kb EcoRl hybridizing fragment common to all clones was mapped and subcloned for sequence determination, Double-stranded DNA sequencing was performed using the dideoxynucleotide chain-termination method (Sanger et el., 1977), and Sequenase (U.S. Biochemicals, Cleveland, OH). Sequencing reactions were performed in both directions to confirm the analysis. Restriction cleavage sites are shown abovethe line. Arrows indicate the direction and extent of sequencing. B, BglIt; E, EcoRI; H, HindlII; M, XmnI; O, Xhol; P, PstI; R, Rsal; S, SacI; U, PvuIl; X, Xbal.

gene is devoid of typical yeast-splicing signals (including the highly conserved 5 ' - T A C T A A C sequence) suggesting that the protein-coding region is uot interrupted by introns. The 5'-flanking region of the gene contains a putative TATA box and the 3'-flanking region contains one potential polyadenylation signal.

(b) Analysis of nucleotide and deduced amino acid sequences Using the computer search programs F A S T A and T F A S T A (Lipman and Pearson, 1985), both the nt and deduced aa sequences were shown to be clearly related to the yeast and bovine A R F sequences, therefore, the gene was designated ARF. Table I compares the coding region, nt sequence and deduced aa sequence of the C. albicans A R F gene to other cloned A R F genes. At the nt level, C. albicans ARF shows between 67-70% identity to other A R F sequences; the highest identity occurs to S. cerevisiae ARFI and the lowest to human AREs. The 5'- and 3'untranslated regions of C. albicans ARF were also compared to the corresponding untranslated regions in the other A R F genes and showed very little or no homology (data not shown). The deduced aa sequence ofC. aibicans A R F was aligned with the sequences of S. cerevisiae, bovine and human A R F using L I N E U P and PRETTY computer programs (Devereux et al., 1984). Compared to the other ARFs, C. albicans ARF shows a 77-83% identity and 89-92% similarity of the deduced aa sequence. Most of these changes occur at the N and C termini and approx. 41-55 % ofthese changes are conservative in nature. In addition, C. albicans A R F is 2 aa shorter at the C terminus (the lysine-rich region of human ARF3) making it 179 aa in length compared TABLE ! Comparison of the nt and deduced aa sequences ofCandida albicansARF to other cloned ARF genes Species

% nt identity'~

% aa identityb

% aa similarity b

S. cerevisiaeARFI S. cerevisiaeARF2 Human ARFI Human ARF3 Human ARF4 Bovine ARFI Bovine ARF2

70 70 67 67 69 70 69

78 77 83 82 77 82 82

89 89 92 92 90 92 91

" % nt identity of the coding regions was calculated using the FASTA program (Pearson and Lipman, 1988). b % identity and similarityof the deduced aa sequence were calculated using the GAP and BESTFIT programs of the University of Wisconsin Genetic Computer Group computer package (Devereux et al., 1984). Identity is defined as identical aa, whereas similarityis defined as a conserved aa substitution.

125 AGATCTCTTATAAATACAAAGTCTTGGTTGTCAACGCTAAACACAGGAACTGCCAATGCCGCTT TCTCTATAGCACCATCGCTACTGCCTTGTTGACTAAACTCCAACTCAATTCCAAATGCCGGTGC ACGTTTTTTCGTAGATGGAGATTTCGGAAATGTAACTGTAATAGACATGATTGGATTACCTAAC AGGCTTGTTGTTGTTTACACTTGCTTGACTTAAAAAAAAACAGCCTATCAACTTCTCGGTTTGT TCAACAAAAAAAAAAGTGTGTACCTCTGGTCGTGTACCATTCCAAACTGTTCACACTTTTTTAG

GTCATGGCACATAATCTTCGTCACACAAATCAACTGATTTTGTCTAACAGCTCTAATCAAGTAT AGTTATCTACATGGGATTAACAATTTCAAAATTATTTGCCAGTCTCTTGGGGAGACGTGAAATG M

