Geranylgeranyltransferase I of Candida albicans: Null ... · boxyl group of the prenyl-cysteine is carboxy-methylated (8). GGTase II makes up a second distinct class of PTases and

JOURNAL OF BACTERIOLOGY,0021-9193/00/$04.0010

Feb. 2000, p. 704–713 Vol. 182, No. 3

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Geranylgeranyltransferase I of Candida albicans: Null Mutantsor Enzyme Inhibitors Produce Unexpected PhenotypesROSEMARIE KELLY,1* DEBORAH CARD,1 ELIZABETH REGISTER,1 PAUL MAZUR,1

THERESA KELLY,1 KEN-ICHI TANAKA,2 JANET ONISHI,1 JOANNE M. WILLIAMSON,1

HONGXIA FAN,1 TOSHIHIKO SATOH,2 AND MYRA KURTZ1

Merck Research Laboratories, Merck and Co., Rahway, New Jersey 07065,1 and Tsukuba Research Institute,Banyu Pharmaceutical Co., Ltd., Tsukuba-City, Japan 300-26112

Received 14 June 1999/Accepted 2 November 1999

Geranylgeranyltransferase I (GGTase I) catalyzes the transfer of a prenyl group from geranylgeranyldiphosphate to the carboxy-terminal cysteine of proteins with a motif referred to as a CaaX box (C, cysteine;a, usually aliphatic amino acid; X, usually L). The a and b subunits of GGTase I from Saccharomyces cerevisiaeare encoded by RAM2 and CDC43, respectively, and each is essential for viability. We are evaluating GGTaseI as a potential target for antimycotic therapy of the related yeast, Candida albicans, which is the major humanpathogen for disseminated fungal infections. Recently we cloned CaCDC43, the C. albicans homolog of S.cerevisiae CDC43. To study its role in C. albicans, both alleles were sequentially disrupted in strain CAI4. NullCacdc43 mutants were viable despite the lack of detectable GGTase I activity but were morphologicallyabnormal. The subcellular distribution of two GGTase I substrates, Rho1p and Cdc42p, was shifted from themembranous fraction to the cytosolic fraction in the cdc43 mutants, and levels of these two proteins wereelevated compared to those in the parent strain. Two compounds that are potent GGTase I inhibitors in vitrobut that have poor antifungal activity, J-109,390 and L-269,289, caused similar changes in the distribution andquantity of the substrate. The lethality of an S. cerevisiae cdc43 mutant can be suppressed by simultaneousoverexpression of RHO1 and CDC42 on high-copy-number plasmids (Y. Ohya et al., Mol. Biol. Cell 4:1017,1991; C. A. Trueblood, Y. Ohya, and J. Rine, Mol. Cell. Biol. 13:4260, 1993). Prenylation presumably occurs byfarnesyltransferase (FTase). We hypothesize that Cdc42p and Rho1p of C. albicans can be prenylated by FTasewhen GGTase I is absent or limiting and that elevation of these two substrates enables them to compete withFTase substrates for prenylation and thus allows sustained growth.

Isoprenylation is a posttranslational modification that in-creases the hydrophobicity of proteins, enabling them to asso-ciate with membranes, and is sometimes required for function(36, 42). Geranylgeranyltransferase I (GGTase I) and farne-syltransferase (FTase) catalyze very similar reactions and com-pose one class of prenyltransferases (PTases). GGTase I uti-lizes the 20-carbon isoprenoid geranylgeranyl diphosphate(GGPP) as a substrate, while FTase utilizes the 15-carbonisoprenoid farnesyl diphosphate (FPP). As a result of the ac-tion of this class of enzymes, the isoprenoid units are covalentlyattached to proteins that end in the C-terminal sequence CaaX(C, cysteine; a, aliphatic amino acid [usually]; X, any aminoacid) via a thioether linkage to the cysteine residue of theCaaX motif. In general, the X residue of the CaaX sequencedetermines if the protein is a substrate for GGTase I or FTase(52). After prenylation, the three C-terminal amino acids ofthe CaaX motif are removed by proteolysis and the free car-boxyl group of the prenyl-cysteine is carboxy-methylated (8).GGTase II makes up a second distinct class of PTases andcatalyzes geranylgeranylation at both cysteines of C-terminalCC or CXC sequences of Rab proteins (12, 52).

GGTase I and FTase have been extensively characterizedbiochemically and genetically in the lower eukaryote Saccha-romyces cerevisiae (36, 42). Both enzymes are zinc-dependent,magnesium-dependent heterodimers comprising an a and a bsubunit. The a subunit is shared by GGTase I and FTase in

both yeast and mammals. RAM2 and CDC43 encode the a andb subunits of S. cerevisiae GGTase I, respectively, and each isessential for viability (2, 13, 15, 25, 34). The b subunit of FTaseis encoded by RAM1, and null mutants of this gene are tem-perature sensitive (15, 20). At low temperatures, GGTase Iprenylates the requisite FTase substrates and alleviates therequirement for FTase (46).

GGTase I prefers substrates in which X of the CaaX motifis leucine. Typically, GGTase I substrates are small GTP-bind-ing proteins involved in cell polarity, cytokinesis, and morpho-genesis (42). Four proteins with CaaL sequences are noted inthe S. cerevisiae Yeast Proteasome Database: Rho1p, Rho2p,Cdc42p, and Bud1p. Rho1p and Cdc42p are essential proteinsand are the most critical substrates of GGTase I in S. cerevisiae,since the lethality of a cdc43 mutant can be suppressed bysimultaneous artificial overexpression of both proteins (35, 46).In this situation, FTase becomes essential and prenylates therequired CaaL-containing substrates. Cdc42p is involved inbud positioning and control of cell polarity in S. cerevisiae (2),while Rho1p is important for bud emergence, actin organiza-tion, and cell wall integrity (17, 18, 21, 32, 49). Rho1p of bothS. cerevisiae and Candida albicans has been shown to be theregulatory subunit of 1,3-b-D-glucan synthase, an essential en-zyme involved in cell wall biosynthesis (10, 22, 26, 39).

