INTRODUCTION

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The pathway by which proteins are transported between membrane-bound intracellular compartments and then out of the cell is highly conserved in all eukaryotes. This secretion (or vesicular transport) pathway has been studied extensively in Saccharomyces cerevisiae, and several dozen S. cerevisiae genes encoding its key components have been cloned and analyzed in some detail (14, 20, 22). The Candida albicans homologs of three essential S. cerevisiae secretion pathway genes have been described. C. albicans SEC18 (19), SEC14 (18), and SEC4 (5) all complemented the corresponding mutations in S. cerevisiae, and their deduced protein products were 50 to 67% identical to the corresponding proteins in S. cerevisiae. However, the functions of these genes have not yet been studied directly in C. albicans, except that efforts to disrupt both chromosomal alleles of C. albicans SEC14 yielded no viable null mutants (18).

In S. cerevisiae, SEC4 encodes a small Ras-like GTPase (Sec4p) that is required for fusion of post-Golgi secretory vesicles to the plasma membrane (23). Because SEC4 is essential in S. cerevisiae, sec4 null mutants are nonviable. However, temperature-sensitive (23) and dominant-negative (27) mutations in S. cerevisiae SEC4 inhibit growth and protein secretion and cause post-Golgi secretory vesicles to accumulate intracellularly. In the present study, we cloned the C. albicans SEC4 homolog by complementing a temperature-sensitive S. cerevisiae sec4 mutant, and we sought to ascertain this gene's functions. Gene disruption studies suggested that C. albicans SEC4 was essential, but they provided no specific information about this gene's functions. Therefore, we used site-directed mutagenesis to construct a mutant sec4 allele analogous to those encoding trans-dominant inhibitors of other ras-like GTPases, and we used this mutant sec4 allele to study the functions of C. albicans SEC4.

	MATERIALS AND METHODS

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Strains and media. S. cerevisiae NY28 (MAT ura3-52 sec4-8) (from P. Novick, Yale University) was cultured in YPD (1% yeast extract, 2% peptone, 2% glucose) or minimal glucose (0.67% yeast nitrogen base without amino acids [YNB], 2% glucose). Wild-type C. albicans SC5314 and its derivative CAI4 (ura3::imm434/ura3::imm434) (from W. Fonzi, Georgetown University) were cultured in YPD, minimal glucose, or minimal galactose (0.67% YNB, 2% galactose). Aspartyl protease secretion by C. albicans was examined in (i) 0.17% YNB without ammonium sulfate and with 0.4% bovine serum albumin (BSA) and either 2% glucose (glucose-BSA) or 2% galactose (galactose-BSA) or (ii) 0.34% YNB without ammonium sulfate and with 0.2% BSA, 0.2% yeast extract, and either 2% glucose (glucose-BSA-YE) or 2% galactose (galactose-BSA-YE). Uracil auxotrophs were selected on 5-fluoro-orotic acid (FOA) medium (minimal glucose, 0.1 mg of uridine per ml, and 0.7 mg of FOA per ml). Solid media were prepared by adding 2% agar.

Libraries and plasmids. A library of genomic DNA fragments from C. albicans WO-1 in the Escherichia coli-S. cerevisiae shuttle plasmid pEMBLYe23 was obtained from P. T. Magee (University of Minnesota). Plasmid p5921, which contains a hisG-URA3-hisG selectable marker (7), was obtained from W. Fonzi. Plasmid pGAL2.7 was constructed by (i) ligating the 2.7-kb XbaI restriction fragment containing C. albicans GAL1 from plasmid pCW31 (9) into the XbaI site in the yeast shuttle plasmid YEp352 and (ii) ligating the 2.7-kb SalI-BamHI restriction fragment from this plasmid into SalI- and BamHI-digested plasmid p1041 (10). The autonomously replicating C. albicans plasmid pVEC was obtained from P. T. Magee. pVEC was constructed by ligating the 3.6-kb NruI-SmaI fragment containing C. albicans ARS2 and URA3 from plasmid pRM1 (21) into the NdeI site in pUC18. pSK was from Stratagene (La Jolla, Calif.).

