Vol. 12, No. 5

MOLECULAR AND CELLULAR BIOLOGY, May 1992, p. 1977-1985

0270-7306/92/051977-09$02.00/0

A G-Protein a Subunit from Asexual Candida albicans Functions in the Mating Signal Transduction Pathway of Saccharomyces cerevisiae and Is Regulated by the al-oc2 Repressor CHANCHAL SADHU,t DENISE HOEKSTRA, MICHAEL J. McEACHERN,4 STEVEN I. REED, AND JAMES B. HICKSt*

Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 92037 Received 25 September 1991/Accepted 4 February 1992

39). The role of GPA2 is not known, but it does not complement the loss of SCG1 for mating response (37). No sexual cycle similar to that of S. cerevisiae has been observed in the opportunistic human pathogen Candida albicans. A number of C. albicans strains show variations of colony morphology and cell types (49, 50). Notable among them are the white and opaque forms of strain WO-1. These forms are so named because of their colony appearance. Cells of the white forms are smaller and elliptical, while the opaque cells are larger and bean shaped. Opaque forms can be maintained as opaque at 25°C, and they can be induced to give rise to white forms by growth at 37°C. There is also an example of a developmental switch in C. albicans between vegetative growth as budding (yeast) and hyphal (mycelial) forms. This switch, often referred to as germ tube formation, can be stimulated in vitro by a number of different experimental regimens, including changes in temperature and pH, but the most consistent effect is in response to serum factors (41). Thus, although no component has so far been characterized at the molecular level, it is likely that signal-sensing and transduction mechanisms are present in C. albicans. A receptor for the adhesive glycoprotein laminin has recently been detected on the surface of C. albicans hyphae (3). In addition, a corticosteroid-binding protein (28, 48) and proteins binding specifically to the mammalian C3bi factor (13) and C3d (43) have been found. No connection has yet been made between the binding proteins and potential receptors described above and specific behavior of C. albicans. We have therefore begun to examine the sensing apparatus of C. albicans, starting at the conserved, G-protein signal transduction mechanism. In this report, we describe the cloning and initial characterization of a Ga-protein gene, CAGI, from C. albicans. The primary

Heterotrimeric guanine nucleotide-binding proteins (G proteins) are important components of signal transduction systems in eucaryotes (4). In conjunction with the serpentine transmembrane receptors, they transduce a wide variety of extracellular signals to the appropriate effectors within the cell. The specificity of G-protein action resides in the a subunit, which interacts directly with the intracellular surface of the receptor and which contains the guanine nucleotide binding site. At rest, the ao subunit contains bound GDP and is complexed with the and y subunits. Upon binding of agonist with the receptor, Ga becomes activated, exchanging GDP for GTP and dissociating from the 13y complex. Activation of the G-protein complex results in subsequent activation of the target effector molecule, which carries the signal to control sites elsewhere in the cell (12, 53). In mammals, a large family of Ga sequences are known, while in the yeast Saccharomyces cerevisiae, two Ga genes, SCGJ (also known as GPAI) (9, 21, 35) and GPA2 (37), have been reported. SCGI (9), STE4, and STE18 (54) encode the a, 1B, and -y subunits, respectively of a G-protein complex that is required for response to mating pheromones. SCGI presumably interacts with the pheromone receptors, STE2 and STE3. The inferred amino acid sequences of these receptors indicate that they are transmembrane proteins, each with seven membrane-spanning domains typical of the receptors associated with G proteins in vertebrates (6, 16,

* Corresponding author. t Present address: ICOS Corporation, 22021 20th Avenue S.E., Bothell, WA 98021.