We are evaluating GGTase I in C. albicans as a potentialtarget for antimycotic therapy. C. albicans is the major oppor-tunistic human fungal pathogen and is the cause of serioussystemic disease in immunocompromised patients and of top-ical infections in healthy individuals (9). S. cerevisiae is fre-quently studied as a model organism for understanding funda-mental processes in C. albicans, a related diploid asexual

* Corresponding author. Mailing address: RY80Y-200, Merck andCo., P.O. Box 2000, Rahway, NJ 07065. Phone: (732) 594-6385. Fax:(732) 594-5468. E-mail: [email protected].

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dimorphic yeast. Rho1p and Cdc42p of C. albicans containCaaL motifs and are likely GGTase I substrates in vivo (22,28). Previously, we reported the purification of C. albicansGGTase I and the cloning and sequence analysis of its a and bsubunit genes (27). Our work showed that C. albicans GGTaseI is also a zinc-dependent, magnesium-dependent heterodimerwhose subunits demonstrated 30% amino acid identity withtheir human counterparts. This relatively low homology sug-gested the possibility of identifying fungus-specific GGTase Iinhibitors.

One important factor regarding the functional requirementfor C. albicans GGTase I in vivo is the prenyl acceptor sub-strate specificity of GGTase I and FTase. We previouslyshowed, using partially purified PTases, that C. albicansGGTase I demonstrated a strong preference for Ras-CaaXsubstrates in which X of the CaaX motif was a leucine, as wastrue for both the Saccharomyces and mammalian GGTase I(27). C. albicans FTase demonstrated strong activity with Ras-CVLM, as does S. cerevisiae FTase, and also farnesylated allRas-CaaL substrates tested at levels ranging from 2 to 20%relative to that observed with Ras-CVLM. Cross-farnesylationof CaaL-containing substrates had been observed before withmammalian and S. cerevisiae FTase (7, 46, 51). Although Sac-charomyces FTase can cross-farnesylate CaaL-containing sub-strates in vitro (7, 46) and has been shown to cross-prenylate invivo (46), GGTase I of S. cerevisiae is still required for viability.We noted that the magnitude of cross-farnesylation of CaaL-containing substrates observed with C. albicans FTase washigher than had been reported with Saccharomyces FTase (7).Therefore, the requirement for C. albicans GGTase I in vivowas investigated.

Here we report that viable strains with no detectableGGTase I activity were recovered upon sequential disruptionof each of the chromosomal homologs containing CaCDC43.We further demonstrate that levels of Cdc42p and Rho1p areelevated in the Cacdc43 mutants as well as in cells treated withGGTase I inhibitors. Our data suggest the existence of a com-pensatory mechanism that is evoked when GGTase I is absentor limiting.

MATERIALS AND METHODS

Strains, media, and culture conditions. The C. albicans strains used in thisstudy are listed in Table 1. Uracil-deficient synthetic medium (SD 2Ura) hasbeen described previously (44). 5-FOA medium contained 1 mg of 5-fluorooroticacid (5-FOA; Toronto Research Chemicals, Inc.)/ml as described by Boeke et al.(5) with 100 mg of uridine/ml in place of uracil. CMS medium contained 0.67%yeast nitrogen base, 0.5% yeast extract, 1.0% peptone, 0.1% glucose, and 0.8 Msorbitol. Cultures were routinely grown at 30°C. Growth comparisons of Ura1

prototrophs cultivated in SD 2Ura broth were made by determining the A600 at

various times. Morphology was assessed by microscopic examination of cellsfrom liquid cultures magnified either 200- or 500-fold with an Optiphot2 invertedmicroscope (Nikon) and photographed with a Nikon FX-35WA camera.

Nucleic acid isolation, hybridization, and sequence analysis. Plasmid DNAwas isolated with Qiagen-tip 500, Qiagen-tip 100, or QIAprep miniprep kits(Qiagen). Genomic DNA from C. albicans was isolated as described by Hoffmanand Winston (16) and further purified with GENECLEAN (BIO 101, Inc.)according to the manufacturer’s instructions. The additional purification step wasnecessary to obtain high-quality DNA from CaCDC43-disrupted strains. South-ern blots were performed with Zeta-ProbeGT-derivatized nylon membranes(Bio-Rad) under stringent hybridization and washing conditions. Hybridizationwas carried out at 65°C for 16 h in 0.5 M Na2HPO4 [pH 7.2]–7% sodium dodecylsulfate (SDS). The blots were washed at 65°C twice with 40 mM Na2HPO4 [pH7.2]–5% SDS and twice with 40 mM Na2HPO4–1% SDS for 30 min per wash.Total RNA was isolated with TRI Reagent (Molecular Research Center), andmRNA was isolated with a PolyATract mRNA kit (Promega) according to themanufacturer’s instructions. mRNA (2 mg) was subjected to electrophoresis on1% agarose formaldehyde gels (41) and transferred to Duralon membranes(Stratagene). The blots were hybridized with QuikHyb solution from Stratagenefollowing the manufacturer’s recommendations. DNA probes for both the South-ern and Northern blots were radiolabeled with [a-32P]dCTP with a PrimeItIIrandom-primed DNA labeling kit (Stratagene). DNA sequence was determinedon a model 377A automated DNA sequencer with a Prism Ready Reactiondyedeoxy terminator cycle-sequencing kit (Applied Biosystems). Sequences wereanalyzed with the Genetics Computer Group software package. Homologysearches were performed with the BLAST network service (3).

Cloning of CDC42 and RHO1 genes from C. albicans. CDC42 and RHO1 wereindependently cloned by amplification with degenerate oligonucleotides prior tothe publications of Mirbod et al. (28) and Kondoh et al. (22), which also describethe cloning of these genes. Genomic fragments with the full coding sequencewere confirmed by DNA sequence analysis. A 2.4-kb XbaI fragment of CandidaDNA containing CDC42 from ATCC 90028 was cloned into pUC19 (50) toconstruct pMK2. A 617-bp fragment containing RHO1 from strain CA124 (43)was cloned into pCR2 (Invitrogen) to construct pTKRBE.