Isolation and properties of C. albicans SEC4. The plasmid library of C. albicans genomic DNA was introduced into S. cerevisiae NY28 by electroporation, and uracil prototrophs were selected on minimal glucose at 25°C, replica plated, and incubated at 37°C. Plasmids from thermotolerant S. cerevisiae transformants were expanded in E. coli and retested for the ability to complement the sec4-8 mutation in S. cerevisiae NY28. Standard methods were used for restriction mapping, subcloning, Southern hybridizations, DNA amplification by PCR, and DNA sequencing. Chromosomal mapping was performed by B. B. Magee (University of Minnesota), as previously described (4).

Targeted disruption of C. albicans SEC4. The method of Fonzi and Irwin (7) was used in an effort to disrupt both chromosomal alleles of C. albicans SEC4. The first step was to construct a gene disruption cassette in which the C. albicans SEC4 open reading frame (ORF) was deleted and replaced with the hisG-URA3-hisG selectable marker from plasmid p5921. To accomplish this, Taq DNA polymerase and primers Nok5SacI (5' GCC GCT GAG CTC TTG GAA CAT TCC TTA TGC AGG C 3') and CA11 (5' GAT GAA TTG TTA ATA CTT GAT ATG TTT TCC 3') were used (i) to amplify 692 bp of 5' untranslated C. albicans SEC4 DNA (nucleotides 173 to 864 upstream from the start codon) from p412a and (ii) to add a SacI restriction site (underlined). The PCR product was digested with SacI and BglII, and it was ligated into the SacI and BglII sites on the left side of the hisG-URA3-hisG selectable marker in p5921, which yielded pL1. Next, primers Nok3Bam (5' GAT GCG GGA TCC CCA TGA ACG AAG AAG AAG AAG AAG AG 3') and Nok3Sal (5' GGC GTG GTC GAC CAA CCA AAC CAG ATA GTG AGA ATT G 3') were used (i) to amplify 820 bp of 3' untranslated C. albicans SEC4 DNA (nucleotides 42 to 861 downstream from the stop codon) from p412a and (ii) to add BamHI and SalI restriction sites (underlined). This PCR product was digested with BamHI and SalI, and it was ligated into the BamHI and SalI sites on the right side of the hisG-URA3-hisG marker in pL1, which yielded pL1R2.

sec4

Construction of plasmids pS28N and pSEC4. To overexpress wild-type and mutant SEC4 alleles in C. albicans, it was first necessary to construct an autonomously replicating plasmid containing a regulable promoter. To accomplish this, we used Taq DNA polymerase, M13 sequencing primer, and primer GAL1 (5' CGG CGG GTC GAC GGA TCC ACT AGT TTA ATT AAG GTA TAA CTC TTT CTT ATA AAA ATC GG 3') (i) to amplify approximately 1.0 kb of the C. albicans GAL1 promoter from plasmid pGAL2.7 and (ii) to add a convenient BamHI restriction site (underlined). The PCR product was digested with XbaI and BamHI, and it was ligated into XbaI- and BamHI-digested pVEC, which yielded plasmid pYM1.

View larger version (38K): [in this window] [in a new window]   FIG. 1.   Plasmid pSEC4 was constructed by inserting into the autonomously replicating C. albicans plasmid pVEC (i) 1.0 kb of genomic DNA immediately 5' to the C. albicans GAL1 start codon and (ii) the ORF and transcription terminator from C. albicans SEC4. pS28N was identical to pSEC4 except that the sec4(S28N) allele replaced wild-type SEC4.

