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Eukaryotic Cell, November 2006, p. 1894-1905, Vol. 5, No. 11
1535-9778/06/$08.00+0     doi:10.1128/EC.00151-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Tcc1p, a Novel Protein Containing the Tetratricopeptide Repeat Motif, Interacts with Tup1p To Regulate Morphological Transition and Virulence in Candida albicans{triangledown} ,{dagger}

Aki Kaneko, Takashi Umeyama, Yuki Utena-Abe, Satoshi Yamagoe, Masakazu Niimi, and Yoshimasa Uehara*

Department of Bioactive Molecules, National Institute of Infectious Diseases, Tokyo 162-8640, Japan

Received 25 May 2006/ Accepted 28 August 2006


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ABSTRACT
 
The transcriptional factor CaTup1p represses many genes involved in intracellular processes, including the yeast-hypha transition, in the human fungal pathogen Candida albicans. Using tandem affinity purification technology, we identified a novel protein that interacts with CaTup1p, named Tcc1p (Tup1p complex component). Tcc1p is a C. albicans-specific protein with a 736-amino-acid polypeptide with four tetratricopeptide repeat (TPR) motifs in the N-terminal portion. Tcc1p formed a protein complex with CaTup1p via the TPR domain of Tcc1p, independently of CaSsn6p-CaTup1p The tcc1{Delta} disruptant showed filamentous growth under conditions inducing the yeast form, as is true of the Catup1{Delta} mutant. Consistent with this result, the common set of hypha-specific genes was negatively regulated by both TCC1 and CaTUP1. These observations will provide new insights into CaTup1p-dependent transcriptional gene regulation in C. albicans.


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INTRODUCTION
 
Gene-specific transcriptionalrepression plays an important role in gene regulation of a broad range of organisms, from prokaryotes to higher eukaryotes. For example, gene repression is involved in timely regulation of growth, spatial restriction in differentiation, or responses to environmental changes. In the gene repression system conserved from lower to higher eukaryotes, the assembly of a multiprotein complex termed a "repressosome" has been under a great deal of study (reviewed in reference 9). In the Saccharomyces cerevisiae model, a central core complex contained in a typical repressosome comprises ScTup1p and ScSsn6p (Cyc8), orthologs of which have been found in humans, flies, worms, slime molds, and fungi (reviewed in reference 26).

ScTup1p and ScSsn6p form a protein complex to act as a global repressor in S. cerevisiae. This complex is targeted to promoters by DNA-binding proteins specific for the different classes of repressed genes (26). ScTUP1 was first identified as a mutant that was able to incorporate deoxythymidine (32). Subsequently, a number of distinct phenotypes of the Sctup1 mutant have been observed, including slow growth, flocculation, loss of mating in alpha strains, poor sporulation, and loss of some aspects of glucose repression. Scssn6 was first identified as a suppressor mutation of the snf1 mutant: Snf1p is required to derepress the expression of many glucose-repressible genes, including the SUC2 invertase gene, and the Scssn6 mutation causes constitutive invertase synthesis (8). The Scssn6 mutations are allelic to the cyc8 mutation (8), which causes increased production of iso-2-cytochrome c (23). Deletion of the ScSSN6 gene results in many phenotypes, most of which are identical to those of the Sctup1 mutant. From the viewpoint of protein structure, ScTup1p contains seven copies of a WD40 repeat, named after two amino acids, tryptophan and aspartic acid, commonly found in the repeat and its length. The seven repeats fold into a propeller-like structure, which is hypothesized to bind the homeodomain protein {alpha}2 (17). ScSsn6p includes 10 copies of the tetratricopeptide repeat (TPR), comprising the 34 amino acids that make up the basic repeat (10), which is related to the interaction of ScSsn6p-ScTup1p (29) or ScSsn6p-{alpha}2 (27). Generally, TPR motifs have been found in a wide variety of proteins from all organisms, from humans to prokaryotes. They mediate molecular recognition and protein-protein interactions. While 22 proteins containing the TPR motif have been found encoded in the yeast genome, only three proteins are involved in transcriptional regulation: Ctr9p, Tfc4p, and ScSsn6p (10). Of the 10 copies of TPRs in ScSsn6p, the first to the third TPR motifs are known to be responsible for ScTup1 binding, whereas combinations of the other TPRs mediate interactions with different repressor proteins specific for each gene family regulated by the ScTup1p-ScSsn6p complex (29).

Recently, studies of Tup1-dependent gene repression in Candida albicans have been undertaken by many scientists. C. albicans is an opportunistic fungal pathogen in humans and can cause either systemic or mucosal infection. In immunocompromised patients, infection with this organism can progress to severe systemic invasion, leading to life-threatening circumstances (20, 21). C. albicans is a polymorphic fungus capable of converting its cell shape from budding yeast to a filamentous form, including pseudohyphae and true hyphae. This morphological transition has been strongly associated with pathogenicity (6).

The C. albicans TUP1 gene was first isolated and disrupted by Braun and Johnson (4). Since then, several research groups have reported that Candida Tup1p represses hypha-specific genes (HSGs) under conditions inducing the yeast form, as suggested by the exclusive filamentation of the gene disruptant. CaTup1p may require the DNA-binding protein CaNrg1p for the repression of hypha-specific genes in a pathway that promotes yeast form growth, because the Canrg1{Delta} mutant displays constitutive filamentation similar to that of the Catup1{Delta} mutant (5, 19). However, whether C. albicans Tup1p and CaNrg1p interact directly with each other remains unknown. The binding partner of CaTup1 has also been thought to be an Ssn6p homolog in C. albicans, analogous to the S. cerevisiae paradigm. However, the phenotypes of the CaSSN6 and CaTUP1 gene disruptants are definitely different (12, 14). A recent, excellent study based on DNA microarray analysis by Garcia-Sanchez et al. (12) has shown that almost no hypha-specific genes that were induced by Catup1 deletion overlapped with any genes that were upregulated by Cassn6 deletion, implying the existence of a CaTup1p-binding partner other than CaSsn6p with respect to morphogenesis regulation.

In this report, we identified a novel protein interacting with Tup1p in C. albicans by using tandem affinity purification (TAP) technology. The protein, termed Tcc1p, a Tup1p complex component, formed a protein complex with CaTup1p independently of the CaSsn6p-CaTup1p complex. Deletion of the TCC1 gene resulted in pseudohyphal morphology under conditions inducing yeast form and attenuated virulence, similar to the phenotype of the Catup1 deletion mutant. These observations will give new insights into Tup1p-dependent transcriptional gene regulation in C. albicans.


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MATERIALS AND METHODS
 
Strains, growth conditions, and basic techniques. Table 1 lists the C. albicans strains used in this study. Cells were grown in yeast-peptone-dextrose (YPD; adjusted to pH 5.6 [Qbiogene Inc.]), SD-Ura (6.7 g liter–1 yeast nitrogen base without amino acids [Difco], 2% glucose, CSM-Ura [Qbiogene Inc.]), or SD-AU (the same as SD-Ura except using CSM-Arg-Ura [Qbiogene Inc.] instead of CSM-Ura) with shaking to induce the yeast form or in YPD (adjusted to pH 7.2) plus 10% serum or Spider medium (1% mannitol, 1% Difco nutrient broth, 0.2% K2HPO4) at 37°C with shaking to induce hyphae. The rate of growth was measured by determining the optical density at 660 nm using a model TN-1506 Biophotorecorder (Advantec, Japan). For filamentous growth on the solid medium, strains were grown for 7 days at 37°C on 10% calf serum solidified with the addition of 2% agar or at 30°C on Spider medium solidified with the addition of 1.4% agar.


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  		TABLE 1. Strains used and constructed in this study

Escherichia coli XL1-Blue and cloning vectors pUC18 and pUC19 were used for DNA manipulation. General recombinant DNA procedures were performed as described by Sambrook and Russell (24). C. albicans was transformed by the method described by Umeyama et al. (30). An Applied Biosystems model 3100 automated capillary sequencer was used for nucleotide sequencing.

Northern hybridization and quantitative real-time reverse transcription (RT)-PCR were performed as previously described (13). All primers used for the amplification of Northern probes and real-time PCR are listed in Table S1 in the supplemental material.

