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ARTICLE

Candida albicans Lacking the Gene Encoding the Regulatory Subunit of Protein Kinase A Displays a Defect in Hyphal Formation and an Altered Localization of the Catalytic Subunit

Alejandro Cassola, Marc Parrot, Susana Silberstein, Beatrice B. Magee, Susana Passeron, Luc Giasson, María L. Cantore
Alejandro Cassola
1Cátedra de Microbiología, Facultad de Agronomía, Universidad de Buenos Aires, IBYF-CONICET, C1417DSE Buenos Aires, Argentina
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Marc Parrot
2School of Dentistry, GREB Laval University, Sainte-Foy, Quebec, Canada G1K 7P4
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Susana Silberstein
1Cátedra de Microbiología, Facultad de Agronomía, Universidad de Buenos Aires, IBYF-CONICET, C1417DSE Buenos Aires, Argentina
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Beatrice B. Magee
3Department of Genetics, Cell Biology and Development, University of Minnesota, St. Paul, Minnesota 55108
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Susana Passeron
1Cátedra de Microbiología, Facultad de Agronomía, Universidad de Buenos Aires, IBYF-CONICET, C1417DSE Buenos Aires, Argentina
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Luc Giasson
2School of Dentistry, GREB Laval University, Sainte-Foy, Quebec, Canada G1K 7P4
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María L. Cantore
1Cátedra de Microbiología, Facultad de Agronomía, Universidad de Buenos Aires, IBYF-CONICET, C1417DSE Buenos Aires, Argentina
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  • For correspondence: cantore@agro.uba.ar
DOI: 10.1128/EC.3.1.190-199.2004
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  • FIG. 1.
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    FIG. 1.

    Restriction map of the C. albicansBCY1 locus. The thick line represents a 7.4-kb EcoRV C. albicans genomic fragment containing the BCY1 gene from fosmid 4A12. The white arrow indicates the position of the C. albicansBCY1 open reading frame. pMP1 and pMP3 refer to the constructions made to delete the C. albicansBCY1 alleles. Endonuclease restriction sites: E, EcoRI; EV, EcoRV; K, KpnI; P, PinAI; S, StyI.

  • FIG. 2.
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    FIG. 2.

    Sequence alignment of the deduced amino acid sequence of C. albicansBCY1 with those of R subunits from several fungal species. Identities are in black boxes. Gaps introduced for alignment are indicated by dashes. The alignment was performed with Clustal W (38) and edited with GeneDoc (version 2.6.002). Potential phosphorylation sites are indicated by an arrowhead (▾). C.a., Candida albicans; S.c., Saccharomyces cerevisiae (accession number M15756 ); B.e., Blastocladiella emersonii (accession number M81713 ); A.n., Aspergillus nidulans (accession number AF043231 ); M.g., Magnaporthe grisea (accession number AF024633 ); N.c., Neurospora crassa (accession number L78009 ).

  • FIG. 3.
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    FIG. 3.

    Effect of TPK2 overexpression on growth of the BCY1 null strain. A C. albicans TPK1/TPK1tpk2/tpk2bcy1/bcy1 mutant was transformed with plasmid pLS4. Four different transformants were tested (•, ⧫, ▴, ▾). The control strain was CAI4 transformed with plasmid pBI (▪). Strains were grown in noninducing medium SD (A) and in CSAA inducing medium (B) at 30°C with agitation (150 rpm). The optical density of the cultures was measured at 600 nm.

  • FIG. 4.
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    FIG. 4.

    Western blot analysis of crude extract from strains CAI4, H2D, and bcy1 tpk2. Equal amounts of protein were subjected to SDS-10% PAGE, transferred to a polyvinylidene difluoride membrane, and reacted with anti-C. albicans Bcy1p antiserum as described in Materials and Methods. The arrow on the left indicates the migration position of Bcy1p.

  • FIG. 5.
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    FIG. 5.

    Protein kinase A activity in crude extracts from CAI4, H2D and bcy1 tpk2 strains. Protein kinase A (PKA) activity was measured in aliquots of crude extracts from the three strains in the absence and in the presence of 10 μM cAMP as described in Materials and Methods. Values are means ± standard deviation from three independent experiments.

  • FIG. 6.
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    FIG. 6.