Gene disruption of CaCDC43. A plasmid containing a deletion of CaCDC43,pCDC40d, was made by replacing an 875-bp BglII-EcoRV fragment frompCaCDC43-40 (27) with a 4.2-kb BglII-PvuII fragment from pMB7 containingthe Ura blaster sequence. The construct was digested with XhoI and XmnI,liberating an ;5.2-kb fragment containing the Ura blaster region with 440 and560 bp of flanking Candida DNA. CAI4 was transformed to Ura1 with thedisrupted DNA according to a lithium acetate protocol described for S. cerevisiaeby Elble (11), and transformants were selected on SD 2Ura medium. Counters-election of the URA3 gene was conducted on 5-FOA-containing medium asdescribed by Fonzi and Irwin (14). A second disruption construct, pCDC43d3,was made by ligating the 1.3-kb XbaI-ScaI URA3 fragment from pJAM11 (45)into the BglII site of pCaCDC43-40. Plasmid pCDC43d3 was linearized withXmnI and XhoI, which released a 3.1-kb fragment containing the URA3 geneflanked by 0.44 and 1.4 kb of Candida DNA and was used to transform CDC43-disrupted heterozygotes to Ura1.

Drug treatment of C. albicans. C. albicans MY1055 was grown overnight inCMS broth, diluted to an A600 of 0.4, and grown to an A600 of 0.6. Compoundsdissolved in 100% dimethyl sulfoxide were added to the cultures, and controlcells were treated with 0.1% dimethyl sulfoxide. After growth for an additional2 h, the cultures were harvested and the cell pellet was resuspended in extractionbuffer composed of 50 mM HEPES [pH 7.5], 0.1 mM MgCl2, 0.1 mM EGTA, 5mM b-mercaptoethanol, and 13 Complete protease inhibitor cocktail (Boehr-inger Mannheim). Cell extracts were prepared and fractionated into P100 andS100 fractions as described in the next section.

Protein extract preparation, fractionation, and partial purification. Cell ex-tracts of Cacdc43 mutants and Ura1 CDC43-disrupted heterozygotes were pre-

TABLE 1. C. albicans strains used in this study

Strain Parental strain Relevant genotype Reference

CAI4 SC5314 CaCDC43a/CaCDC43b 145 CAI4 Cacdc43aD::hisG-URA3-hisG/CaCDC43b This study5a 5 Cacdc43aD::hisG/CaCDC43b This study5a-1 5a Cacdc43aD::hisG/Cacdc43b::URA3 This study5a-5 5a Cacdc43aD::hisG/Cacdc43b::URA3 This study8 CAI4 CaCDC43a/Cacdc43bD::hisG-URA3-hisG This study8a 8 CaCDC43a/Cacdc43bD::hisG This study8a-1 8a Cacdc43a::URA3/Cacdc43bD::hisG This study8a-2 8a Cacdc43a::URA3/Cacdc43bD::hisG This studyCa-L1 CAI4 CaCDC43a/CaCDC43b 1 pJAM11(URA3)a 45MY1055 Clinical isolate CaCDC43/CaCDC43 1ATCC 90028 Clinical isolate CaCDC42/CaCDC42 31

a Integrated at URA3 locus.

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pared from cultures grown overnight in SD 2Ura broth and from CAI4 and the5-FOA-resistant Ura2 heterozygotes grown with uridine (100 mg/ml) added tothe medium. The following day, the cells were diluted to an A600 of 0.4 andallowed to double before being harvested. An equal volume of chilled lysis buffercontaining 50 mM bis-tris propane–HCl [pH 7.0], 1 mM b-mercaptoethanol, 13Complete protease inhibitor cocktail, and 1 mM Pefabloc protease inhibitor(Boehringer Mannheim) was added to the cell pellet (;1 g [wet weight]). Thecell slurry was homogenized in a Mini-Bead-Beater (Biospec) with 0.5-mm-diameter glass beads eight times for 30 s each time, with intermittent cooling onice. The crude extract was collected and centrifuged at 100,000 3 g at 4°C for 1 hto produce pellet (P100) and supernatant (S100) fractions. The pellet was re-suspended to a volume equal to the supernatant fraction (;1 ml). GGTase I andFTase were partially purified from S100 fractions (from 200 ml of cell culture) bychromatography on a 1-ml HiTrap Q-Sepharose column (Pharmacia). The S100fraction (0.7 to 0.8 ml; 6.5 to 10.5 mg/ml) was loaded onto the column equili-brated in 20 mM bis-tris propane–HCl [pH 7.0], 0.02 mM ZnCl2, and 1 mMMgCl2 (buffer A). The column was washed in buffer A and eluted with a 20-mlgradient of NaCl (0 to 0.75 M) in buffer A at a flow rate of 1 ml/min, andfractions (0.5 ml) were collected. The protein concentration was determined bythe method of Bradford (6) with Coomassie Plus protein assay reagent (Pierce)with bovine serum albumin as a standard.

Protein PTase assays. Protein PTase activities were determined by an acidquench-filtration assay as previously described (27). The conditions for theGGTase I assay were 50 mM HEPES [pH 7.5], 1 mM dithiothreitol (DTT), 10mM MgCl2, 0.1 mM ZnCl2, 0.05% dodecyl maltoside, 20 mM Ras-CVIL (or 10mM CaCdc42p), and 4 mM 3H-GGPP (or 4 mM 3H-FPP as described in thelegend to Fig. 4). FTase assays were performed with 50 mM HEPES [pH 7.5], 1mM DTT, 1 mM MgCl2, 0.05 mM ZnCl2, 0.05% dodecyl maltoside, 20 mMRas-CVLM, and 4 mM 3H-FPP. All reactions were performed in duplicate andwere initiated by the addition of PTase activity (2.5 to 5 ml) to a final volume of25 ml and incubated at 30°C for 30 to 60 min. All other details of the assay andthe origin of the Ras substrates were described previously (27).