Immunostaining was performed as described previously (30). Microscopic observation was performed using a conventional fluorescence microscope (model IX81; Olympus, Japan) equipped with a model DP70 digital camera (Olympus, Japan).

Animal experiments were performed as described previously (30). For each group, five male CD-1 (ICR) mice aged 4 weeks (Charles River, Japan) were inoculated with 106 CFU by intravenous injection. Kaplan-Meier survival curves were compared using the log rank test. A P value of <0.05 was considered significant.

Plasmid and strain construction. All primers used in this study are listed in Table S1 in the supplemental material.

(i) p3HA-ARG4 vector. The p3HA-ARG4 plasmid was designed to fuse a protein with three tandem repeats of the hemagglutinin (3xHA) tag at the C terminus and to contain the ARG4 marker. A KpnI-SacI fragment digested from plasmid pRS-Arg4{Delta}SpeI (33) was cloned into the KpnI-SacI sites of pUC18 to yield pUC18-ARG4. Then, a 3.8-kb HindIII-PvuI fragment containing the ARG4 marker of pUC18-ARG4 and a 3.2-kb HindIII-PvuI fragment containing the 3xHA tag and the ACT1 terminator of p3HA-ACT1 were ligated to yield p3HA-ARG4.

(ii) pMyc-SAT1 and p3HA-SAT1 vectors. The p3Myc-SAT1 plasmid was designed to facilitate the constitutive expression of a protein fused with a three-tandem repeat of the Myc (3xMyc) tag at the C terminus and to contain a nourseothricin resistance marker, SAT1. To generate the DNA fragment 3xMyc-A, which encodes the N-terminal portion of the tag sequence, two oligonucleotides, 3xMyc-1 and 3xMyc-4, were annealed by boiling them for 3 min and then allowing them to cool to room temperature. To generate the DNA fragment 3xMyc-B, which encodes the C-terminal portion of the tag sequence, two oligonucleotides, 3xMyc-2 and 3xMyc-3, were annealed by the above-described method. The two DNA fragments 3xMyc-A and 3xMyc-B were then ligated, gel purified, and inserted into the XhoI-SphI sites of pFLAG-ACT1 (31) to yield p3Myc-ACT1 (30). A HindIII-PstI fragment digested from plasmid pSFS1A (22) was cloned into the HindIII-PstI sites of pUC18 to yield pUC18-SAT1. Then, a 2.9-kb HindIII-PvuI fragment containing an SAT1 marker of pUC18-SAT1 and a 3.2-kb HindIII-PvuI fragment containing a 3xMyc tag and an ACT1 terminator of p3Myc-ACT1 were ligated to yield p3Myc-SAT1. p3HA-ACT1 was used instead of p3Myc-ACT1 to yield p3HA-SAT1.

(iii) p6HF-Met3 vector. The p6HF-Met3 plasmid was designed to facilitate the conditional expression of a protein fused with a six-histidine-FLAG (HF) tag at the C terminus. A 5.6-kb XhoI-EcoRI fragment containing the MET3 promoter of p3HA-MET3 (30) and a 1-kb XhoI-EcoRI fragment containing the HF tag and the ACT1 terminator of p6HF-ACT1 (16) were ligated to yield p6HF-Met3.

(iv) TCC1 disruption. Gene disruption of TCC1 was performed using a method similar to that described previously (13). Briefly, two fragments, disTCC1-A and disTCC1-B, were amplified using primers disTCC1-1 and -2 and disTCC1-3 and -4, respectively, and used as a flanking homology region for a gene disruption cassette. The PCR-amplified disruption cassette containing an hph200-URA3-hph200 or ARG4 marker was transformed into the TUA4 arg4– ura3– strain (hph200 represents a 200-bp portion of hph). Finally, both alleles of the TCC1 locus were replaced with hph200 and ARG4, yielding strain TCC103. Direct-colony PCR and genomic PCR were performed to verify the strain construction in each step.

(v) TCC1 revertant. For a complementation test, the DNA fragment TCC1comp was amplified from TUA4 chromosomal DNA using primers disTCC1-1 and TCC1comp-C. A mixture of the amplified DNA fragments TCC1comp and fragTCC1-HF (used for HF tagging, as described below) was introduced into TCC103 to generate TCC107, in which one allele has the wild-type open reading frame tagged with His6-FLAG at the C terminus and the other allele is replaced by an ARG4 marker. At first, as a negative control, the DNA fragment TCC1comp-nega-C was amplified using primers TCC1comp-nega5' and TCC1comp-C and TUA4 chromosomal DNA as a template. Next, the DNA fragment TCC1comp-nega was amplified using primers disTCC1-1 and TCC1comp-C and DNA fragments disTCC1-A and TCC1comp-nega-C. A mixture of the resultant DNA fragments TCC1comp-nega and fragTCC1-HF was introduced into TCC103 to generate TCC106, in which one allele has an incomplete TCC1 open reading frame with the region between methionine-1 and proline-704 deleted and the other allele is the ARG4 marker. Direct-colony PCR and genomic PCR were performed to verify the strain construction in each step.

(vi) TUP1, SSN6, and NRG1 disruptions. Gene disruption of TUP1, SSN6, and NRG1 was performed in a manner similar to that described above. A primer set consisting of disTUP1-1, -2, -3, and -4 or disNRG1-1, -2, -3, and -4 for the TUP1 or the NRG1 gene, respectively, was used for the creation of a disruption cassette. A disruption cassette for SSN6 was amplified using 120-mer-long primers, disSSN6-5' and iSSN6-3'. Both alleles of each of the TUP1, SSN6, or NRG1 genes were replaced with the ARG4 marker and a Ura-blaster cassette, hph200-URA3-hph200 (for SSN6) or hisG200-URA3-hisG200 (for TUP1 and NRG1), in strain TUA4, to generate the homozygous mutant TUP102, SSN602, or NRG102, respectively. SSN602 cells were plated on 5-fluoroorotic acid-containing medium to isolate the ura– segregants (SSN603). Direct-colony PCR and genomic PCR were performed to verify the strain construction in each step.

(vii) HGC1 disruption. Single or double disruptions of HGC1 and/or TCC1 were performed using a method similar to that described above. Briefly, for the first allele, the two fragments disHGC1-A and disHGC1-B were amplified using primers disHGC1-1 and -2 and disHGC1-3 and -4, respectively, and used as flanking homology regions for a gene disruption cassette with pC220-URA3 (30). The PCR-amplified disruption cassette containing the C220-URA3 marker was transformed into the TUA4 arg4– ura3– strain or TCC103 (tcc1{Delta}), yielding HGC101 or HGC111, respectively. Then, strain HGC101 and HGC111 were plated on a medium containing 5-fluoroorotic acid to isolate HGC102 and HGC112, respectively. For another allele of HGC1, the DNA fragment disHGC1-C was amplified using primers disHGC1-5 and disHGC1-6; we used disHGC1-A and disHGC1-C as a gene disruption cassette with pUC19-URA3KX (30). A second transformation using the amplified DNA led to the isolation of an hgc1{Delta} single mutant (HGC104) or an hgc1{Delta} tcc1{Delta} double mutant (HGC114). Direct-colony PCR and genomic PCR were performed to verify the strain construction in each step.

(viii) TCC1 deletion series. To generate plasmids expressing the full length of Tcc1p, a DNA fragment was amplified by primers TCC1-FL-5' and TCC1-FL-3', using the TUA4 chromosome as a template, digested with PstI and XhoI, and cloned into the PstI-XhoI sites of p6HF-MET3, yielding p6HF-MET3-TCC1. To generate plasmids expressing a deletion series of Tcc1p, a DNA fragment corresponding to amino acid positions 1 to 475 or 1 to 250 of Tcc1p was amplified by primers TCC1-FL-5' and TCC-N1-3' or by TCC1-FL-5' and TCC1-N2-3', using the p6HF-MET3-TCC1 plasmid as a template, digested with PstI and XhoI, and cloned into the PstI-XhoI sites of p6HF-ACT1, yielding p6HF-ACT1-TCC1-N1 or p6HF-ACT1-TCC1-N2, respectively. A DNA fragment corresponding to amino acid positions 251 to 736 or 476 to 736 of Tcc1p was amplified by primers TCC1-C1-5' and TCC-FL-3' or by TCC1-C2-5' and TCC1-FL-3', using the p6HF-MET3-TCC1 plasmid as a template, digested with PstI and XhoI, and cloned into the PstI-XhoI sites of p6HF-MET3, yielding p6HF-MET3-TCC1-C1 or p6HF-MET3-TCC1-C2, respectively.