: Present address: Department of Microbiology and Immunology, University of California at San Francisco, San Francisco, CA 94143. 1977

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We have isolated a gene, designated CAGI, from Candida albicans by using the G-protein a-subunit clone SCG1 of Saccharomyces cerevisiae as a probe. Amino acid sequence comparison revealed that CAG1 is more homologous to SCG1 than to any other G protein reported so far. Homology between CAG1 and SCG1 not only includes the conserved guanine nucleotide binding domains but also spans the normally variable regions which are thought to be involved in interaction with the components of the specific signal transduction pathway. Furthermore, CAG1 contains a central domain, previously found only in SCG1. cagi null mutants of C. albicans created by gene disruption produced no readily detectable phenotype. The C. albicans CAGJ gene complemented both the growth and mating defects of S. cerevisiae scgl null mutants when carried on either a low- or high-copy-number plasmid. In diploid C. albicans, the CAG1 transcript was readily detectable in mycelial and yeast cells of both the white and opaque forms. However, the CAG1-specific transcript in S. cerevisiae transformants containing the C. albicans CAGi gene was observed only in haploid cells. This transcription pattern matches that of SCGI in S. cerevisiae and is caused by al-a2-mediated repression in diploid cells. That is, CAGi behaves as a haploid-specific gene in S. cerevisiae, subject to control by the al-ac2 mating-type regulation pathway. We infer from these results that C. albicans may have a signal transduction system analogous to that controlling mating type in S. cerevisiae or possibly even a sexual pathway that has so far remained undetected.

1978

Strain

IWD .. CSl .. CS4 .. CS9 .. CS10 .. CS7 .. CS8 .. CS11 ... CS12 .. Dlll .. CS13 ..

SADHU ET AL.

MOL. CELL. BIOL.

TABLE 1. S. cerevisiae strains used Genotypea MA AITa/AM Ta [trpl leu2 his3 ura3-52 mal scgl::URA3] AIATa, pYEp2l3-CAGI; haploid segregant of IVYD Same as CS1 MA4Tot, pYEp2l3-CAGI; haploid segregant of IVYD Same as CS9 MATa, pYEpl3-SCGI; haploid segregant of IVYD Same as CS7 MATa, pYEpl3-SCG1; haploid segregant of IVYD Same as CSl1 MAATaMAL4Tot SCGI/scgl::LEU2 [ade2-11 his3-11, 15 leu2-3,112 trpl-1 ura3-1 canl-100] MA4Ta scgl::LEU2, pYCp5O-CAGI; haploid segregant of Dlll

sequence of this gene shows a close relationship to the Saccharomyces SCG1 gene, including a segment of the Saccharomyces gene not found in the mammalian counterparts. The C. albicans CAG1 gene can provide nearly complete mating function to haploid Saccharomyces strains lacking the SCGI gene. In addition, measurement of transcript levels has revealed not only that the C. albicans CAG1 gene is transcribed efficiently from its own promoter in S. cerevisiae transformants but that it is subject to the same regulation as is SCGJ.

MATERUILS AND METHODS Strains, media, and plasmids. Genotypes of the S. cerevisiae strains are listed in Table 1; the Candida strains used are listed in Table 2. S. cerevisiae strains harboring plasmids were cultured in appropriate selective medium. All other cultures were routinely grown in YPD medium. Yeast genetic manipulations were performed as described previously (46). Plasmids YEp13 and YEp213 are Escherichia coli-S. cerevisiae shuttle vectors containing the 2,um origin of replication and the LEU2 gene as a selective marker. YCp5O is also an E. coli-S. cerevisiae shuttle vector and contains yeast autonomously replicating and centromeric sequences and the URA3 gene (46). For the construction of plasmid YEp213-CAG1, Sall linkers was attached to the 1.9-kbp TABLE 2. Candida strains used

Species

Strain

3153a

WO-1 32032 32033 32077 18814 B4201 B4365 B4252 CK CG

C. C. C. C. C. C. C. C. C.

albicans albicans albicans albicans stellatoidea claussenii albicans stellatoidea stellatoidea C. krusei C. glabrata

Strain

no. or

reference

ATCC 28367 50 ATCC 32032 ATCC 32033 ATCC 32077 ATCC 18814 25 ATCC 20408 ATCC 11006 ATCC 34135 ATCC 34138

1000

500

Hpal

S

S

16

&N4

56

-p

Sau3A

2000

1500

Hpal

HIndlil BamHI| Clal

--4

p

6

I

-

Rsal

FIG. 1. Partial restriction map and sequencing scheme of the CAGI clone. DNA sequencing was performed by using a combination of restriction fragment subclones and synthetic oligonucleotides. Numbers indicate distances in base pairs.