Effects of pS28N and pSEC4 in C. albicans. Plasmids pS28N and pSEC4 were introduced into C. albicans CAI4 by the lithium acetate method, and uracil prototrophs were selected on minimal glucose. pS28N-transformed C. albicans was cured of plasmids by culturing for 40 generations in YPD and by plating on FOA medium. Absence of pS28N after curing was verified by PCR, using primers BL3 (5' CCA ATG CTT AAT CAG TGA GGC AAC 3') and BL5 (5' AGT ATT CAA CAT TTC CGT GTC GCC 3') from the -lactamase gene. Plasmid copy numbers were estimated by Southern hybridization, using a 0.85-kb probe for -lactamase DNA that was generated with primers BL3 and BL5 and a 0.75-kb probe for C. albicans ADE2 DNA that was generated with PCR primers CaADE2Spe3 (5' TTG AGC ACT AGT CAT TTC AAC ACC GAA AAT ACC ACA C 3') and CaADE2Pac15 (5' TGG ATG GTT AAT TAA GAA GCA GCA CAT AGA TTG AAT ATC 3').

Nucleotide sequence accession number. The GenBank accession number for C. albicans SEC4 is AF015306.

	RESULTS

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Isolation and properties of C. albicans SEC4. When S. cerevisiae NY28 was transformed with the C. albicans genomic DNA library, plasmids isolated from 10 of 17 thermotolerant transformants contained similar 6.9-kb inserts. One of these plasmids (p412a) again conferred thermotolerance when it was reintroduced into S. cerevisiae NY28. A 1,269-bp portion of the insert from p412a was sequenced in both directions and contained a 630-bp intronless ORF flanked on its 5' end by four potential TATA elements and on its 3' end by an S. cerevisiae-type transcription termination sequence (30). This ORF encoded a deduced protein of 210 amino acids and 23 kDa that had 63% identity with S. cerevisiae Sec4p and 45% identity with human Rab3 protein (Rab3p). The deduced protein also contained (i) four consensus guanine nucleotide interaction domains, (ii) a membrane attachment sequence at its C terminus, and (iii) conserved amino acids in positions where single amino acid substitutions cause temperature-sensitive and dominant-negative mutations in S. cerevisiae SEC4 and other Ras-like GTPases (Fig. 2).

View larger version (24K): [in this window] [in a new window]   FIG. 2.   The deduced Sec4p's of C. albicans (Ca) and S. cerevisiae (Sc) were 61% identical (:). Conserved GTP-binding (*) and membrane attachment (+) domains are shown, as are the positions where a G147D substitution causes a temperature-sensitive mutation () and where S34N and N133I substitutions cause dominant-negative mutations () in S. cerevisiae Sec4p.

Targeted disruption of C. albicans SEC4. Genomic Southern analyses showed that the insert from p412a hybridized to a single-copy gene on SfiI fragment M of C. albicans chromosome R (data not shown). Therefore, we attempted to construct C. albicans sec4 null mutants by homologous gene targeting. The genotypes of 20 uracil prototrophs obtained when C. albicans CAI4 was transformed with the sec4::hisG-URA3-hisG gene disruption cassette were analyzed by allele-specific PCR and by Southern hybridization.

View larger version (39K): [in this window] [in a new window]   FIG. 3.   Targeted disruption of C. albicans SEC4. (A) Restriction maps of the C. albicans SEC4 locus (top) and the sec4::hisG-URA3-hisG gene disruption cassette (bottom). Primers used for allele-specific PCR and the probe used for Southern hybridizations are shown. Abbreviations: G, BglII; B, BamHI; N, NdeI; A, polymorphic ApaI site. (B) When C. albicans CAI4 was transformed with the linearized sec4::hisG-URA3-hisG gene disruption cassette, PCR with primers 23 and G1 generated a product only if the gene disruption cassette integrated homologously (YM5 and YM6, but not wild-type CAI4 or ectopic integrant YM7). Also, primers 5 and 10 generated a 1.2-kb PCR product from wild-type SEC4, no additional product from the large sec4::hisG-URA3-hisG cassette (YM5 and YM6), and a 1.5-kb product when recombination between flanking hisG repeats generated a smaller sec4::hisG allele (YM5F and YM6F). When YM5F or YM6F (SEC4/sec4::hisG) was transformed again with sec4::hisG-URA3-hisG DNA, homologous (YM5F1, YM5F2, YM5F3, YM6F6, YM6F7, and YM6F8) and ectopic (YM6F5) integrations were observed. However, 20 of 20 transformants analyzed retained a wild-type SEC4 allele. (C) When C. albicans CAI4 genomic DNA was digested with ApaI (A), NdeI (N), or both enzymes and hybridized with the probe shown in panel A, the presence of a polymorphic ApaI restriction site showed that the sec4::hisG-URA3-hisG cassette could replace either of the two SEC4 alleles in C. albicans CAI4 (compare YM6 to YM5). Since URA3 contains an NdeI site, the hybridized fragments from the sec4::hisG alleles in YM6F and YM5F (first knockout after FOA) were larger than those from the sec4::hisG-URA3-hisG alleles in YM5 and YM6. When the sec4::hisG-URA3-hisG cassette was reintroduced into YM6F, it replaced the sec4::hisG allele, but not the remaining wild-type SEC4 allele (see YM6F7). When the gene disruption cassette was reintroduced into YM5F, more complex integration events occurred (see YM5F2), but allele-specific PCR (see YM5F2 in panel B) showed that all transformants retained at least one wild-type SEC4 allele.