To generate Ura-auxotrophic strain iTUP1-HA-ARG4 as a host for the TCC1 deletion series, a DNA fragment encoding a 3xHA tag and an ARG4 marker was amplified by primers iTUP1-5' and iTUP1-3', using p3HA-ARG4 as a template, and introduced into TUA4. To verify the strain construction, direct-colony PCR was performed, after which the nucleotide sequence of the PCR fragment was confirmed. Plasmid p6HF-ACT1, p6HF-MET3-TCC1, p6HF-ACT1-TCC1-N1, p6HF-ACT1-TCC1-N2, p6HF-MET3-TCC1-C1, or p6HF-MET3-TCC1-C2 was introduced into iTUP1-3HA-ARG4 to yield DelTCC1VEC, DelTCC1-W, DelTCC1-N1, DelTCC1-N2, DelTCC1-C1, or DelTCC1-C2, respectively.

(ix) Epitope tagging. To tag CaTup1p with the HF epitope in the genomic locus, a DNA fragment containing the 3' region of TUP1, the HF tag sequence, the ACT1 terminator, the URA3 marker, and the downstream region of TUP1 was amplified by PCR with primers iTUP1-5' and iTUP1-3', using p6HF-ACT1 (16) as a template, and introduced into TUA4 to generate iTUP1-HF. Similarly, in order to tag Tcc1p or CaSsn6p with the HF epitope, primers iTCC1-5' and iTCC1-3' or primers iSSN6-5' and iSSN6-3' were used for the amplification of fragTCC1-HF or fragSSN6-HF to yield the iTCC1-HF or iSSN6-HF DNA cassette, respectively.

To generate strain iTCC1-HA, a DNA fragment containing a 3xHA tag, an ACT1 terminator, and a URA3 marker was amplified by primers iTCC1-5' and iTCC1-3', using p3HA-ACT1 as a template, and introduced into TUA4.

To generate strain iSSN6-HA, a DNA fragment containing a 3xHA tag, an ACT1 terminator, and a URA3 marker was amplified by primers iSSN6-5' and iSSN6-3', using p3HA-ACT1 as a template, and introduced into TUA4.

To generate strain iSSN6-Myc, a DNA fragment containing a 3xMyc tag, an ACT1 terminator, and an SAT1 marker was amplified by primers iSSN6-5' and iSSN6-3', using p3Myc-SAT1 as a template, and introduced into TUA6. Nourseothricin-resistant clones were selected on YPD medium containing 200 µg/ml clonNAT (WERNER BioAgents, Germany).

Strain 3TAG-TTS was designed to simultaneously express three tagged proteins, CaTup1p-HF, Tcc1p-HA, and CaSsn6p-Myc. A DNA fragment containing a 3xHA tag and an ARG4 marker was amplified by primers iTCC1-5' and iTCC1-3', using p3HA-ARG4 as a template, and introduced into iTUP1-HF to yield 2TAG-TT. Next, a DNA fragment containing a 3xMyc tag and an SAT1 marker was amplified by primers iSSN6-5' and iSSN6-3', using p3Myc-SAT1 as a template, and introduced into 2TAG-TT to yield 3TAG-TTS.

Strain D2T06ACT1 was designed to simultaneously express two tagged proteins, CaTup1p-HF and Tcc1p-HA, in an ssn6{Delta} genetic background. The same PCR-amplified DNA cassette as that used for the iTUP1-HF construction was introduced into SSN603 to generate D2T05. The p6HF-TCC1 plasmid was digested with PstI and XhoI and cloned into the PstI-XhoI sites of p3HA-SAT1 to yield p3HA-SAT1-TCC1. The StuI-digested p3HA-SAT1-TCC1 plasmid was transformed into D2T05 to yield D2T06ACT1, in which Tcc1p-HA is expressed from the ACT1 promoter.

Strain TUP112 was designed to express Tcc1p-HA protein in a strain in which the CaTUP1 gene can be repressed in the presence of methionine and cysteine. To generate Ura-auxotrophic strain iTCC1-HA-ARG4 as a host for CaTUP1 depletion, a DNA fragment encoding a 3xHA tag and an ARG4 marker was amplified by primers iTCC1-5' and iTCC1-3', using p3HA-ARG4 as a template, and introduced into TUA4. Gene replacement of the first allele of CaTUP1 was performed in a manner similar to that described above. A primer set consisting of disTUP1-1, -2, -3, and -4 was used to create a disruption cassette, which was amplified using pUC18-SAT1 as a template. The first allele was replaced with the SAT1 marker in strain iTCC1-HA-ARG4 to yield TUP111. To construct pCaDis-TUP1, DNA fragment TUP1-A was amplified with primers TUP1-N and TUP1-SacI-3', DNA fragment TUP1-B was amplified with primers TUP1-SacI-5' and TUP1-400-3', and each DNA fragment was gel purified. A 400-bp portion of CaTUP1 containing an artificial SacI site was amplified by mixing the two DNA fragments (TUP1-A and TUP1-B) and the two primers (TUP1-N and TUP1-400-3'), digested by BamHI and SphI, and then cloned into the BamHI-SphI sites of pCaDis (7). The SacI-digested pCaDis-TUP1 was transformed into a heterozygous strain, TUP111, to generate TUP112.

To verify the strain construction, direct-colony PCR was performed, after which the nucleotide sequence of the PCR fragment was confirmed.

Yeast two-hybrid assay. The MATCHMAKER two-hybrid system 3 (Clontech) was used for the yeast two-hybrid assay.

A DNA fragment corresponding to amino acid positions 1 to 130 of CaTup1p was amplified by primers THTUP1-5' and THTUP1-3', using TUA4 chromosomal DNA as a template, digested with BamHI and PstI, and cloned into the BamHI-PstI sites of pGADT7 (Clontech), yielding pGADT7-TUP1-N.

A DNA fragment corresponding to amino acid positions 1 to 475 of the Tcc1p codon, optimized for S. cerevisiae, where two CUG codons were changed to TCG, was obtained by a three-step PCR. First, three DNA fragments were amplified using p6HF-TCC1-N as a template with primers THTCC1-5' and THTCC1-mut1-3', primers THTCC1-mut1-5' and THTCC1-mut2-3', and primers THTCC1-mut2-5' and THTCC1-3' to generate THTCC1-NT-A, THTCC1-NT-B, and THTCC1-NT-C, respectively. Each fragment was purified by agarose gel electrophoresis to prevent contamination of the template DNA. A second PCR was performed with two DNA fragments, THTCC1-NT-A and THTCC1-NT-B, and two primers, THTCC1-5' and THTCC1-mut2-3', in the same tube to generate THTCC1-NT-AB. A third PCR was performed with two DNA fragments, THTCC1-NT-AB and THTCC1-NT-C, and two primers, THTCC1-5' and THTCC1-3'. A resultant DNA fragment containing the substitutions at Ser182 and Ser258 (changed from CTG to TCG) was digested with EcoRI and PstI and cloned into the EcoRI-PstI sites of pGBKT7 (Clontech) to generate pGBKT7-TCC1-N.

As shown in Fig. 4, both pGBKT7 and pGADT7 derivative plasmids were simultaneously introduced into S. cerevisiae AH109 (Clontech), and the transformants were checked for the expression of MEL1, which encodes {alpha}-galactosidase, according to the manufacturer's protocol. Plasmids pGADT7-T and pGBKT7-53 were supplied as positive controls in the MATCHMAKER two-hybrid system 3 (Clontech).