Expression and purification of C. albicans Cdc42p. Histidine6-CaCdc42p fu-sion protein was expressed from plasmid pMK11 (T. Satoh, unpublished data)and purified as follows. A 1-liter culture of E. coli BL21(DE3)(pMK11) in Luriabroth containing 100 mg of ampicillin/ml was grown at 37°C to an A600 of 0.8. Theculture temperature was reduced to 18°C, IPTG (isopropyl-b-D-thiogalactopyr-anoside) was added to a final concentration of 1 mM, and the cells were har-vested following a 21-h incubation at 18°C. The cell paste (5.4 g [wet weight]) wasresuspended in 40 ml of 50 mM Na-phosphate, 20 mM Tris-HCl [pH 8], 200 mMNaCl, 50 mM GTP (lysis buffer) containing 1 mM Pefabloc protease inhibitor,and 1 mM benzamidine (Sigma). All subsequent steps were performed at 4°Cunless otherwise indicated. The cells were lysed by sequential treatment withlysozyme (0.75 mg/ml; 20 min at 25°C), sonication, and DNase (2.5 mg/ml; 20min). The crude lysate was centrifuged (15,000 3 g; 15 min), and the supernatantsolution was applied in batch format (20 min with rocking) to Talon metal affinityresin (4 ml; Clontech) equilibrated in lysis buffer. The resin was batch washedtwice with lysis buffer (40 ml), transferred to a column, washed with lysis buffer(40 ml) and eluted with lysis buffer containing 100 mM imidazole at a flow rateof 2 ml/min. The Talon eluate was pooled (35 ml) and dialyzed overnight against3 liters of 20 mM Tris-HCl [pH 8.5]–50 mM GTP. The dialyzed sample contained37.5 mg of protein estimated as .90% CaCdc42 fusion protein by SDS-poly-acrylamide gel electrophoresis. The histidine tag was cleaved by digestion over-night with thrombin (Jones Medical Industries) at 16°C (2.5 U of thrombin/mg offusion protein) and removed by passage over Talon resin. The cleaved CaCdc42pwas concentrated on a Centriprep-10 (Amicon) and purified to homogeneity bygel filtration on a Superdex-200 HR 10/30 column (Pharmacia) equilibrated indialysis buffer containing 100 mM NaCl and 1 mM DTT. The integrity of theCaCdc42p samples was verified by mass spectroscopic analysis.

Preparation of antisera and Western blot analysis. Preparation of anti-Rho1psera will be described elsewhere (T. Satoh, unpublished data). Anti-C. albicansCdc42p polyclonal antiserum was generated in four rabbits (Covance ResearchProducts) with purified recombinant CaCdc42p as the antigen. The antiserumdetected recombinant CaCdc42p and a protein of similar size, ;21 kDa, inmicrosomes of C. albicans by Western blotting. A faint band migrating slightlymore slowly than Cdc42p was detected and may represent a different, modifiedform of the protein as suggested by Ziman et al. (53), who have found thatantiserum raised against S. cerevisiae Cdc42p recognizes multiple proteins similarin size. Polyclonal antiserum against S. cerevisiae plasma membrane H1 ATPase,Pma1p, was produced in rabbits by using native Pma1p purified from S. cerevisiaemicrosomes as the antigen (the identity of the purified Pma1p was verified byamino acid sequencing) (P. Mazur and W. Baginsky, unpublished data). InWestern blots of C. albicans and S. cerevisiae microsomes, the anti-Pma1p serumcross-reacted with a protein band of similar size (ca. 100 kDa). Protein samples(2.5 to 25 mg) were suspended in Laemmli sample buffer, boiled for 5 min, andseparated by electrophoresis on either 12 or 15% precast mini-SDS-polyacryl-amide gels (Bio-Rad) followed by transfer to a nitrocellulose membrane. Anti-Rho1p antibody was reacted at a 1:1,000 dilution followed by horseradish per-oxidase-conjugated anti-mouse immunoglobulin G secondary antibody at a1:5,000 dilution. Anti-Cdc42p antibody was used at a dilution of 1:1,000, andanti-Pma1p antibody was used at a 1:10,000 dilution, followed by horseradishperoxidase-conjugated anti-rabbit immunoglobulin G secondary antibody at a

dilution of 1:16,000. The antigen-antibody complexes were detected with an ECLchemiluminescence detection system (Amersham) under conditions recom-mended by the manufacturer.

RESULTS

CaCDC43 is not essential for growth in C. albicans CAI4. Tostudy the role of Cdc43p in C. albicans, both copies of theCaCDC43 gene were disrupted. A plasmid containing a dele-tion of CaCDC43 was constructed, and a linear fragment wasused to transform CAI4 to Ura prototrophy as described inMaterials and Methods. This Ura blaster strategy (14) shouldresult in deletion of ;60% of the coding sequence ofCaCDC43 on one chromosomal homolog. To confirm that agene replacement occurred, transformants were analyzed bySouthern blot hybridization. The CaCDC43 locus of CAI4 hasan allelic HindIII polymorphism which can be used to distin-guish which of the two chromosomal homologs is disrupted.For our purposes, the 2.7-kb HindIII-HindIII fragment pro-duced by digestion of genomic DNA from strain CAI4 definesthe CaCDC43a allele and the 13-kb HindIII-HindIII fragmentdefines the CaCDC43b allele (Fig. 1 and 2). If CaCDC43a isdisrupted, the 2.7-kb HindIII fragment will be replaced bynovel 5.0- and 1.1-kb fragments, and if the other allele isdisrupted, the 13-kb HindIII fragment will be replaced by frag-ments of 5.0 and 11.4 kb. Transformants in which either of thetwo alleles is disrupted are diagrammed in Fig. 1, and Southernblotting results are shown in Fig. 2. Transformants 5 and 8 havethe a and b alleles disrupted, respectively. As expected, the5.0-kb fragment in each of the heterozygotes also hybridized toa URA3 probe (data not presented). The appropriate excisionof the URA3 gene via recombination between the hisG repeatsof the Ura blaster sequence was confirmed by the reduction ofthe 5.0-kb fragment to 2.0-kb in each of the heterozygotes,as exemplified by transformants 5a and 8a. To inactivate an-other allele of CaCDC43, another disruption construct wasmade that contained an insertional inactivation of CaCDC43with no hisG sequence (described in Materials and Methods).If disruption of CaCDC43 was a lethal event, we expectedstrong selection for targeted integration to occur in the chro-mosome that was already disrupted; therefore, a construct thathad no sequence bias towards integration at this locus waspreferable. Integration of the disrupted plasmid DNA into theundisrupted allele of strain 5a would result in the replacementof the 13.0-kb HindIII fragment with a 14.3-kb fragment, andthe 2.0- and 1.1-kb fragments indicative of disruption ofCaCDC43 would remain, as exemplified by transformants 5a-1and 5a-5. A double disruption of strain 8a would contain a4.0-kb fragment instead of the 2.7-kb fragment; 2.0- and11.4-kb fragments indicative of the disruption of CaCDC43bwould remain, as shown by transformants 8a-1 and 8a-2. Allseven transformants of 8a analyzed and 8 of 11 transformantsof 5a had a hybridization pattern consistent with disruption ofCaCDC43 on both chromosomal homologs.