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GACTTGCCGCGGTGAAACTACCACTAATTAAGAAATAAACCCCACAGAAGAATTTCAACATAAC GTCCCCTCAGACAATGAAAAAAAATATATATATAAACTCCATCTCTTATCTCTATTTTTCTTTA TCAAATTCACCTAAAGTATCAAAACTATTATACACAATGTCCGACGAAGAAGCCGATGTCTTGT ACGAAGTCAGAGATAGAACTGCTATCATCACCTTCAATATTCCTTCCAAATTAAATGCCTTAAA TGGTGAACAATATTTAAAGTTGGGTAAATTCATGGAAAGGGCCAACGAAGAAGAAGGCACAGTC ATGACTTTGATTCAATCTACAGGTAGATTTTTCTCTGCTGGTGCCAACTTTGCCGACAACGGAT TGATGGATGTTGACCCCCAATTGTTATTTACTCACGAATATTGGTTGGGAAAATTTGTTGCTAG AAACGTTTGGTTGACCAACTTGTTCAATGACCACAAGAAGATTTTGGTTGCTGCAGTCAACGGT CCTTCCATTGGGTTGAGTACTGGGTTGTTGATGTTGGTCGATTTGGTTTACGTCCACGATTTGA GCAAATTCTATTTATTGGCACCATTTGCCAACTTGGGTTTGGTTGCTGAAGGTGCTGCCTCTGC CACCTTGTTTGCCAGGTTAGGATGGTCCAAAGCTTCAGAAGCTTTATTGTTGGCCAAGCCAATT AGTGGTGCTGACTGCTACAATGTTGGGTTGATCAACAAACACTACGACGGTAAGTTCAAGTCCA CAGAAGAATTC 1473

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Fig. 2. The nt sequenceof the C. albicansARF gene. Only the nontranscribed DNA strand is shown. Numberingis in the 5'-+ 3' direction. The deduced aa sequence for the correct reading frame is shown below. A possible TATA box and one potential polyadenylation site are single and double underlined, respectively.The GenBank accession No. for the C. albicm~sARF sequence is M54910. Asterisk, stop codon. to all other ARFs which are 181 aa in length (Bobak et al., 1989; Price et al., 1988; Sewell and Kahn, 1988). Surprisingly, C. albicans ARF is slightly more similar to the mammalian than to the S. cerevisiae aa sequence. C. aibicans A R F shares several long stretches of perfect aa identity with other yeast and mammalian ARFs and with GTP-binding proteins (Fig. 3). The most striking of these are the proposed consensus aa sequences for GTP binding and hydrolysis (Allende, 1988; Halliday, 1984; Masters et al., 1986). These regions include the Gly-XaaGly-Xaa-Xaa-Gly-Lys sequence at aa positions 24-30, Asp-Xaa-Xaa-Gly at positions 67-70, and Asn-Lys-XaaAsp at positions 126-129. All of the ARFs are identical in these putative GTP-binding domains and are homologous with other GTP-binding proteins, except that all ARFs

have an Asp at position 26 instead of Gly. On this basis, the ARFs are clearly related to the heterotrimeric G proteins and also to the small G proteins (Freissmuth et al., 1989; Schultz et al., 1989). In addition, each ARF sequence contains a single potential glycosylation site, Asn-Xaa-Ser/ Thr at aa positions 60-62. C. albicans ARF also contains the consensus sequence of N-terminal myristoylation, Metinit-Gly-Xaa-Xaa-Xaa-Ser/Ala/Thr (Buss et al., 1987; Towler et al., 1987), as was found in other ARF proteins. The 77-83 % aa homology exhibited between C. albicans ARF and the S. cerevisiae and mammalian ARFs is within the range of aa homologies shown for other C. albicans genes that have been sequenced: p-tubulin is 75% identical to chicken f~-tubulin and 82% identical to S. cerevisiae p-tubulin (Smith et al,, 1988), cyclophilin is 68% identical