Figure 4
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  		FIG. 4. Expression profile and nuclear localization of Tcc1p. (A) Western blotting for the detection of Tcc1p-HA. 3TAG-TTS cells expressing CaTup1p-HF, Tcc1p-HA, or CaSsn6p-Myc from each native promoter were used. Each lane was processed using a total cell extract with the following culture conditions: AS, asynchronous cells cultured for 3 h at 30°C; –Glc, unbudded cells collected in yeast nitrogen base medium (without glucose); HU, synchronized cells with 0.1 M hydroxyurea at the G1/S phase; NOC, synchronized cells with 20 µg/ml nocodazole at the G2/M phase; Ser, hyphal cells cultured in YPD containing 10% serum for 1 or 3 h (represented by 1 and 3); Spi, hyphal cells cultured in Spider medium for 1 or 3 h (represented by 1 and 3). After cells were grown under each condition, Western blotting using anti-FLAG, anti-HA, or anti-Myc antibody was performed. Western blotting using anti-PSTAIRE antibody was performed as a loading control. (B) Fluorescence microscopy for the detection of Tcc1p-HA. TUA4 cells expressing HA-tagged Tcc1p, CaTup1p, and CaSsn6p were grown overnight at 30°C, inoculated into fresh YPD (pH 5.6) medium, fixed in 3% formaldehyde, treated with anti-HA for a primary antibody and anti-rabbit immunoglobulin G antibody conjugated with Alexa Fluor 594 for a secondary antibody, and viewed with a fluorescence microscope and differential interference contrast optics. Immunostained, 4',6'-diamidino-2-phenylindole (DAPI)-stained, and differential interference contrast images in the same field of view are shown. Bar, 10 µm. (C) Subcellular fractionation was analyzed by Western blotting with anti-HA and anti-histone H4 antibodies. Cell extracts from strains CAF2-1 (not tagged; lanes 1 and 2) and iTCC1-HA (Tcc1p-HA; lanes 3 and 4) were separated into cytoplasmic fractions (lanes 1 and 3) and nuclear fractions (lanes 2 and 4).

Preparation of total cell lysates, purification, and Western blotting. Cells were collected and disrupted with glass beads in NP-40 buffer (10 mM Tris HCl [pH 8], 1 mM EDTA, 150 mM NaCl, 10% glycerol, 1% NP-40) using Bead Shocker (Yasui Kikai, Japan). After centrifugation at 10,000 x g for 10 min, the supernatant was extracted for Western blotting and purification. Tandem affinity purification was performed as described by Kaneko et al. (16). Western analysis was performed as described by Umeyama et al. (30). Anti-FLAG M2 monoclonal antibody and agarose were purchased from Sigma. Anti-HA F-7 (monoclonal; horseradish peroxidase conjugate), anti-Myc 9E10 (monoclonal; horseradish peroxidase conjugate), and anti-PSTAIRE (polyclonal) antibodies were purchased from Santa Cruz. Anti-HA and anti-Myc agarose were purchased from Sigma, and anti-histone H4 polyclonal antibody was purchased from Upstate.

Subcellular fractionation. Cells were harvested and treated to obtain spheroplasts with Zymolyase 100T in Zymolyase buffer (50 mM Tris HCl [pH 7.5], 10 mM MgCl2, 1 M sorbitol, 1 mM dithiothreitol) at 30°C for 40 min with mild shaking. The spheroplast suspension was introduced drop by drop into a beaker containing Ficoll buffer (18% [wt/vol] Ficoll-400, 10 mM Tris HCl [pH 7.5], 20 mM KCl, 5 mM MgCl2, 3 mM dithiothreitol, 1 mM EDTA). The diluted solution was centrifuged at 20,000 x g for 20 min at 4°C, and the supernatants were used for Western analysis as a cytoplasmic fraction. The resultant pellets were resuspended in Ficoll buffer in a volume equal to the supernatant and used for Western analysis as a nuclear fraction.

Nucleotide sequence accession number. The newly determined sequence for TCC1 was deposited in GenBank under accession number AB252688 [GenBank] .


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RESULTS
 
Identification of proteins interacting with CaTup1p. In the budding yeast, S. cerevisiae, Tup1p forms a protein complex with Ssn6p to act as a global repressor. Until a few years ago, it had been believed that C. albicans Ssn6p would form a complex with CaTup1p to regulate its morphogenesis, on the basis of the S. cerevisiae paradigm. However, recent reports (12, 14) have shown that morphological phenotypes and morphology-related gene regulation of the Cassn6 mutant barely overlap those of the Catup1 mutant, indicating that CaTup1p and CaSsn6p act independently on morphological transition. Therefore, to identify a CaTup1p-binding partner involved in transcriptional repression for a hyphal program other than CaSsn6p, we performed TAP. Crude extracts prepared from a strain in which CaTup1p was tagged with the His6-FLAG epitope sequence were subjected to TAP procedures, leading to the detection by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins composing a CaTup1p complex (Fig. 1A). We identified three major proteins of the purified protein complex by peptide mass fingerprinting using matrix-assisted laser desorption ionization-time of flight mass spectrometry. Two proteins approximately 60 kDa and 160 kDa in size were expected to be CaTup1p and CaSsn6p, respectively. There is the possibility that a gel band corresponding to CaTup1p includes tagged or native protein. Peptide fingerprinting of the 80-kDa protein demonstrated that this band was presumed to be a novel protein corresponding to CaO19.6734 and Ca19.14026 (http://www-sequence.stanford.edu/group/candida). Since no protein homologous to this novel protein was found in the S. cerevisiae genome database (http://www.yeastgenome.org/) or other fungal genome databases, we called Tcc1p the Tup1p-complex component. Reciprocally, TAP procedures with strain iTCC1-HF or iSSN6-HF, in which a single genomic locus for TCC1 or CaSSN6 was tagged with His6-FLAG, identified CaTup1p as a binding protein (data not shown). At the start, we used the database sequence of CaO19.6734 to construct a C terminus-tagged protein. However, we could not detect a tagged protein by Western blotting. In fact, experimental nucleotide sequencing of a PCR-amplified DNA fragment corresponding to CaO19.6734 revealed that the sequence that we had determined had one base pair insertion compared to the coding sequences of CaO19.6734 in the Candida genome database; this frameshift leads to different lengths of the deduced polypeptides (Fig. 1B). Since we were able to detect an appropriate size for the Tcc1p protein epitope tagged at the C terminus based on the newly determined sequences (accession no. AB252688 [GenBank] ), we used this sequence for further analysis.


Figure 1
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  		FIG. 1. Identification of a protein encoded by Orf19.6734 as a component of the CaTup1p complex. (A) The CaTup1p complex was purified by tandem affinity purification (– or + TAP-tag) using anti-FLAG and Ni-nitrilotriacetic acid (Ni-NTA) agarose. Protein fractions of crude extracts (lanes 1 to 4) and purified samples (lanes 5 to 8) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by silver staining. Open arrowheads indicate the component of the CaTup1p complex identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Yeast cells (Y) of strains CAF2-1 (lanes 1 and 5) and iTUP1-HF (lanes 3 and 7) were grown at 30°C for 4 h in YPD medium (pH 5.6). Hyphal cells (H) of strains CAF2-1 (lanes 2 and 6) and iTUP1-HF (lanes 4 and 8) were grown at 37°C for 4 h in YPD medium (pH 7.2) containing 10% calf serum. M, molecular size marker. (B) Differences in nucleotide sequences between the database sequence (19.6734) and the analyzed sequence (AB252688). The altered nucleotide is indicated as boxed characters. The nucleotide positions of each open reading frame are indicated on the right. (C) Schematic diagram showing the sequence conservation of CaSsn6p and Tcc1p. (D) Sequence alignment of the TPR motifs of CaSsn6p and Tcc1p. The numbering on the right side refers to the position of each sequence in the protein. The TPR consensus sequence (3) is shown below the alignment, with the motif numbering indicated below the sequence. Eight amino acid residues (W/L/F, L/I/M, G/A/S, Y/L/F, A/S/E, F/Y/L, A/S/L, and P/K/E) show a high frequency of conservation. Boxed residues in the alignment indicate amino acids matching the consensus sequence shown below.

On the basis of the nucleotide sequences that we determined, the deduced open reading frame encoded a polypeptide of 736 amino acids with a calculated molecular mass of 80,177 Da. Tcc1p contains four copies of the TPR motif in the N-terminal portion and glutamine-rich regions in the C-terminal portion (Fig. 1C). In general, the TPR motif is involved in protein-protein interaction (10). C. albicans Ssn6p also has nine copies of the TPR motif; the amino acid alignment of the TPR motif in the sequences of Tcc1p and CaSsn6p is shown in Fig. 1D with TPR consensus sequences. In addition, although Tcc1p was predicted to be a nuclear protein because it interacted with CaTup1p, it was found to contain neither nuclear localization signals nor nuclear exporting signals. Thus, Tcc1p, which was identified as a CaTup1p-binding partner, is a novel C. albicans-specific protein with a TPR motif.