The growth in liquid culture of null mutants 5a-1 and 8a-1was compared to that of heterozygotes 5 and 8 and a Ura1

transformant of CAI4, Ca-L1. No substantial differences werefound until late log phase, when the growth of the null mutantswas reduced to ;70% of that of the CDC43 heterozygote andthe wild-type parent strain (data not shown). The morphologyof the null mutant cells differed from that of the parent strainsseveral hours before the onset of late-log-phase growth; themutant cells were rounder and swollen, were substantiallyclumped, and had significantly fewer buds than the heterozy-gote or parent strain, as shown in Fig. 3.

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Cacdc43 null mutants are devoid of GGTase I activity. Toconfirm that GGTase I activity had been abolished in the cdc43mutants, cell extracts were prepared from wild-type, CDC43-disrupted heterozygous strains, and the cdc43 null mutants. Asshown in Fig. 4, with GGPP and Cdc42p as the prenyl donorand acceptor, respectively, the specific activity of geranylgera-nylation from heterozygotes 5 and 5a was approximately 50%of the activity observed for wild-type CAI4. Null cdc43 mutants5a-1 and 5a-5 had essentially no GGTase I activity. The specificactivity of farnesylation of Cdc42p was not increased in thesestrains, as indicated in Fig. 4. Additional null cdc43 mutants,8a-1 and 8a-2, were also devoid of GGTase I activity (data notpresented). GGTase I activity was measured in the samestrains with Ras-CVIL as a prenyl acceptor. The specific ac-tivity of geranylgeranylation in the heterozygotes was againapproximately half of the value obtained for the wild type. Thenull cdc43 mutants, 5a-1, 5a-5, 8a-1, and 8a-2, contained ap-proximately 3% of the activity of wild-type cells.

We suspected that the residual activity remaining in thecdc43 mutants when assayed with Ras-CVIL was due to gera-nylgeranylation by FTase, since we have previously observed avery low level of C. albicans FTase-catalyzed geranylgeranyla-

FIG. 1. Diagram illustrating construction of Cacdc43 null mutants by sequential gene disruption, starting with strain CAI4 (not drawn to scale). The plasmids aredescribed in Materials and Methods. Relevant HindIII fragments in all of the strains are noted. H, HindIII; B, BglII; E, EcoRV.

FIG. 2. Autoradiogram of Southern blot hybridization of genomic DNAfrom C. albicans cdc43 null mutants and parent strains (illustrated in Fig. 1 anddescribed in Table 1). The probe was a radiolabeled 2.7-kb CaCDC43 HindIIIfragment.


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tion of this acceptor (27). To confirm this point, GGTase I andFTase activities were partially resolved by anion-exchangechromatography, and column fractions were assayed for PTaseactivity with Ras-CVIL and GGPP and with Ras-CVLM andFPP. As shown in Fig. 5A, a gene dosage-dependent loss ofGGTase I activity was evident and a small peak of residualgeranylgeranylation activity was observed in the cdc43 mutant5a-1, which is shifted two fractions relative to peak GGTase Iactivity in the wild type and the CDC43-disrupted heterozy-gote. The resolution of GGTase I and FTase in each of thethree strains is shown in Fig. 5B, from which is appears that thepeak of residual GGTase I activity is coincident with the FTaseactivity.

Distribution of GGTase I substrates in Cacdc43 mutants.Since prenylation affects protein hydrophobicity, we expectedthe subcellular localization of GGTase I substrates to changein the cdc43 mutants. It had previously been shown for S.cerevisiae ts cdc43 mutants that soluble levels of either Cdc42por Rho1p were elevated at the restrictive temperature, de-pending upon the specific ts allele (33, 54). We fractionatedextracts into particulate and soluble fractions and determinedthe subcellular distribution of Cdc42p and Rho1p by Westernblot analysis. Both Rho1p and Cdc42p localized predominantly

to the membrane or pellet fraction in wild-type CAI4 and theCDC43-disrupted heterozygotes, 5 and 5a (Fig. 6). In thecdc43 mutants 5a-1 and 5a-5, the distribution of Rho1p andCdc42p was shifted, with the bulk of these proteins localizingto the cytosolic fraction. We also noted what appeared to be anincrease in the total amount of Cdc42p and Rho1p in the cdc43mutants. The protein migrating slightly faster than Rho1p instrain 5a-1 was found occasionally in the null mutants only andis likely to be a degradation product of Rho1p. The distribu-tion of Rho1p and Cdc42p was also shifted from the membra-nous fraction to the cytosolic fraction in Cacdc43 null mutants8a-1 and 8a-2, and levels of these proteins were elevated (datanot presented). No changes were observed in the distributionor levels of an integral membrane protein that is not a GGTaseI substrate, Pma1p, the plasma membrane H1 ATPase, asshown in Fig. 6.