HpaI fragment of the CAGI gene and inserted into the Sall site of plasmid YEp213. YCp5O-CAG1 was constructed by subcloning the 5.1-kbp EcoRI fragment in the EcoRI site of plasmid YCp5O. Plasmid YEp13-SCG1 carries a 5.5-kbp Sau3AI fragment of S. cerevisiae genomic DNA containing the SCGI gene in the BamHI site of plasmid YEp13. Isolation of the CAGI clone. In an effort to isolate a gene for a Ga protein from C. albicans, we screened a library of EcoRI fragments from strain WO-1 in the A phage vector EMBIA, using SCG1 as a probe. Hybridization was carried out at 60°C in 0.5 M sodium phosphate buffer. Recombinant phage DNA was prepared from seven purified positive plaques and digested with EcoRI. All seven contained a 5.1-kbp fragment which yielded a strong signal when blotted and probed with SCG1. The cloned EcoRI fragment was transferred to the Bluescript plasmid vector (Stratagene Inc., La Jolla, Calif.) and digested with additional restriction enzymes, yielding the partial map shown in Fig. 1. We located the CAG1 sequence on the plasmid by hybridizing the SCGI probe to blots of the gels used to map restriction sites. The sequence of the CAGI clone was determined by the dideoxy-chain termination method (42), using a combination of subclones and synthetic oligonucleotide primers. All other recombinant DNA procedures were carried out according to the published protocols (30). RNA isolation. C. albicans cells were grown in YPD or in Lee's medium (27) and harvested at different stages of growth. Cells were washed in water and resuspended in RNA isolation buffer (0.1 M NaCl, 0.1 M Tris [pH 8.0], 0.05 M EDTA, 1.0% sodium dodecyl sulfate [SDS]). The cell suspension was vortexed thoroughly after the addition of glass beads (1.5 g/ml) and an equal volume of phenolchloroform (1:1, vol/vol). After centrifugation, the aqueous phase was collected and the organic phase was reextracted with RNA isolation buffer. The aqueous phases were pooled and extracted repeatedly with phenol-chloroform (1:1) until there was no interphase. The aqueous phase was made to 2 M LiCl, and RNA was precipitated overnight at 4°C (1). The RNA precipitates were centrifuged at 10,000 rpm in a Sorvall HB4 rotor, washed in 2 M LiCl, and dissolved in water. RNA was reprecipitated by the addition of 2 volumes of ethanol in the presence of 0.3 M sodium acetate (pH 5.2) at -20°C. The precipitates were washed with 70% (vol/vol) ethanol in water and dissolved in water. Concentrations of RNA solutions were determined by measuring A260. Northern (RNA) blot analysis. Aliquots of the RNA samples were denatured by heating at 65°C in a mixture of 6% formaldehyde, 30% formamide, 1 x morpholinepropanesulfonic acid (MOPS) buffer, and 5% glycerol containing 0.04% bromophenol blue and xylene cyanol (30). The sam-

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CS14 .. Same as CS13 CS15 .. MATot scgl ::LEU2, pYCp5O-CAGI; haploid segregant of Dlll CS16 .. Same as CS15 CS26 .. MA4Ta SCG1; haploid segregant of Dlll CS27 .. MATa SCG1; haploid segregant of Dlll a Markers within brackets are homozygous.

0 Hindll

VOL. 12, 1992

1979

a 14-min switch time; and 80 V for 12 h with an 18-min switch time. Nucleotide sequence accession number. The nucleotide sequence data reported in this paper has been deposited in the EMBL, GenBank, and DDBJ data bases under the accession number M88113.