Tcc1p and CaSsn6p interact independently with CaTup1p. To confirm that CaTup1p and Tcc1p bind to each other and to analyze the relationships among CaTup1p, Tcc1p, and CaSsn6p, we constructed a strain in which CaTup1p, Tcc1p, and CaSsn6p were tagged with disparate epitope tags (His6-FLAG, 3xHA, and 3xMyc, respectively) and performed immunoprecipitation, followed by Western blotting. We also constructed three strains, in each of which His6-FLAG-tagged CaTup1p (iTup1p-HF), 3xHA-tagged Tcc1p (iTcc1p-HA), or 3xMyc-tagged CaSsn6p (iSsn6p-Myc) was expressed under the control of each native promoter. Western analysis confirmed that each protein was expressed with each epitope tag as expected (Fig. 2A, lanes 1 to 4). Cell extracts from each strain were then subjected to tandem affinity purification. Western blotting using anti-FLAG, anti-HA, and anti-Myc antibodies with the immunoprecipitant from each strain demonstrated that Tcc1p-HA and CaSsn6p-Myc were detected in the fraction of strain 3TAG-TTS (Fig. 2A, lane 5), indicating that the CaTup1p binds to Tcc1p and CaSsn6p. When a similar immunoprecipitation using anti-HA antibody agarose was performed, only CaTup1p-HF and Tcc1p-HA were detected and CaSsn6p-Myc was below the detectable level (Fig. 2A, lane 9). Similarly, an immunoprecipitation experiment using anti-Myc antibody agarose showed interaction between CaTup1p-HF and CaSsn6p-Myc and no interaction of Tcc1p-HA (Fig. 2A, lane 13). These reciprocal experiments indicated that CaTup1p-Tcc1p and CaTup1p-CaSsn6p were independent complexes.


Figure 2
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  		FIG. 2. Tcc1p interacts with CaTup1p independently of CaSsn6p. (A) Tcc1p-CaTup1p and CaSsn6p-CaTup1p are contained in different complexes. Immunoprecipitation (IP) and Western blotting (IB) were performed. Cells from strain 3TAG-TTS (lanes 1, 5, 9, and 13), iTUP1-HF (lanes 2, 6, 10, and 14), iTCC1-HA (lanes 3, 7, 11, and 15), and iSSN6-Myc (lanes 4, 8, 12, and 16) were cultured at 30°C for 3 h in YPD medium (pH 5.6). CaTup1p-HF, Tcc1p-HA, and CaSsn6p-Myc were expressed under the control of each native promoter in one cell of the 3TAG-TTS strain. Blots for total proteins (a, e, and i) and immunoprecipitated fractions (b, c, d, f, g, h, j, k, and l) were probed with anti-FLAG (a, b, c, and d), anti-HA (e, f, g, and h), and anti-Myc (i, j, k, and l) antibodies. Immunoprecipitation was performed with anti-FLAG agarose, followed by Ni-NTA agarose (b, f, and j), anti-HA agarose (c, g, and k), and anti-Myc agarose (d, h, and l). Panels e and h and panels i and k were originally derived from the same blot. (B) Tcc1p forms a complex with CaTup1p in Cassn6{Delta} cells. Cells from 2TAG-TT (lane 1), iTCC1-HA (lane 2), and SSN622 (lane 3) were cultured at 30°C for 3 h in YPD medium (pH 5.6). Then, tandem affinity purification and Western blotting were performed. Blots for total proteins (upper panels) and purified fractions (middle panels) were probed with anti-HA antibody. Blots for purified fractions were probed with anti-FLAG antibody (lower panels).

To verify this independence more clearly, we investigated Tcc1p-CaTup1p interaction in a Cassn6 deletion mutant background. For this purpose, tandem affinity purification was performed using the wild type and Cassn6{Delta} cells expressing Tcc1p-HA and CaTup1p-HF (Fig. 2B). Western blotting using anti-HA antibody revealed that Tcc1p-HA was copurified with CaTup1p-HF, even in the absence of CaSSN6 (Fig. 2B, lane 3), suggesting that CaSsn6p might have no effect on CaTup1p-Tcc1p complex formation.

The TPR domain of Tcc1p contributes to interaction with CaTup1p. To determine which region in Tcc1p is necessary for CaTup1p binding, we used an immunoprecipitation experiment and a yeast two-hybrid system. For immunoprecipitation, a series of His6-FLAG-tagged deletion mutants of the Tcc1p protein were expressed from the ACT1 or MET3 promoter in the C. albicans cells that contain HA-tagged CaTup1p. By performing TAP, the Tcc1p mutant that contains TPR motifs corresponding to the amino acid positions 1 to 475 (Tcc1-N1) or 1 to 250 (Tcc1-N2) was detected as a protein interacting with HA-tagged CaTup1p (Fig. 3B, lanes 3 and 4, respectively). CaTup1p-HA was not detected in the immunoprecipitated complex with the C-terminal domains of Tcc1p, corresponding to amino acid positions 251 to 736 (Tcc1-C1) and 476 to 736 (Tcc1-C2), although only a small amount of the Tcc1p mutant was present (Fig. 3B, lanes 5 and 6). Even when three times as much crude extract as indicated in Fig. 3B was used for purification, no signals of CaTup1p-HA were detected (data not shown). The faint signals of the Tcc1-N2, Tcc1-C1, and Tcc1-C2 deletion mutants could possibly be due to protein instability. These results suggest that the first two TPR domains of Tcc1p might serve an important role in binding to CaTup1p, although the possibility that the C-terminal glutamine-rich region can bind to CaTup1p could not be denied. To confirm the importance of the TPR domain in the CaTup1p-Tcc1p interaction in vivo, we designed a yeast two-hybrid system. The effect of the repressive activity of CaTup1p was avoided by using the portion of CaTup1p comprising amino acids 1 to 130, which contains no WD repeats, because the full-length CaTup1p fused to the Gal4 DNA-binding domain itself could repress the reporter activity. The CaTup1p mutant without this N-terminal portion could not bind to Tcc1p in the above-described immunoprecipitation experiment (our unpublished results). It is important to note that TCC1 contained two CUG codons, which encode serine in C. albicans but leucine in S. cerevisiae (25), in the sequence coding for amino acid positions 1 to 475. To express TCC1 functionally in S. cerevisiae, we changed both these codons into TCG by PCR-mediated mutagenesis, and the N-terminal portion of codon-optimized Tcc1p was fused to the Gal4-activating domain. When the Gal4-binding-domain-fused N-terminal portion of CaTup1p (amino acids 1 to 130) and the Gal4-activation domain-fused N-terminal portion of Tcc1p (amino acids 1 to 475) were simultaneously expressed in a host strain, AH109, that possesses an {alpha}-galactosidase MEL1 gene as a reporter, the constructed strain took on a blue color on agar medium containing X-{alpha}-Gal (5-bromo-4-chloro-3-indolyl-{alpha}-D-galactopyranoside) (Fig. 3C), indicating an in vivo protein-protein interaction. Integrating the results of immunoprecipitation and the yeast two-hybrid assay suggests that the TPR domain of Tcc1p and the N-terminal glutamine-rich domain of CaTup1p contribute to Tcc1p-CaTup1p binding.