Northern blot analysis was performed on the cdc43 nullmutants to assess whether the increased levels of Rho1p andCdc42p could be attributed to an increase in the levels of theirrespective transcripts. No differences were found between theRHO1 or CDC42 mRNA levels in the wild type, the CDC43-disrupted heterozygote, and the cdc43 null mutants, as shownin Fig. 7.

FIG. 3. Morphologies in mid-log phase of C. albicans Cacdc43 null mutants compared to those of parent strains at high and low magnifications. (A and D) Wild-typeCa-L1; (B and E) CDC43-disrupted heterozygote 5 (C and F) Cacdc43 null mutant 5a-1.


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GGTase I inhibitors have similar effects on cellular locationof substrate proteins. J-109,390 is a potent inhibitor of C.albicans GGTase I with a 50% inhibitory concentration (IC50)of 120 nM, but it demonstrates no antifungal activity againstwild-type C. albicans when tested at concentrations as high as

300 mM (T. Satoh, unpublished data). We treated wild-typeMY1055 with J-109,390 and analyzed Rho1p and Cdc42p inthe particulate and soluble fractions by Western blot analysis.As shown in Fig. 8A, the distribution of Rho1p changed, withmore Rho1p found in the soluble fraction than in the partic-ulate fraction in cells treated with 5.0 and 25 mM J-109,390. Itis also evident that the total amount of Rho1p increased.Cdc42p was elevated in both the supernatant and pellet frac-tions. J-109,390 did not affect the distribution or levels of thecontrol protein, Pma1p. Results obtained with another potentGGTase I inhibitor, L-269,289, which has an IC50 of 100 nMagainst C. albicans GGTase I (J. Williamson, unpublisheddata), are shown in Fig. 8B. Treatment with L-269,289 resultedin a moderate increase in Rho1p and a shift in distribution tothe soluble fraction at the highest concentration tested.Cdc42p began to appear in the soluble fraction at 10 mML-269,289. No growth inhibition was obtained with 50 mML-269,289 under the conditions of this experiment.

3-Hydroxy-3-methyl-glutaryl–coenzyme A (HMG-CoA) syn-thase catalyzes an early step in the formation of mevalonate,which in turn is a precursor in the biosynthesis of GGPP.Inhibition of HMG-CoA synthase should reduce the pool ofGGPP and consequently reduce the level of prenylation byGGTase I. The results obtained from treatment with HMG-CoA synthase inhibitor, L-659,699 (C. albicans IC50, 30 nM)(37), are shown in Fig. 8C. Treatment with L-659,699 caused ashift in the distribution of Rho1p and an increase in the pro-tein. We also observed an increase in the total amount ofCdc42p at all concentrations tested and an increase in solubleCdc42p. No effect on growth was found under the conditions ofthis experiment.

We also tested several control compounds with differentmodes of action. Each compound was titrated with a range of

FIG. 4. PTase specific activity in Cacdc43 null mutants and parent strains.PTase specific activities were determined on S100 fractions as described inMaterials and Methods and assayed with Cdc42p substrate (10 mM) as the prenylacceptor and either GGPP (solid bars) or FPP (hatched bars) at 4 mM as a prenyldonor. The data are averages of duplicate determinations, and the error barsindicate the differences between replicates.

FIG. 5. Residual geranylgeranylation activity in Cacdc43 null mutants is due to FTase. GGTase I and FTase activities were partially resolved by chromatographyof S100 fractions as described in Materials and Methods. The samples loaded were CAI4, 5.4 mg; 5a, 7.8 mg; and 5a-1, 7.4 mg. Individual column fractions were assayedfor GGTase I and FTase activities with Ras-CVIL (20 mM) and GGPP (4 mM) or Ras-CVLM (20 mM) and FPP (4 mM), respectively. (A) GGTase I activity in CAI4(open triangles), 5a (solid triangles), and 5a-1 (diamonds). (B) Comparison of GGTase I (circles) and FTase (squares) profiles in CAI4, 5a, and 5a-1.


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concentrations, starting with a level that did not inhibit growthand including at least one concentration that was growth in-hibitory. The results obtained with anisomycin, a protein syn-thesis inhibitor, are shown in Fig. 8D. Anisomycin did notaffect the distribution or levels of either Rho1p or Cdc42p atany of the concentrations tested. Similar results were obtainedwith cerulenin, brefeldin A, and nikkomycin Z (data not pre-sented).

Some variability in the amounts of Rho1p and Cdc42p de-tectable in the supernatant and pellet fractions was noted fromexperiment to experiment. However, the patterns we obtainedwith each drug and each antibody were reproducible. Othershave also found that polyclonal antibodies raised against pre-nylated GTP-binding proteins yield variable results (38).

DISCUSSIONWe disrupted the C. albicans homolog of an essential S.

cerevisiae gene, CDC43, and found that it is not necessary forviability of the pathogen. C. albicans strains with no GGTase Iactivity were isolated upon sequential disruption of each ofthe alleles of CaCDC43. The possibility that the CaCDC43locus of CAI4 was aneuploid was ruled out at the onset byusing a HindIII restriction site polymorphism to distinguishwhich allele was disrupted. A similar allelic restriction sitepolymorphism was instrumental in showing that the initial at-tempts to disrupt the C. albicans URA3 gene were performedin a strain tetraploid at the URA3 locus (19). CaCDC43 is notthe first example of a dispensable C. albicans gene with acorresponding essential Saccharomyces homolog. SLN1, en-coding a required histidine kinase osmosensor in S. cerevisiae,was recently disrupted in C. albicans, and null mutants areviable (30).

The viability of the Cacdc43 mutants is consistent with ourprevious in vitro data demonstrating cross-prenylation ofGGTase I substrates by C. albicans FTase and the poor anti-Candida activity of potent GGTase I inhibitors known to pen-etrate the cell. The growth rate of the Cacdc43 mutants iscomparable to that of the parent strains until late log phase,and then it differs only marginally. Despite this, the null mu-tants do show a phenotype. The mutant cells are rounder andlarger, clump together, and have substantially fewer buds com-pared to the parent strain. This phenotype is reminiscent of S.cerevisiae ts cdc43 mutants (2) and C. albicans treated withaculeacin A, which inhibits cell wall biosynthesis (48). Our

results suggest that a cell wall defect is tolerated to some extentin C. albicans. We provide evidence to suggest a compensatorymechanism that allows for sustained growth of the Cacdc43mutants and wild-type C. albicans treated with GGTase I in-hibitors.