RESULTS Identification of a heteromeric G-protein gene in C. albicans. When Southern blots containing EcoRI or HindIIIdigested C. albicans genomic DNA from two different strains, WO-1 and 3153a, were probed with 32P-labelled SCG1, distinct bands were observed at both the reduced and normal stringency of hybridization, indicating the presence of a close homolog. In strain WO-1, the probe detected a single EcoRI fragment of 5.1 kbp, while in 3153a, two fragments of 1.8 and 3.2 kbp appeared. The HindIll digestion patterns for the two strains were identical, showing fragments of 1.0 and 1.4 kbp (data not shown). We cloned the 5.1-kbp EcoRI restriction fragment identified by this probe from strain WO-1 as described in Materials and Methods. As the nucleotide sequence was accumulated and translated, it was clear that the gene encoded a protein very similar to SCG1, and the primary amino acid sequences fell into nearly perfect register over long stretches of the sequence. The resulting sequence, containing a continuous reading frame of 429 amino acids, along with 514 bp of 5' flanking sequence and 80 bp at the 3' end, is presented in Fig. 2. Structural comparison of CAG1 and SCG1. Figure 3 shows an alignment of the inferred amino acid sequences for CAG], SCG1, and the gene for a human Got subunit (32). Gaxs was chosen for contrast because, of the published Ga sequences, it is the least similar to SCG1. The regions of conservation among all three genes in the figure are thus the most likely conserved in the GaL family. It is apparent that CAG1 contains stretches of sequences along its entire length (labeled Gl through G5) that have been postulated to be involved in GTP binding (Fig. 3). Overall, the primary amino acid sequence of CAG1 is 47% homologous to that of Gas (including the conservative substitutions). Homology is particularly pronounced around the regions implicated in GTP binding (5, 17). Similar regions were also found to be conserved between CAG1 and the S. cerevisiae G protein SCGL. However, sequence similarity between CAG1 and SCG1 extends beyond these regions. CAG1 exhibits 65% amino acid sequence identity (77% counting conservative replacements) to SCGL. The sequence conservation between CAG1 and SCG1 is most striking in regions that are least conserved among the nonyeast members of the family. These regions are boxed in Fig. 3 and include the amino-terminal 33 residues, the carboxy-terminal 61 residues, and part of a central segment found only in SCG1 and CAG1. The amino terminus of the heteromeric G-protein a subunit is thought to be necessary for binding to the f subunit and is also one of the more variable regions (53). Between CAG1 and SCG1, the aminoterminal 40 residues are 60% conserved, compared with 27% for the next-best candidate. No other Ga subunit, including the product of the Saccharomyces GPA2 gene (37), shows more than 23% conservation in this segment. In the proposed receptor binding region at the carboxy terminus (19, 20, 31, 53), 46 of 53 residues are conserved (39 of 53 are identical) between SCG1 and CAG1. None of the nonyeast

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ples were electrophoresed on 1.2% agarose gel containing 6% formaldehyde in lx MOPS buffer. Electrophoresis was conducted at room temperature in lx MOPS buffer. After electrophoresis, RNA was blotted overnight on a Genescreen (NEN Research Products) membrane by capillary transfer using lOx SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate). RNA was UV cross-linked to the membrane (23) and prehybridized in 7% SDS-0.5 M Na2HPO4 (pH 7.2) for 5 min at 60°C (8). Hybridization was in the same solution with a 32P-labelled probe prepared by random priming (11). After hybridization, the membrane was washed twice in 5% SDS-50 mM Na2HPO4 at 60°C for 1 h each time and autoradiographed. Quantitation of mating efficiency. Quantitative determination of mating efficiency was carried out as described previously (38). S. cerevisiae cells (106) were mixed with the standard tester (ilv) a or a cells (107) and collected on a GF/C membrane. The membrane was then incubated on a YPDagar plate at 30°C for 2 h. Subsequently, the cells were resuspended by dipping the membrane in 10 ml of minimal medium (SD) and vortexing vigorously. Samples were plated on SD-agar plates to determine the number of prototrophs and on ILV dropout plates to determine the number of cells whose efficiency was being measured. The data were normalized by assuming the efficiency of mating of the cells carrying only the chromosomal SCGI gene to be 1. Disruption of the CAGI gene. The CAG1 gene was disrupted by inserting an approximately 2.5-kbp BglII-ClaI fragment of the C. albicans ADE2 gene from pMC2 (24) into the BamHI (position 859) and ClaI (position 1038) sites of the CAG] gene. An ade2 strain of WO-1, WO-1 3/6 (J. Schmidt, University of Iowa), was then transformed, with the resultingADE2-interrupted CAG1 gene excised as a SalI-linkered HpaI fragment. Sixteen of the resulting transformants were then screened by transverse alternating-field electrophoresis (TAFE) analysis for the presence of the ADE2 gene on one of the two chromosomes carrying the CAG] gene. Fifteen of the sixteen clones were found to contain the ADE2 gene in one or the other chromosome carrying the CAGI gene. Southern blotting analysis of EcoRI-digested genomic DNA from several of these clones confirmed that one of the two copies of CAG] was indeed interrupted. The interruption was rendered homozygous by subjecting one of the heterozygous clones to a brief pulse of UV light to induce mitotic recombination. Approximately 1,000 freshly plated cells were exposed for 5 s to a single 15-W General Electric G15T8 germicidal light bulb at a distance of 30 cm, resulting in approximately 10% cell killing. Approximately 1% of the resulting colonies contained one or more red sectors. These red sectors contain ade cells formed via the mitotic recombinational loss of the single functional ADE2 gene. Since mitotic recombination is expected to be a reciprocal event, the white parts of these sectored colonies should contain cells that have two ADE2-disrupted CAG1 genes. Ten such isolates were examined by Southern blotting of EcoRI-restricted DNA and by Southern blotting of TAFE-separated chromosomes, and seven were found to be homozygous for the disruption. Pulsed field gel electrophoresis. TAFE was carried out on a Beckman (Palo Alto, Calif.) Geneline apparatus, using the Tris acetate buffer recommended by Beckman on 1.0% agarose. Electrophoresis was done in six stages as follows: 100 V for 12 h with a 2-min switch time; 100 V for 36 h with a 4-min switch time; 100 V for 24 h with a 7-min switch time; 80 V for 24 h with an 11-min switch time; 80 V for 24 h with