Figure 3
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  		FIG. 3. TPR domain within Tcc1p is necessary for binding to CaTup1p. (A) Diagram of Tcc1p TPR motif in the wild type and the deletion derivatives used for interaction assays. (B) Immunoprecipitation of CaTup1p-HA by the Tcc1p deletion mutant. Cells from strains DelTCC1-W (Tcc1-W; lane 1), DelTCC1VEC (vector; lane 2), DelTCC1-N1 (Tcc1-N1; lane 3), DelTCC1-N2 (Tcc1-N2; lane 4), DelTCC1-C1 (Tcc1-C1; lane 5), and DelTCC1-C2 (Tcc1-C2; lane 6) were cultured at 30°C for 4 h in YPD medium (pH 5.6). Crude extracts (lanes 1 to 3, 300 µg; lane 4, 600 µg; lanes 5 and 6, 1,000 µg each) were subjected to tandem affinity purification using anti-FLAG agarose and Ni-NTA agarose. Total protein (upper panel) and purified fractions (middle panel) were probed with anti-HA antibody. The same purified fractions were immunoblotted (IB) with anti-FLAG antibody (lower panel). The signals for the deletion mutants (W, N1, N2, C1, and C2) are indicated by arrowheads, while the 75-kDa nonspecific signal is indicated by an asterisk. (C) The interaction between the N-terminal portion of the codon-optimized Tcc1p and the N-terminal portion of CaTup1p was detected in the yeast two-hybrid system. Cell suspensions of strains harboring pGADT7 and pGBKT7 derivative plasmids were spotted onto an X-{alpha}-Gal plate assay. Tup1-N is a pGADT7 derivative plasmid that contains the DNA fragment corresponding to amino acid positions 1 to 130 of CaTup1p. Tcc1-N is a pGBKT7 derivative plasmid that contains the codon-optimized DNA fragment corresponding to amino acid positions 1 to 475 of Tcc1p. The pGADT7-T and pGBKT7-53 plasmids, which encode the simian virus 40 large T antigen and the murine p53 protein, respectively, served as positive controls.

Expression profile and nuclear localization of Tcc1p. To determine whether Tcc1p expression alters in response to nutrient depletion, cell cycle toxins, or hyphal induction and whether the expression profile overlaps that of CaTup1p, crude extracts were prepared from yeast or hyphal cells of strain 3TAG-TTS in which CaTup1p, Tcc1p, or CaSsn6p was separately tagged in one cell for Western blotting analysis. Western blotting using anti-FLAG antibody demonstrated that CaTup1p was expressed under all conditions examined (Fig. 4A). Tcc1p-HA was not detected in the G1 phase but was expressed under both yeast and hyphal growth conditions. The expression levels reached a peak when cells were arrested with nocodazole at the G2/M phase. Analysis by anti-Myc antibody Western blotting showed that the profile of the CaSsn6p-Myc expression was similar to that of Tcc1p-HA. Furthermore, we analyzed the CaTup1p protein complex immunoprecipitated from the samples under each condition. Western blotting with Tcc1p-HA and CaSsn6p-Myc showed that the alterations of protein interacting with CaTup1p were similar to the profiles of expression (data not shown). That is, the independent complexes CaTup1p-Tcc1p and CaTup1p-CaSsn6p could exist in similar stages of the cell cycle.

To examine the cellular localization of Tcc1p, CaTup1p, or CaSsn6p, each protein was tagged with three tandem repeats of HA at the C terminus, and cells expressing HA-tagged proteins were immunostained with anti-HA antibody. CaTup1p-HA and CaSsn6p-HA localized in the nucleus as expected (Fig. 4B). Immunostaining with anti-HA antibody also demonstrated nuclear localization of Tcc1p (Fig. 4B). Under conditions that induce hyphae, while obvious nuclear localization of CaTup1p-HA and CaSsn6p-HA was observed, no signal of Tcc1p-HA was detected (data not shown), suggesting that the accumulation of Tcc1p in the nucleus might be dependent on morphology. To confirm the nuclear localization biochemically, subcellular fractionation was performed. A successful fractionation was verified by Western blotting probed with anti-histone H4 antibody. Tcc1p-HA was detected in both the nuclear fraction and the cytoplasmic fraction (Fig. 4C). To investigate whether the accumulation of Tcc1p in the nucleus depends on CaTup1p, HA-tagged Tcc1p was expressed in a CaTUP1 conditional mutant, in which CaTUP1 lies under the regulation of the MET3 promoter, and subcellular fractionation was performed. The repression of CaTUP1 in the presence of methionine and cysteine was verified by microscopic observation and quantitative RT-PCR of CaTUP1 and ECE1 mRNA (data not shown). In the presence of methionine and cysteine (with the MET3 promoter off), CaTUP1 expression was 3% of that of the wild type and, as a consequence, a hypha-specific gene, HYR1, was elevated significantly. Even if the expression of CaTup1p was depressed, Tcc1p-HA was detected in both the nucleus and the cytoplasm (data not shown), indicating that the nuclear localization of Tcc1p may not depend on CaTup1p.

tcc1{Delta} disruptant shows cell elongation phenotype. To investigate the cellular functions of Tcc1p in C. albicans, we deleted both copies. If Tcc1p functions as a CaTup1p-mediated transcriptional repressor, phenotypes of the tcc1{Delta} mutant would at least partly overlap those of the Catup1{Delta} mutant. The two copies of TCC1 were sequentially replaced with ARG4 and a 200-bp portion of hph (hph200) in strain TUA4. The ability to generate a viable tcc1/tcc1 null mutant strain indicates that TCC1 is not an essential gene in C. albicans. There are no significant differences between the wild type and the tcc1{Delta} mutant in their susceptibilities to fluconazole, calcofluor white, sodium chloride, or hydrogen peroxide (data not shown), indicating that TCC1 might not play an important role in drug or stress resistance. To confirm that the loss of the TCC1 function was responsible for any of the observed phenotypes, a PCR-amplified fragment containing a TCC1 complete open reading frame was used to replace the hph200 locus of the tcc1{Delta} mutant TCC103 to generate the reconstituted strain TCC107, in which Tcc1p is tagged with His6-FLAG at the C terminus. As a negative control strain, a PCR-amplified fragment containing only a 100-bp C-terminal portion of the TCC1 gene was used to replace the hph200 locus of TCC103 to generate null mutant TCC106. Both the null mutant TCC106 and the reconstituted strain TCC107, which have a single copy of URA3 at their respective TCC1 loci, were used for the following experiments to compare phenotypes of morphology and virulence.

The effect of the TCC1 deletion on the phenotype of the tcc1{Delta} mutant was studied under conditions that promote both yeast and hyphal growth in C. albicans (Fig. 5A). On a serum agar medium that induces hyphal growth, there were no significant differences among the wild type, the tcc1{Delta} null mutant, and the revertant. On a YPD agar medium adjusted to pH 5, under conditions in which the wild type and the revertant exhibit smooth colonies and the Catup1{Delta} and Canrg1{Delta} mutants should show filamentous growth, as reported previously (4, 5, 19), the TCC1 disruptant exhibited rough colonies, indicating more filamentous growth in the disruptant. The filamentous phenotype of the tcc1{Delta} mutant was enhanced by an alkaline condition. In addition, on Spider medium that induces filamentation, the growth zone of the tcc1{Delta} mutant that indicates filamentation was larger than that of the wild type. These observations suggest that the deletion of TCC1 may enhance the filamentation of C. albicans under the conditions examined in this study.


Figure 5
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  		FIG. 5. Morphology of C. albicans strains grown on solid agar medium (A) and in liquid medium (B). (A) Cells from strains TUA6 (TCC1/TCC1), TCC106 (tcc1/tcc1), and TCC107 (tcc1/tcc1 TCC1) were grown overnight at 30°C. Then, 106 cells were spotted onto the indicated agar plate and grown for 7 days at 30°C on YPD medium and Spider medium and at 37°C on agar medium containing 10% serum. (B) Cells from strains TUA6 (TCC1/TCC1), TCC106 (tcc1/tcc1), and TCC107 (tcc1/tcc1 TCC1) were grown for 2 h under the conditions indicated at the left. Bar, 10 µm.

We then observed cell morphology in liquid yeast- and hypha-inducing media (Fig. 5B). In the YPD medium that supports yeast growth, cell elongation was observed with tcc1{Delta} cells, whereas the length of tcc1{Delta} cells was shorter than the previously reported lengths of Catup1{Delta} and Canrg1{Delta} cells (4, 5, 19). Calculation of the axial growth rates (length/width) supported the fact that tcc1{Delta} cells exhibited the cell elongation phenotype during yeast growth (TUA6, 1.18 ± 0.31 [n = 204]; TCC106, 2.47 ± 0.85 [n = 221]; and TCC107, 1.44 ± 0.26 [n = 206]). Additionally, tcc1{Delta} cells did not exhibit a Catup1{Delta}-like constitutive filamentation without constriction. There were no significant differences between the wild type and the tcc1{Delta} mutant grown in Spider medium or in serum medium, which induces hyphal growth. The phenotypes described above were restored in the reconstituted strain TCC107, indicating that filamentous phenotypes were caused solely by the deletion of the TCC1 gene.