The absence of GGTase I activity in the Cacdc43 mutants invitro correlates with the effects on prenylation in vivo, as seenby the pronounced shift in distribution from the particulate tothe soluble fraction of two GGTase I substrates, Rho1p andCdc42p. This phenotype is also observed in S. cerevisiae tscdc43 mutants grown at the nonpermissive temperature andresults from the lack of the hydrophobic prenyl group (33, 54).Earlier experiments had shown that if the cysteine of the CaaXbox of S. cerevisiae Cdc42p was mutated to serine, precludingprenylation, Cdc42p was found almost exclusively in the solu-ble fraction (53). Direct characterization of prenyl group for-mation in vivo in S. cerevisiae is difficult, since it does not takeup exogenous mevalonate or other precursors of the prenylgroup well enough for radiolabeling studies to be performed.We have also found that mevalonate is incorporated poorly inC. albicans (data not presented).

Our data suggest that the inhibition or absence of C. albicansGGTase I results in a compensatory mechanism based on thenet accumulation of at least two of its substrates, Cdc42p andRho1p. This endogenous compensation is similar to the cor-rection of the lethal growth defect of an S. cerevisiae cdc43 nullmutant by simultaneous artificial overexpression of bothCDC42 and RHO1 (35, 46). Levels of Cdc42p and Rho1p wereelevated in the Cacdc43 mutants and in wild-type C. albicanstreated with potent GGTase I inhibitors. The GGTase I inhib-itors J-109,390 and L-269,289 resulted in a significant increasein the Rho1p detected, as did treatment with L-659,699, anHMG-CoA synthase inhibitor which is expected to reduce thecellular pool of GGPP and secondarily to affect GGTase I.Treatment with J-109,390 and L-659,699 also led to an eleva-tion of Cdc42p, detected by Western blot analysis. Accumula-tion of these GGTase I substrates in C. albicans is likely due toa posttranscriptional mechanism, as we observed no apparentincreases in mRNA levels of RHO1 or CDC42 in the Cacdc43mutants. Although the Western analysis was not quantitative,we estimate that the amount of Rho1p and Cdc42p is morethan twofold greater in the Cacdc43 null mutants than in thewild type. We would have been able to detect an increase as

FIG. 6. Western blot analysis of subcellular distribution of Rho1p andCdc42p in Cacdc43 null mutants and parent strains. Extracts were fractionatedinto P100 (microsomal; P) and S100 (cytosolic; S) fractions as described inMaterials and Methods. To assess the relative amount of protein in each fraction,equal volumes of the supernatant and particulate fractions of each strain wereloaded on an SDS–15% polyacrylamide gel for detection of Rho1p and Cdc42pand on an SDS–12% polyacrylamide gel for detection of Pma1p. Equal amountsof microsomal protein (2.5 mg for Pma1p detection and 25 mg otherwise) of eachstrain were loaded. The gels were transferred to a nitrocellulose membrane andanalyzed with anti-Cdc42p, anti-Rho1p, and anti-Pma1p antibodies as describedin Materials and Methods.

FIG. 7. Autoradiogram of Northern blot hybridization of C. albicans CDC42and RHO1 to mRNA from Cacdc43 null mutants and parent strains. A PMA1probe was used as a control for RNA loading and transfer. The gel blot washybridized first to the RHO1 and PMA1 probes and was subsequently strippedand rehybridized to the CDC42 probe. The probes were a 670-bp SpeI-XbaIfragment containing CDC42 isolated from pMK2, a 610-bp EcoRI-BglII RHO1fragment from pTKRBE, and a 2.0-kb EcoRV PMA1 fragment isolated from p37(29).


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small as 1.5- to 2-fold in the levels of the corresponding mRNAtranscripts. We have described the changes in levels of Rho1pand Cdc42p in relative terms, as we cannot precisely quantitateRho1p and Cdc42p by using the data from the Western blots.Immunoprecipitation of Cdc42p and Rho1p should allow exactquantitation of these GGTase I substrates, but it may not bepossible with existing antibodies. We have recently learnedthat more consistent immunoprecipitations are obtained if pre-nylated GTP-binding proteins are epitope tagged and immu-noprecipitated with antiserum raised against the epitope (38).

Saccharomyces cdc43 mutants in which Rho1p and Cdc42pare overexpressed grow slowly, but their growth is improved ifthe CaaX boxes of the overproduced proteins are altered tothat of an FTase substrate (35). In the absence of Saccharo-myces GGTase I, FTase becomes essential, and in fact, growthof the strains is enhanced when FTase is elevated, suggestingthat FTase prenylates the required GGTase I substrates (46).Similarly, we propose that in response to limiting GGTase I inC. albicans, elevated levels of Rho1p and Cdc42p enable theseproteins to compete with FTase substrates for prenylation byFTase, resulting in sustained growth. This hypothesis explainsboth the viability of the Cacdc43 mutants and the poor anti-fungal activity of potent, but specific, GGTase I inhibitors thatwe know are capable of penetrating the cell (J. Onishi, unpub-lished data; T. Satoh, unpublished data). Our hypothesis isapplicable to more than one C. albicans strain, as the compen-satory mechanism was observed in strains derived from CAI4and in MY1055, a clinical isolate that we routinely use inmouse models for pathogenesis.