126

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Fig. 3. Comparison of deduced aa sequence of C. albica,s, S. cerevisiae, bovine and human ARF genes. The aa identities, compared with the deduced sequence of C. albica,s ARF, are indicated by a dash, aa differences are shown with the corresponding single-letter aa code. The last digit of numerals is aligned with the corresponding aa. References for ARF nt sequences are as follows: $. cerevisiaeARFI and ARF2 (Seweil and Kahn, 1988; Stearns et al,, 1990b), bovine ARFi (Price ~t al., 1988), bovine ARF2 (Sewell and Kahn, 1988), human ARFI and ARF3 (Bobak et ai., 1989) and human ARF4 (Monaco et al., 1990).

to the human T-cell cyclophilin and 81 ~o identical to the cytosolie S. cerevisiae cyclophilin (Koser et al., 1990), aspartyl proteinase is 85% identical to S. cerevfsiae aspartyl proteinase (Lott et al,, 1989), and lanosterol 14~-demethylase is 62?/0 identical to S. cerevisiae lanosterol 14~demethylase (Lai and Kirsch, 1989). Although the nt and aa homologies strongly suggest that the gene sequence presented in this report encodes a C. albicans ARF, in the absence of biochemical data, it should be noted that other possibilities exist, since a number of other ARF-related genes and pseudogenes have been identified (Alsip and Konkel, 1989; Monaco et al., 1990; Tamkun et al., 1991).

(c) Southern-blot analysis A Southern blot of C. albicans and S. cerevisiae genomic D N A was probed with the 450-bp Rsal-XhoI fragment of the C. aibicans A R F gene (Fig. 4). Under low-stringency conditions, two hybridizing bands are seen in EcoRI digest of S. cerevisiae DNA, consistent with the presence of two A R F genes in this organism (Sewell and Kahn, 1988; Stearns et al., 1990b). The blot also shows two hybridizing bands in each of several restriction enzyme digests of C. a/bicans DNA. This suggests that there may be two A R F genes in this organism, since none of the enzymes used have a restriction site within the fragment that was era-

127

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C. albicans I

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other organisms i n . w h i c h A R F has been described similarly have multiple A R F genes.

REFERENCES

kb 8.5-6.4-4.3 --

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Fig. 4. Southern-blot analysis of the C. aibicans ARF gene. C. albicans strain stn-I DNA (10/~g) was digested with restriction endonucleases as described by the manufacturer and electrophoresed on 0.7 % agarose gels in Tris.acetate/EDTA buffer (Maniatis e: ai., 1982). After electrophoresis, gels were denatured in 0.5 M NaOH/I.5 M NaCI for I h and neutralized in 1 M "Iris pH 8.0/1.5 M NaCI for I h before transferring the DNA to nitrocellulose (Maniatis et al., 1982). After a 24-h transfer in 2 x SSC, the filters were baked for 2 h at 80°C. The biters were prehybridized in 4 x SSC/I x Denhardt's/10% dextran sulfate/20 mM Tris pH 7.4/40% formamide/denatured salmon sperm DNA (20pg/ml) for 4 h at 37°C. The blots were then probed with the 32p-labeled RsaI-Xhol fragment for 24 h at 37°C. The blots were washed in 2 x SSC/0.1 ~o SDS three times for 15 min at 24°C, then ence for 15 rain at 45°C before being exposed to Kodak XAR film at -70°C. None of the enzymes used cut within the fragment. S.c., S. cerevisiae. Molecular size standards in kb are shown on the left.

ployed as the probe. A n alternative interpretation would be that the b a n d i n g pattern results from an R F L P . However, it is unlikely t h a t an R F L P for each o f the five different enzymes would be present.

(d)

Conclusions (1) T h e C. albicans A R F gene h a s been cloned a n d se-

quenced. T h e gene is highly conserved showing a 6 7 - 7 0 % identity to the S. cerevisiae and m a m m a l i a n A R F genes. At the aa level, the C. albicans A R F is 7 8 - 8 3 % identical to other A R F s . (2) C. albicans m a y contain a s e c o n d A R F gene. All

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