C. ALBICANS G PROTEIN

MOL. CELL. BIOL.

SADHU ET AL.

1980

-514 AAGCTTCATAGATTTATCAAAGAACAGGATGACGAGCATATGGAACAGAAACA

CAGI 1

sOG1

1 1

HMGC GA SVPVDDDEIDPFLQDKRINDAIEQSLQLRQQNSKK M GCTVSTQTIGDESDPFLQNKRANDVIBQSLQL EKQRDKN NGCLGNSKT- EDQRNEEKAQREANKKIEKQLQKDKQVYRA

-461 TTTAGACATTTCGTTACCAACTTCATCCTCCTCAAATGCATATTCACTACCCAGCTCCATACCCCAGTACA

Gms

-390 CATTTACACAATCTTCAAGACCACAATTTGTCACCAAATTCAATAATACAAGACTAGGAAAAATTTACATA

G1 CAM 41 GV 1 L L L L GAGES G K ST V L K Q L K L L H KG G F TQ Q - ERR Q YS H SOG1 1 EIKLLLLGAGESGKSTVLKQLKLLHQGGFSHQ-ERLQYAQ G=s 40 THRLLLLGAGESGXSTIVKQMRILHVNGFNGDSEKATKVQ

-319

TTAAACAAGAGACGACTCTTTACATTTATAAAGAGATACAGTTTAGTCAGTTTTTGCAAAAAAGTTTGAT

-248

GGTGGGTATGCTGGTGGCCAGCATTTACGTACGAGGATTGGGTTAACTTGTATTGAGAGTAGACCATATTT

XOOOOGXGXXGKS

-177 TTTTTTTTGATGTGATTTTTAACATGGCTGCGGGAGTAAGCAGAAGGAAACGTTGATGTTTCAGATTTCAC -106 CACAAAGTGTAGAGAAGAAAAAAAGGAAAGATATTTTGGGGTTTTTTCTTAATGTACATTAAAATCTGTCT -35

TTTAGTTTACCTTTTTTTAATACCAGTATTCAATC ATG GGT TGT GGC GCT AGT GTT CCG GTT MET Gly Cys Gly Ala Ser Val Pro Val GAT GAT GAT GAA ATT GAT CCA TTT CTT CAA GAT AAA CGT ATA Asp Asp Asp Glu Ile Asp Pro Phe Leu Gln Asp Lys Arg Ile