Effects of TCC1 disruption on the transcription of hypha-specific genes. To determine whether the transcription of HSGs is induced under yeast growth conditions by TCC1 disruption as well as by CaTUP1 or CaNRG1 disruption, we compared the expression levels of HSGs such as HWP1 (28), ECE1 (2), HYR1 (1), and HGC1 (34) by Northern hybridization and quantitative real-time RT-PCR. If Tcc1p served as a transcriptional repressor for HSGs, as does CaTup1p, HSGs would be upregulated by Tcc1p depletion. The wild type, the disruptant, and the reconstituted strain were cultured under yeast or hyphal growth conditions and subjected to Northern blotting analysis (Fig. 6A). A comparison of yeast growth (Fig. 6A, lane 1) and hyphal growth (Fig. 6A, lane 2 or 3) confirmed the successful detection of HSGs. There were no significant differences between the wild type and the tcc1{Delta} disruptant in the HWP1, the ECE1, or the HGC1 gene expression levels, based on Northern analysis. The expression of HYR1 was decreased by TCC1 deletion under hyphal growth conditions, consistent with previous data indicating that HYR1 mRNA in the Catup1{Delta} mutant under such conditions was lower than that in the wild type (15). This implies that the CaTup1p-Tcc1p complex might regulate the activation or repression of a transcriptional repressor in the HYR1 gene during filamentous growth. However, Northern analysis indicated that the yeast growth conditions did not seem to induce any significant HSGs.


Figure 6
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  		FIG. 6. Expression of hypha-specific genes in the tcc1{Delta} mutant. (A) Northern blotting. Cells were grown for 3 h in the indicated media (Y, YPD, pH 5.6, at 30°C; Se, YPD, pH 7.2, plus 10% calf serum at 37°C; Sp, Spider medium at 37°C). RNA was prepared from each strain, and Northern analysis was performed using probes for the indicated genes. The ethidium bromide-stained rRNAs are presented below the hybridization patterns to demonstrate comparable loading. (B) Quantitative real-time RT-PCR analysis of hypha-specific mRNAs. Strains TUA6 (the wild type), TCC106 (tcc1/tcc1), SSN602 (ssn6/ssn6), TUP102 (tup1/tup1), and NRG102 (nrg1/nrg1) were used. Values normalized to ACT1 mRNA and fold induction relative to the values of the TUA6 wild-type strain are shown for four HSGs (HWP1, ECE1, HYR1, and HGC1). Data are shown as means ± standard deviations of results from at least three experiments.

As described above, the filamentous phenotype of the tcc1{Delta} mutant was less than that of the Catup1{Delta} or Canrg1{Delta} mutant (Fig. 5). Therefore, since it remains a possibility that derepression by TCC1 deletion might be weaker than that by CaTUP1 or CaNRG1 deletion, we attempted to detect the expression of HSGs by quantitative real-time RT-PCR, which has a higher sensitivity than Northern analysis. Compared to the results reported so far, the HWP1 and ECE1 mRNA were derepressed in Catup1{Delta} and Canrg1{Delta} cells but not in the Cassn6 mutant (Fig. 6B), consistent with the report by Garcia-Sanchez et al. (12). Interestingly, HYR1 mRNA and HGC1 mRNA were derepressed in Catup1{Delta} and Canrg1{Delta} cells and even in the Cassn6{Delta} cells. However, Garcia-Sanchez et al. (12) reported that HYR1 mRNA was not elevated in Canrg1{Delta} and Cassn6{Delta} cells and that HGC1 mRNA was not elevated in Cassn6{Delta} cells. These discrepancies are probably caused by an experimental difference between microarray analysis and quantitative RT-PCR. These results indicate that HWP1/ECE1 and HYR1/HGC1 might be transcriptionally regulated in a different manner and that CaSsn6p may regulate HYR1 and HGC1 negatively. Moreover, all HSGs tested were derepressed by the deletion of TCC1, but the expression level of the TCC1 mutant was lower than that of the Catup1{Delta} or Canrg1{Delta} mutant (Fig. 6B). Therefore, these results further support that Tcc1p, accompanied by CaTup1p, might function as a transcriptional repressor involved in the morphological transition of C. albicans.

The filamentous phenotype of the tcc1{Delta} mutant is restored by HGC1 disruption. In the previous paragraph, we demonstrated that Tcc1p might repress the expression of the hypha-specific G1 cyclin Hgc1p under conditions that induce yeast growth. The concept that HGC1 acts downstream of CaTup1p regulation has been supported by the previous report that constitutive filamentation is blocked by the deletion of HGC1 (34). To examine whether Tcc1p is also linked to the function of HGC1, we deleted HGC1 together with TCC1. The observation that the hgc1{Delta} single mutant was deficient in hyphal formation (Fig. 7) was consistent with a previous report (34). The double mutant did not show filamentation on a serum medium (Fig. 7), in that the deletion of TCC1 did not induce filamentation of hgc1{Delta} cells and the deletion of HGC1 reduced filamentation of tcc1{Delta} cells. On a YPD agar medium, the deletion of HGC1 eliminated the filamentous phenotype of tcc1{Delta} cells as well as that of Catup1{Delta} cells. This reinforced the idea that CaTup1p and Tcc1p might coordinate in regulating the function of HGC1, followed by the repression of hyphal formation.


Figure 7
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  		FIG. 7. Filamentation of the tcc1{Delta} mutant was restored by HGC1 disruption. Cells from strains TUA6 (TCC1/TCC1 HGC1/HGC1), TCC106 (tcc1/tcc1 HGC1/HGC1), HGC104 (TCC1/TCC1 hgc1/hgc1), and HGC114 (tcc1/tcc1 hgc1/hgc1) were grown overnight at 30°C. Then, 106 cells were spotted onto the indicated agar plate and grown for 7 days at 30°C on YPD medium and at 37°C on agar medium containing 10% serum. Photographs of colony edges were taken with a phase-contrast microscope at x20 magnification.

Attenuated virulence in the tcc1{Delta} disruptant. CaTUP1 or CaNRG1 disruption causes reduced virulence in a mouse model of systemic candidiasis (19). To determine whether Tcc1p plays an important role in pathogenicity, mice were intravenously injected with the wild type (TUA6), the tcc1{Delta} null mutant (TCC106), and the revertant (TCC107) and monitored for survival. We found that mice infected with 106 CFU of the tcc1{Delta} mutant did not die until 31 days postinfection, with a sustained-survival curve, whereas the same-size inocula of the wild-type or the reconstructed strain killed all infected mice within 11 days (Fig. 8). The mean survival times for the mice infected with the wild type, the null mutant, and the revertant were 7.5 ± 3.78 days, 21.2 ± 8.07 days, and 9 ± 2.35 days, respectively (TCC106 versus TUA6, P < 0.01; TCC106 versus TCC107, P < 0.02). This demonstrates that TCC1 is involved in C. albicans virulence and supports, in part, the concept that Tcc1p might function with CaTup1p in C. albicans.


Figure 8
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  		FIG. 8. The tcc1{Delta} mutant exhibits markedly reduced virulence. C. albicans cells from TUA6 (TCC1/TCC1), TCC106 (tcc1/tcc1), and TCC107 (tcc1/tcc1+TCC1) were grown overnight in YPD. Each mouse was injected via the tail vein with 106 CFU and monitored for survival.