Consistent with the above hypothesis, the GGTase I inhib-itor J-109,390 (C. albicans GGTase I IC50, 120 nM; MIC, .300mM) is at least 2,500-fold less potent against C. albicans FTase(T. Satoh, unpublished data). Two lines of evidence indicatedthat the failure of J-109,390 to inhibit growth was not due to aninability to penetrate the cell. Previously it was shown thatJ-109,390 inhibited b-1,3 glucan synthase activity in vivo andshifted the distribution of Rho1p from the membranes to thecytosol (T. Satoh, unpublished data). We confirmed thatJ-109,390 caused an increase in soluble Rho1p and additionallycaused an elevation of this protein. Interestingly, although we

detected an increase in Cdc42p upon treatment with J-109,390,we did not see a shift in the distribution of Cdc42p from theparticulate to the soluble fraction. It is possible that someinhibitors, such as J-109,390, may preferentially prevent pre-nylation of a subset of substrates. Similar substrate discrimi-nation has been reported by Ohya et al., who observed thatsome ts mutations in CDC43 of S. cerevisiae GGTase I led to anincrease in soluble Cdc42p whereas another resulted in anincrease in soluble Rho1p (33). The other GGTase I inhibitortested, L-269,289, affected the distribution of both substrates.

In agreement with our hypothesis that FTase farnesylatesrather than geranylgeranylates GGTase I substrates whenGGTase I is absent in C. albicans, the in vitro specific activityfor farnesylation of either substrate tested, Cdc42p (1.1 pmol/min/mg) or Ras-CVIL (21.4 pmol/min/mg), was 36- to 112-foldhigher than the specific activity of geranylgeranylation (0.03and 0.19 pmol/min/mg, respectively) in crude extracts preparedfrom the Cacdc43 null mutants 5a-1 and 5a-5 (Fig. 4 and datanot shown). Interestingly, in extracts from wild-type CAI4, thespecific activity of farnesylation of Ras-CVIL was threefoldhigher than the specific activity of geranylgeranylation (datanot shown). However, the determination of which prenyl groupis added in vivo in C. albicans when GGTase I is limiting maydepend upon the relative pools of FPP and GGPP in the cell atany given growth state. It is also conceivable that under normalconditions a small percentage of either Rho1p or Cdc42p isprenylated by FTase. Chemical analysis of the prenyl groupsformed in vivo will be necessary to address these issues, and itawaits development of the appropriate radiolabeling tech-niques.

Recent studies with mammalian farnesyltransferase inhibi-tors (FTIs) describe a similar phenomenon; proteins that arenormally FTase substrates may become geranylgeranylatedwhen FTase activity is limiting. Treatment of mammalian cellswith the FTI SCH 44342, SCH 56582, B956, or B957 led to thegeranylgeranylation of N-Ras, K-Ras, or both. These proteinsare normally farnesylated in vivo (40, 47). Similarly, FTIL-739,749 resulted in a decrease in farnesylated RhoB con-comitant with an increase in geranylgeranylated RhoB (23).

FIG. 8. Western blot analysis of subcellular distribution of Rho1p and Cdc42p in wild-type C. albicans treated with the indicated concentrations of drugs. Extractswere made and the samples were processed as described in the legend to Fig. 6. The results are representative of data from a minimum of two experiments for eachcondition. (A) J-109,390; (B) L-269,289; (C) L-659,699; (D) anisomycin.


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RhoB is unusual in that it is normally both farnesylated andgeranylgeranylated in vivo.

It is possible that FTase is upregulated in response to lim-iting GGTase I. In some cases a modest elevation (#1.5-fold)in Ras-CVLM farnesylation was observed for the Cacdc43mutants (Fig. 5 and data not shown). However, a similar in-crease in farnesylation of the alternate substrate Cdc42p (Fig.4) or Ras-CVIL (data not shown) was not detected, suggestingthat wild-type levels of FTase are sufficient in the Cacdc43mutant. Further studies will be needed to determine if thesynthesis or activity of FTase is upregulated in response tolimiting GGTase I.

Perhaps some or all of the Rho1p and Cdc42p remaining inthe pellet fraction when GGTase I is limiting is not prenylatedand becomes membrane associated by mass action. There isevidence in the literature that nonprenylated proteins retainsome function. Prenylation of mammalian RhoB is requiredfor cell transformation but not for its ability to activate serumresponse element-dependent transcription (24). In Schizosac-charomyces pombe, overexpression of a constitutively activemutant allele of Rho1p that is prenylated resulted in a fourfoldincrease in 1,3-b-D-glucan synthase activity which became GTPindependent (4). If the CaaX box of the constitutively ex-pressed Rho1p was mutated to preclude prenylation, glucansynthase activity was still enhanced but the increase was justtwofold, and only 50% of the activity was GTP dependent.

It seems unlikely that the viability of the C. albicans Cacdc43null mutants is due to prenylation of GGTase I substrates byGGTase II, since the substrate specificities of GGTase IIscharacterized to date differ significantly from that of GGTaseI (12, 52). To our knowledge, there are no examples of cross-prenylation of GGTase I substrates by GGTase II. However,we cannot rule out this possibility entirely, since GGTase IIfrom C. albicans has not yet been characterized. Alternatively,C. albicans could possess a second GGTase I not detectable byour current in vitro assay conditions. Southern blot hybridiza-tion under high-stringency conditions did not reveal aCaCDC43 homolog (E. Register and R. Kelly, unpublishedobservations). To prove conclusively that prenylation ofGGTase I substrates by FTase is responsible for the viability ofthe Cacdc43 mutants, it will be necessary to disrupt the bsubunit of FTase in the Cacdc43 mutants. If this double mutantis nonviable, as we expect, a dual GGTase I-FTase I inhibitorcan be considered for antimycotic therapy.

ACKNOWLEDGMENTS

We are grateful to John Rosamond for providing the CA124 C.albicans genomic DNA library. We thank Jennifer Nielsen, MariaMeinz, and Doug Johnson for helpful discussions. We are grateful toTracey Klatt for mass spectroscopic analysis and John Polishook forhelp with microscopy. We appreciate the critical reading of the manu-script by Cameron Douglas, Michael Justice, Suzanne Mandala, andJennifer Nielsen.

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Geranylgeranyltransferase I of Candida albicans: Null ... · boxyl group of the prenyl-cysteine is carboxy-methylated (8). GGTase II makes up a second distinct class of PTases and