AAT GAT GCT ATT Asn Asp Ala Ile

GAA CAA AGT TTA CAA TTG CGT CAA CAA AAC TCG AAA AAG GGA GTC AAG TTG TTG Glu Gln Ser Leu Gln Leu Arg Gln Gln Asn Ser Lys Lys Gly Val Lys Leu Leu TTG TTG GGT GCT GGT GAA AGT GGT AAA TCA ACA GTT TTA AAA CAA TTG AAA TTA Leu Leu Gly Ala Gly Glu Ser Gly Lys Ser Thr Val Leu Lys Gln Leu Lys Leu TTA CAT AAA GGT GGG TTT ACC CAA CAG GAG AGA AGA CAA TAT TCT CAT GTC ATT Leu His Lys Gly Gly Phe Thr Gln Gln Glu Arg Arg Gln Tyr Ser His Val Ile

TGG TGT GAC GTT ATT CAA TCA ATG AAA GTT TTA ATC ATT CAA GCA AGA AAG TTG Trp Cys Asp Val Ile Gln Ser Met Lys Val Leu Ile Ile Gln Ala Arg Lys Leu TCA TTA ATT CCT TAT Ser Leu Ile Pro Tyr

AAG

CAG

Lys Gln

ATT ATA TTA CGA AGC GAT CCT TTA AAA CAA ATA GAT GCT AGT GTT GCT GGT GGT Ile Ile Leu Arg Ser Asp Pro Leu Lys Gln Ile Asp Ala Ser Val Ala Gly Gly ACA GAT TTC CTA AAT GAT TTT GTT GTC AAG TAT AGT GAA GAA AAC AAG AAC AAG Thr Asp Phe Leu Asn Asp Phe Val Val Lys Tyr Ser Glu Glu Asn Lys Asn Lys

AGA CGG TTG AAG AGT ACT GGG ACA ACA GAT ATA TGG GGT AAA GAT GAC GAT TCC Arg Arg Leu Lys Ser Thr Gly Thr Thr Asp Ile Trp Gly Lys Asp Asp Asp Ser AAT ATC AAT TCA GAT GCA ATT AAT CAA GCT TTG GAA CTG TCT TTG AAT AAA GAT Asn Ile Asn Ser Asp Ala Ile Asn Gln Ala Leu Glu Leu Ser Leu Asn Lys Asp

TCT GAA CAG TTT ACT CGT CTT TCC ATA GCT GAA GCA ATC CAT AAA TTA TGG AAG Ser Glu Gln Phe Thr Arg Leu Ser Ile Ala Glu Ala Ile His Lys Leu Trp Lys

TTG GAC TCG GGT ATT AAA AAG TGT TTT GAC AGG TCA AAT GAG TTC CAA TTG GAA Leu Asp Ser Gly Ile Lys Lys Cys Phe Asp Arg Ser Asn Glu Phe Gln Leu Glu

GGT AGT GCT GAT TAT TAT TTC GAT AAT GTC GTC AAC TTT GCT GAT ACA AAT TAT Gly Ser Ala Asp Tyr Tyr Phe Asp Asn Val Val Asn Phe Ala Asp Thr Asn Tyr 730

TTA TCT ACT GAT TTG GAT ATT TTA AAA GGG AGA ATT AAG ACT ACT GGT ATC ACT Leu Ser Thr Asp Leu Asp Ile Leu Lys Gly Arg Ile Lys Thr Thr Gly Ile Thr

784

GAG ACA GAT TTT TTA ATT AAA TCG TTT CAA TTT AAA GTG TTA GAT GCT GGT GGA Glu Thr Asp Phe Leu Ile Lys Ser Phe Gln Phe Lys Val Leu Asp Ala Gly Gly

838

CAA CGG TCA GTA CGT AAA AAA TGG ATC CAT TGT TTT GAA GAC ATC ACT GCT GTT Gln Arg Ser Val Arg Lys Lys Trp Ile His Cys Phe Glu Asp Ile Thr Ala Val

892

TTA TTT GTT TTG GCT ATC TCT GAA TAC GAT CAA AAC CTA TTT GAA GAT GAA CGG Leu Phe Val Leu Ala Ile Ser Glu Tyr Asp Gln Asn Leu Phe Glu Asp Glu Arg