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DISCUSSION
 
In this study, we identified proteins that form complexes with CaTup1p, a global transcriptional repressor. Analogous to the S. cerevisiae homolog, CaSsn6p has long been regarded as a CaTup1p-binding partner for mediation of the negative regulation of yeast-hypha morphological transition in C. albicans. However, whether CaSsn6p actually interacts with CaTup1p remains unproven. Furthermore, a recent report (12) in which DNA microarray analysis was performed to compare the wild type and the Cassn6 mutant indicates that genes repressed by CaTup1p do not necessarily correspond to those repressed by CaSsn6p. In other words, it is highly possible that CaSsn6p may not be a CaTup1p-binding partner, at least in the morphological transition of C. albicans. In order to isolate a real binding partner that chiefly regulates morphogenesis, we used the so-called TAP technique, which we previously used for the purification of a septin complex (16). We found that complexes purified by the TAP technique contained CaSsn6p (Fig. 1). Moreover, immunoprecipitation/Western analysis of strains expressing CaTup1p and CaSsn6p tagged with a different epitope demonstrated that CaSsn6p interacted with CaTup1p (Fig. 2). Therefore, we were able to provide evidence that CaTup1p and CaSsn6p of C. albicans formed a protein complex, as did those of S. cerevisiae.

To identify a binding partner that actually regulates the morphology of C. albicans together with CaTup1p, we purified a protein complex including CaTup1p. One of the components contained in the CaTup1p complex was identified as TCC1, a Tup1p-complex component. In addition, we demonstrated that CaTup1p independently interacted with Tcc1p or CaSsn6p. However, Tcc1p and CaSsn6p share common properties and behaviors as described below. Their first similarity is the existence of a TPR domain: Tcc1p contains four copies of the TPR motif, and nine TPRs are located within the C. albicans Ssn6p polypeptide. Since both Tcc1p and CaSsn6p possess the TPR motif, Tcc1p probably interacts with CaTup1p in a mode similar to that of CaSsn6p. Actually, we demonstrated that the TPR domains of Tcc1p are responsible for CaTup1p binding by using immunoprecipitation and yeast two-hybrid analysis (Fig. 3). Their second behavior in common is their expression profiles. The expression levels of Tcc1p and CaSsn6p, which were tagged with different epitopes in one cell, were altered in response to cell cycle toxins and glucose depletion: the expression levels reached a peak when cells were arrested at the G2/M phase and decreased to an undetectable level in the unbudded G1 cells (Fig. 4A). In addition, the complex formation with CaTup1p in response to cell cycle toxins was no different between Tcc1p and CaSsn6p (data not shown). Their third similarity is localization to the nucleus. Immunostaining with anti-HA antibody demonstrated that Tcc1p and CaSsn6p localize to the nucleus (Fig. 4B), although both were predicted to contain no nuclear localization signals. Whereas subcellular fractionation further supports the nuclear localization of Tcc1p, Tcc1p was also detected in the cytoplasmic fraction. Tcc1p localization to the nucleus was not altered in a CaTUP1-depleted cell, suggesting that Tcc1p localization may not depend on CaTup1p. Since the nuclear localization of Tcc1p was not observed in hyphal cells, shuttling between the cytoplasm and the nucleus would be regulated by some signaling associated with morphology. Despite these common characteristics, Tcc1p and CaSsn6p seem to function differently: the phenotypes of the mutants and the transcriptional control of HWP1 and ECE1 were contradictory between Tcc1p and CaSsn6p. One of the reasons that the functions of Tcc1p and CaSsn6p have different effects is assumed to be because they might require disparate sequence-specific DNA-binding proteins. Nevertheless, derepression of HYR1 and HGC1 mRNA in tcc1{Delta} and Cassn6{Delta} cells might indicate the existence of a common DNA-binding protein. Future global transcriptional analysis using a DNA microarray might give clues for solving the question of why the depletion of each protein resulted in opposing phenotypes, despite the common binding partner, the similar expression profiles, and the nuclear localization. Also, study of a double deletion of TCC1 and CaSSN6 would explain how the two products share roles.

The Catup1{Delta} null mutant demonstrates constitutive filamentation even under conditions that induce the yeast form (4). The Cassn6{Delta} homozygous mutant shows a higher rate for phenotype switching (12) and no filamentation on serum or Spider agar medium (data not shown). When both alleles of TCC1 identified in this study were deleted, the disruptant grew in a pseudohyphal form under conditions that induce the yeast form. Obviously, the morphological phenotype of tcc1{Delta} is more similar to that of Catup1{Delta} than to that of Cassn6{Delta} from the viewpoint of the cell elongation phenotype. Taken together with the results of the immunoprecipitation experiment and morphological observation, it is highly possible that the protein complex consisting of CaTup1p and Tcc1p might behave independently of CaTup1p-CaSsn6p. However, the filamentous phenotype of the tcc1{Delta} mutant was not as severe as that of the Catup1{Delta} or Canrg1{Delta} mutant, and the tcc1{Delta} mutant showed a relatively mild cell elongation. This reduced severity is probably associated with the degrees of HSG repression. For example, the elevation of derepressed HSG transcription by the deletion of TCC1 was not stronger than that in the Catup1{Delta} or Canrg1{Delta} mutant (Fig. 6). In addition, although we did not compare them directly, the virulence demonstrated by the tcc1{Delta}, Catup1{Delta}, or Canrg1{Delta} mutant indicates a decrease or attenuation: all tcc1{Delta} mutant-infected mice were killed within 31 days (Fig. 8), while almost no mice inoculated with the strain from which CaTUP1 or CaNRG1 was deleted were dead within the time period examined (4, 5, 19). These results suggest that the degree of cell elongation, the increased quantity of HSG transcription, and the attenuated virulence, which are caused by gene disruption, are closely connected to each other. While the Catup1{Delta} mutant was demonstrated to be more sensitive to hydrogen peroxide than the wild type (18), the tcc1{Delta} mutant did not show significant differences in drug or stress resistance (data not shown), enhancing the significance of the effect of Tcc1p on morphogenesis. Moreover, deletion of the CaTup1p-binding partner gene, TCC1, did not result in more severe filamentation than did deletion of TUP1, suggesting that a relationship of CaTup1p and Tcc1p is not as essential for transcriptional repression but that Tcc1p would play a supportive role for the CaTup1p function.

We have identified Tcc1p as a novel factor that interacts with CaTup1p. No homolog of Tcc1p could be identified in S. cerevisiae, suggesting the possibility that Tcc1p has C. albicans-specific functions. Considering the phenotypes of the tcc1{Delta} mutant, it would not be unreasonable to think about a relationship with CaNrg1p, a DNA-binding protein that represses hypha-specific genes during the yeast phase. Evidence that proves direct binding between Tup1p and Nrg1p in C. albicans has not yet been reported. Based on genes regulated by TCC1, a hypothesis is proposed that DNA-binding protein CaNrg1p recruits a Tcc1p-CaTup1p protein complex to promoter regions of hypha-specific genes, including HGC1, to repress transcription of these genes. A mechanical relationship among CaTup1p, Tcc1p, and CaNrg1p remains unproven. We are now investigating the relationship between CaNrg1p and CaTup1p-Tcc1p, although it is difficult to deal with the CaNrg1p protein because of its low-level expression and fragility (our unpublished results).

To conclude, we have presented a deletion analysis of TCC1, a novel C. albicans gene encoding a protein with TPR motifs. Tcc1p forms a protein complex with CaTup1p, although direct binding between the two proteins remains unproven. Many genes have been reported to date whose deletion results in constitutive filamentation similar to that in the Catup1{Delta} mutant. An analysis of detailed relationships, including the transcriptional regulation of, or the protein-protein interaction resulting from, these genes that maintain the yeast form, including Tcc1p, will provide a clue to the elucidation of the mechanisms of the yeast-hypha transition and virulence in C. albicans.


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ACKNOWLEDGMENTS
 
We are grateful to J. Morschhauser for generously providing plasmids.

A. Kaneko was supported by the Cooperative System for Supporting Priority Research, Japan Science and Technology Corporation. This work was supported in part by grant KH53315 from the Japan Health Sciences Foundation. It was also supported by Health Science Research Grants for Research on Emerging and Re-emerging Infectious Diseases of the Ministry of Health, Labor and Welfare of Japan.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Bioactive Molecules, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. Phone: 81 3 5285-1111. Fax: 81 3 5285-1175. E-mail: yuehara{at}nih.go.jp. Back

{triangledown} Published ahead of print on 22 September 2006. Back

{dagger} Supplemental material for this article may be found at http://ec.asm.org/. Back


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Eukaryotic Cell, November 2006, p. 1894-1905, Vol. 5, No. 11
1535-9778/06/$08.00+0     doi:10.1128/EC.00151-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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