Effects of sec4(S28N) overexpression on growth and viability. Although the gene disruption studies summarized above suggested that C. albicans SEC4 was essential, they provided no specific information about this gene's functions. Therefore, we examined the effects on growth and viability of C. albicans transformants of GAL1-regulated expression plasmids encoding wild-type Sec4p (pSEC4) or a mutant Sec4p analogous to known trans-dominant inhibitors of several other Ras-like GTPases (1, 2, 8, 10, 21-23) (pS28N). Seven of seven pSEC4 transformants tested grew well (i.e., OD₆₀₀ of >15 at 24 h) in repressing (minimal glucose) and inducing (minimal galactose) media. Nine of nine pS28N transformants tested also grew well in minimal glucose, but six of these transformants grew slowly in minimal galactose.

View larger version (22K): [in this window] [in a new window]   FIG. 4.   Effects of sec4(S28N) overexpression on growth. Wild-type C. albicans (SC5314) and pSEC4-transformed C. albicans CAI4 (SEC4) grew well in minimal glucose (Glu) and minimal galactose (Gal). pS28N-transformed C. albicans CAI4 also grew well in minimal glucose (S28N/Glu), but these transformants grew very slowly when they were transferred to minimal galactose (S28N/Gal).

Effects of sec4(S28N) overexpression on protein secretion. We next examined the abilities of the pSEC4- and pS28N-transformed C. albicans strains to secrete Sap when they were incubated in glucose or galactose medium at high cell densities (OD₆₀₀ = 10) that permitted minimal cell growth. In one experiment, the cell-free supernatants of pS28N- and pSEC4-transformed C. albicans that had been incubated in glucose-BSA or galactose-BSA for 2 and 7 h contained large amounts of residual BSA and little or no immunoreactive Sap. After 20 h, however, the cell-free supernatants of the galactose-incubated pS28N transformants contained much more residual BSA and much less immunoreactive Sap than the supernatants of the glucose-incubated pS28N transformants or the glucose- or galactose-incubated pSEC4 transformants (Fig. 5). Since the final cell density of the galactose-incubated pS28N transformants was only slightly lower (OD₆₀₀ = 31) than that in the controls (OD₆₀₀ = 49 for glucose-incubated pSEC4 transformants, OD₆₀₀ = 57 for galactose-incubated pSEC4 transformants, and OD₆₀₀ = 46 for glucose-incubated pS28N transformants), the marked differences in Sap secretion could not be ascribed to differences in final cell densities.

View larger version (40K): [in this window] [in a new window]   FIG. 5.   Effects of sec4(S28N) overexpression on protein secretion. (A) pS28N transformants incubated in galactose-BSA at 30°C for 20 h degraded much less extracellular BSA than pS28N transformants incubated in glucose-BSA or pSEC4 transformants incubated in glucose-BSA or galactose-BSA. Reducing SDS-polyacrylamide gel electrophoresis with cell-free supernatants from 0.1 OD600 unit per lane and Coomassie blue staining is shown. (B) Western blotting showed that the galactose-incubated pS28N transformants secreted much less immunoreactive Sap than glucose-incubated pS28N transformants, glucose-incubated pSEC4 transformants, or galactose-incubated pSEC4 transformants (0.5 OD600 unit per lane).