946

GTA AAT AGA ATG CAT GAG TCT ATT GTC TTG TTT GAT TCA TTG TGC AAC TCC AAA Val Asn Arg Met His Glu Ser Ile Val Leu Phe Asp Ser Leu Cys Asn Ser Lys

1000 TGG TTT GCA AAC ACC CCA TTC ATA TTA TTT TTG AAC AAA ATC GAT ATT TTC GAA Trp Phe Ala Asn Thr Pro Phe Ile Leu Phe Leu Asn Lys Ile Asp Ile Phe Glu 1054 AAC ARO ATC AAA AAG AAT CCG CTA AAG AAT TAT TTC CCA GAC TAT GAT GGC AAA Asn Lys Ile Lys Lys Asn Pro Leu Lys Asn Tyr Phe Pro Asp Tyr Asp Gly Lys 1108 CCA GAC GAT ACT AAT GAA GCA ATC AAG TTT TTT GAG ACA AAT TTT TTG AAA ATA Pro Asp Asp Thr Asn Glu Ala Ile Lys Phe Phe Glu Thr Asn Phe Leu Lys Ile 1162 AAT CAA ACC AAT AAA CCT ATC TAT GTT CAT CGA ACG TGT GCT ACA GAT TCA AAA Asn Gln Thr Asn Lys Pro Ile Tyr Val His Arg Thr Cys Ala Thr Asp Ser Lys 1216 TCA ATG AAA TTT GTC TTG AGT GCT GTT ACC GAC ATG ATT GTA CAA CAA AAC TTG Ser Met Lys Phe Val Leu Ser Ala Val Thr Asp Met Ile Val Gln Gln Asn Leu 1287 1270 AAA AAG AGT GGT ATT ATG TAG TTGCAAGAAATAGGCGATATCTTTTTTACTTTACTATTAATGT Lys Lys Ser Gly Ile Met ---

1334 TCAGTTTAARATTTTTTGAGTTTATATCTATTT 1366

FIG. 2. DNA and deduced amino acid sequences of the CAGI clone. The putative promoter element (-169 to -152) and the translational stop codon (1287 to 1289) are underlined with solid and broken lines, respectively.

gene

products show greater than 50% conservation in this

region.

Both CAGI and SCGI encode unusually large Ga subunits of 429 and 472 amino acids, respectively, compared with a range of 325 to 375 amino acids in nonyeast genes. It has been shown that for SCG1, nearly all of the size difference resides in a 109-amino-acid segment between residues 126 and 235 that is not found in any of the nonyeast homologs (9, 35). Likewise, CAG1 exhibits a 73-amino-acid insert in the identical location that shows significant amino acid sequence similarity (60%) to the SCG1 insert over half of its length (residues 125 to 173). The other Ga protein from S. cerevisiae, GPA2, also contains an unusual sequence of 83 amino

CAG1 118 IILRSDPLKQIDASVAGGTDFLNDFVVKYSEENKNKRRLK SOG1 120 |ILLKAKALDYINASVAGGSDFLNDYVLKYSERYETRRRVQ Gas 120 VMNVPDF

- - - - - - - - - - -

C4G1 158 STGTTDIWGKDDDSSNIINSDAINQAIELSLNKDSEQFTRLS SCG1 160 STGRAKAAF- DEDGNI SNVKSDTDRDAETVTQNEDVDRNN GQS

CAG1

SOG1

----------------------------------------

-_ __--_ __-_ __-_ __--

199 SSRINLQDICKDLNQEGDDQMFVRKTSKEIQGQNRRNLIH

GaS 1Z7 - -

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

DFP

C4C1 S9G1

198 -- IAEAIHKLWKLDSGIKKCFDRSNEFQLBGSADYYFDNV 239 EDIAKAIRQLWNNDKGIKQCFARSNEFQLEGSAAYYFDNI Gas 130 PEFYEHAKALWE- DEGVRACYERSNEYQLIDCAQYFLDKI

G2

G3

C4G1 236 VNF AD TNY L ST DL D I L KG RI K T T G I T B T D F L I K S F QF K V L 50G1 279 EKFASPNYVCTDEDILKGRIKTTGITETEFNIGSSKFKVL GOs 169 DVI K Q A DY VP SD Q DL L R CR V L T S G IF ET K F Q V D K V N F H M F OJOO D -(X )n- T G3 276 C0G1 DAGGQ RSVRIKWIHCFEDITAVLPVLAISEYDQNLFEDER SO1 319 DAGGQRSERKXWIHCFBGITAVLFVLAMSEYDQMLPEDER Gas 209 DVGGQRDERRKWIQCFNDVTAI IFVVASSSYNMVIREDNQ DXAGJX

G4 SOG1 359 VN RH H ES IM L F D T L L N SKW F K D T P F IL FL N KID L FE EK V Gs 249 TNRLQEALNLFKSIWNNRWLRT ISVILFLNKQDLLAEKVL

CA1 316 VNRMHESIVLFDSLCHSWWFANTPFILFLNKIDIPENKI OOOOHKXD

C4C1 355 -KKNPLKN|YFPDY DGKPDDTNEA S0G1 398 -KSMPIRKYFPDYQGRVGDAEAG -

- - - - - - - - - - - - - -I - - - - - - - - - - - - - - - L

GOs 289 AGKSKIEDYFPEFARYTTPEDATPEPGEDPRVTRAKYFIR G5 C081 378 KFFETNFLKINGTNKPIYVHRTCATDSKSMKFVLSAVTDM 0111 421 KYFEK IFLSLNKTNKPIYVKRTCATDTOTMKFVLSAVTDL Gas 329 DEFLRISTASGDGRHYCYPHFTCAVDTENIRRVFNDCRDI

TCATDT Q V

C0G1 418 IVQHNLKKSGIN 429 SCG1 461 IIQQNLKKSGII 472

as 369 IQRMHLRQYELL 380

FIG. 3. Comparison of amino acid sequences of G-protein a subunits. The single-letter amino acid code is used. Sequence identity or conservative replacements between CAG1 and SCG1 are in boldface; identities or conservative replacements shared by Goas are shown in boldface in that sequence. Conservative amino acids are grouped as follows: A,G,P,S,T; D,E,N,Q; F,W,Y; H,K,R; and I,L,M,V. Stretches of amino acids (Gl to G5) presumably involved in GTP binding and hydrolysis are overlined, and the corresponding consensus sequences are given below, with X indicating any amino acid and 0 and J representing hydrophobic and hydrophilic amino acids, respectively (5). Sequences which are otherwise variable but conserved between CAG1 and SCG1 are boxed.

acids but is located in a different region of the protein, very near the amino terminus (37). The overall similarity between the deduced amino acid sequences of the C. albicans CAGI and S. cerevisiae GPA2 genes is 57%, including the conservative replacements (37). A similar degree of homology was observed between CAG1 and the inferred amino acid sequence of the Schizosaccharomyces pombe Ga gene (40). Chromosomal location and copy number. A Southern blot of restriction enzyme-digested DNA from two C. albicans strains probed with the 2.2-kbp HpaI fragment containing CAGl is shown in Fig. 4A. The cloned fragment corresponds to a single EcoRI fragment of the expected size in WO-1 and the two fragments previously seen in 3153a. HindlIl, BglII, and XbaI digestion patterns are identical in the two strains, with the later two yielding a single band. This result strengthens our conclusion that CAGI is present in only one chromosomal location and thus would be represented twice in a

Downloaded from http://mcb.asm.org/ on June 28, 2016 by guest

AAA ATC AAA TTA GAT TGT GAT CAG CCT AAT AAT Lys Ile Lys Leu Asp Cys Asp Gln Pro Asn Asn

CAM1 83 VIWCDVIQSMKVLIIQARKLKIKLDCDQPNNSLIP- -YKQ SO31 83 VIRADAIQSMKILIIQARXLGIQLDCDDPINNKDLFACKR Gns 83 DIKNNLKEAIETIVAAMSNLVPPVELANPENQFRVDY ILS

VOL. 12, 1992

A WO-1 IB C E H Xl

B

3153a lB C E H

Xl

1