Effects of sec4(S28N) overexpression on ultrastructural morphology. There was marked intracellular accumulation of secretory vesicles when a galactose-inhibited pS28N transformant was incubated in minimal galactose for 6.5 h. These vesicles were approximately 90 nm in diameter (which is similar to the size of the post-Golgi vesicles that accumulate in S. cerevisiae sec4-8 mutants subjected to restrictive temperatures), and they were localized mostly in the region of the bud or adjacent to the plasma membrane. In contrast, there was no intracellular accumulation of transport vesicles in glucose-incubated pS28N transformants or in glucose- or galactose-incubated pSEC4 transformants (Fig. 6).

View larger version (112K): [in this window] [in a new window]   FIG. 6.   Effects of sec4(S28N) overexpression on ultrastructural morphology. Typical post-Golgi secretory vesicles accumulated in pS28N-transformed C. albicans cells incubated in minimal galactose for 6.5 h (A) but not in pS28N transformants incubated in glucose (B), pSEC4 transformants incubated in galactose (C), or plasmidless C. albicans SC5314 incubated in galactose (D).

	DISCUSSION

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C. albicans causes more serious infections than any other fungus, but classical genetics are seldom useful for studying this organism because of its diploid genome and its lack of sexual reproduction. Recently, however, powerful molecular methods have been developed for studying C. albicans biology and pathogenesis. For example, many C. albicans genes have been cloned and sequenced, and integrative and episomal DNA transformation systems are well established. Moreover, SfiI macrorestriction maps of each C. albicans chromosome are available (4), and more than 100 genes have been assigned to specific SfiI fragments. Also, a detailed physical map of chromosome 7 has recently been constructed (3), and efforts to sequence the complete C. albicans genome are nearing completion.

Powerful methods for using the rapidly expanding body of C. albicans DNA sequence data to understand the biology of C. albicans and the pathogenesis of C. albicans infections have also been developed. The development of a technically straightforward method for deleting specific genes from a C. albicans ura3 strain whose virulence can be restored to wild-type levels by reintroducing URA3 (7) was a major advance, and this and similar methods have been used to ascertain directly the importance of several proposed C. albicans virulence factors (8, 12, 15, 24). Although this experimental approach is extremely useful, one of its major limitations is that disruption of essential genes causes loss of viability. Therefore, many fundamental questions about the biology and virulence of C. albicans cannot be answered simply by cloning and disrupting individual genes. In S. cerevisiae and other model fungi, the functions of essential genes can be studied by generating and analyzing conditional mutants or by overexpressing dominant-negative mutant alleles (6, 13, 17, 27). To our knowledge, the only example in which either of these approaches has been used with medically important fungi was that temperature-sensitive mutations were introduced by homologous gene targeting into the essential NMT genes (encoding myristoyl-coenzyme A:protein N-myristoyl transferase) of Cryptococcus neoformans (16) and C. albicans (28).

We chose to study C. albicans SEC4 because (i) Ras-like GTPases are highly conserved even among distantly related eukaryotes and (ii) single amino acid substitutions in conserved domains of S. cerevisiae Sec4p and related Ras-like GTPases cause temperature-sensitive and dominant-negative mutations. The first step was to clone and sequence a C. albicans gene that complemented the temperature-sensitive sec4-8 mutation in S. cerevisiae, and we found that the gene of interest was highly homologous to S. cerevisiae SEC4 and identical to the C. albicans SEC4 gene recently reported by Clement et al. (5). The first chromosomal SEC4 allele was readily disrupted in C. albicans CIA4, but efforts to disrupt the wild-type SEC4 allele in three C. albicans SEC4/sec4::hisG strains yielded no viable null mutants. One possible explanation for the inability to disrupt the second chromosomal allele of any gene may be that there is insufficient similarity between the gene disruption cassette and the second allele to facilitate homologous recombination. In the case of C. albicans SEC4, however, the presence of a polymorphic ApaI restriction site allowed us to show that the sec4::hisG-URA3-hisG gene disruption cassette we used was capable of replacing either of the two SEC4 alleles in C. albicans CAI4. Southern analyses also demonstrated homologous integration of the gene disruption cassette within the wild-type SEC4 locus of a SEC4/sec4::hisG-URA3-hisG strain, but allele-specific PCR showed that all of the resulting transformants retained a wild-type SEC4 allele. Since these results suggested that SEC4 is essential in C. albicans, it was clear that other approaches would be needed to define this gene's functions.

Small Ras-like GTPases in the Rab/SEC4/YPT1 family contain conserved domains required for guanine nucleotide binding, GTP-GDP exchange, and GTP hydrolysis. The S17N substitution in mammalian Ras protein alters GTP-GDP exchange-dependent signalling and results in dominant oncogenic transformation. Similarly, S. cerevisiae Ypt1p, mammalian Rab1p, and mammalian Rab3p with SN substitutions at analogous positions are potent trans-dominant inhibitors of protein transport (1, 2, 11, 13, 25-27). Therefore, we reasoned that it might be possible to define the functions of C. albicans SEC4 by (i) introducing a mutation encoding the S28N substitution and (ii) overexpressing the resulting sec4(S28N) allele in wild-type C. albicans. We found that autonomously replicating GAL1-regulated plasmids expressing sec4(S28N) (pS28N) had no discernible effects on growth or Sap secretion in C. albicans transformants incubated in glucose, but these plasmids markedly inhibited growth and Sap secretion in galactose-incubated transformants. In addition, typical post-Golgi secretory vesicles accumulated intracellularly in galactose-incubated pS28N transformants but not in glucose-incubated controls. We concluded from these results that C. albicans SEC4 (like S. cerevisiae SEC4) (i) is required for growth and protein secretion and (ii) functions at a later step in the protein secretion pathway than the formation of post-Golgi secretory vesicles.

Two unexpected findings were that sec4(S28N) overexpression did not inhibit growth and protein secretion in a minority of galactose-incubated transformants and that the growth inhibition phenotype was lost after prolonged incubation in galactose. Plasmid curing studies clearly established that inhibition of growth and protein secretion in galactose was mediated by pS28N, and Southern hybridization studies did not support the hypothesis that variable growth phenotypes were caused by marked differences in plasmid copy number. Therefore, the most likely explanation for the instability of the galactose inhibition phenotype is that the deleterious effects of GAL1-regulated sec4(S28N) overexpression selected for clones in which (i) a loss-of-function mutation occurred in one or more key elements in pS28N [e.g., the sec4(S28N) coding sequence, the GAL1 promoter, or ARS2) or (ii) the S28N mutation itself was lost, either by reversion or gene conversion.

The fact that the sec4 mutant phenotype could be induced despite the presence of two wild-type SEC4 alleles implies that sec4(S28N) functioned as a dominant-negative mutant allele. To our knowledge, C. albicans SEC4 is the first essential gene of any medically important fungus whose functions have been elucidated by constructing and overexpressing a dominant-negative mutant allele. It should be noted that construction of the sec4(S28N) allele required prior knowledge of the sequences of trans-dominant inhibitors of several other Ras-like GTPases. Thus, dominant-negative alleles may be most useful for ascertaining the functions of highly conserved genes required for fundamental cellular processes such as protein secretion, cell cycle control, and signal transduction. Also, further studies will be needed to determine why the sec4 mutant phenotype was unstable and to develop methods for maintaining the stability of dominant-negative alleles in C. albicans. Despite these limitations, the general approach used in this study may prove to be useful for studying the functions of additional essential genes in organisms like C. albicans, whose diploid genomes and lack of sexual reproduction preclude the use of more traditional genetic methods.