    Growth of strains CAI4, H2D, and bcy1 tpk2. Cells were grown in liquid YPD supplemented with uridine (50 μg/ml). The optical density at 600 nm (OD600) of the cultures was adjusted to 0.1 with the same medium, and 5-μl aliquots from the cultures and from 10-fold serial dilutions were spotted onto YPD plates supplemented with uridine (50 μg/ml). Plates were incubated at 30°C for 2 days.

  • FIG. 7.
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    FIG. 7.

    Germinative behavior of strains CAI4, H2D, and bcy1 tpk2. Stationary-phase yeast cells were induced to germinate for 2 h at 37°C in MnCl2/imidazole medium in the presence of 10% horse serum or 10 mM GlcNAc. Bar, 5 μm.

  • FIG. 8.
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    FIG. 8.

    Coimmunoprecipitation of Tpk1-GFP with Bcy1p. S-100 fractions from strains CAI4, H2D, bcy1 tpk2, ASM1, ASM2, and ASM3 containing the same amounts of protein were subjected to immunoprecipitation with anti-C. albicans Bcy1p antiserum as described in Materials and Methods. Immunoprecipitates were resolved in SDS-7.5% PAGE, transferred to polyvinylidene difluoride membranes, and developed with a monoclonal anti-GFP antibody. Lanes: 1, parental strain CAI4; 2, mutant H2D strain; 3, bcy1 tpk2 double mutant strain; 4, ASM1 strain; 5, ASM2 strain; 6, ASM3 strain. The positions of molecular mass markers are indicated on the left. The arrow indicates the position of the 96-kDa fused protein band. The low-molecular-mass band observed in all lanes corresponds to a nonspecific reaction of the antibody.

  • FIG. 9.
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    FIG. 9.

    Subcellular localization of Tpk1-GFP in wild-type and mutant strains. Localization of the fusion protein was visualized by fluorescence microscopy in strains ASM1, ASM2, and ASM3 (upper panels). Nuclei were visualized by DAPI staining (lower panels). Bar, 5 μm.

Tables

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  • TABLE 1.

    Strains and plasmids used in this study

    Strain or plasmidCharacteristicsSource or reference
    C. albicans strains
        CAI4 ura3::λimm434/ura3::λimm43411
        H2D ura3::λimm434/ura3::λimm434 Δtpk2::cat/Δtpk2::cat7
        bcy1 tpk2 ura3::λimm434/ura3::λimm434 Δtpk2::cat/Δtpk2::cat bcy1/bcy1This study
        ASM1 ura3::λimm434/ura3::λimm434 TPK1/TPK1-GFP-URA3This study
        ASM2 ura3::λimm434/ura3::λimm434 Δtpk2::cat/Δtpk2::cat TPK1/TPK1-GFP-URA3This study
        ASM3 ura3::λimm434/ura3::λimm434 Δtpk2::cat/Δtpk2::cat bcy1/bcy1 TPK1/TPK1-GFP-URA3This study
    Plasmids
        pBI-1 PCK1 promoter in URA3-marked CARS2 vectorJ. F. Emst
        pLS4 PCK1 promoter controlling a His-tagged TPK2 in pRC2312E. Setiadi J. F. Ernst
  • TABLE 2.

    Results of transformation experiments performed to delete both alleles of BCY1 in C. albicans strain CAI4

    Expected genotype (plasmid)aNo. of transformants tested by Southern analysisNo. of transformants of the expected genotype
    BCY1/Δbcy1::cat-URA3-cat (pMP1)92
    Δbcy1::cat-URA3-cat/Δbcy1::cat (pMP1)1130
    bcy1::cat-URA3-cat/Δbcy1::cat (pMP3)200
    BCY1/Δbcy1::cat-URA3-cat Δtpk2::cat/Δtpk2::cat (pMP1)165
    Δbcy1::cat-URA3-cat/Δbcy1::cat Δtpk2::cat/Δtpk2::cat (pMP1)319
    • ↵ a The transformations were performed with the indicated plasmid.

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Candida albicans Lacking the Gene Encoding the Regulatory Subunit of Protein Kinase A Displays a Defect in Hyphal Formation and an Altered Localization of the Catalytic Subunit
Alejandro Cassola, Marc Parrot, Susana Silberstein, Beatrice B. Magee, Susana Passeron, Luc Giasson, María L. Cantore
Eukaryotic Cell Feb 2004, 3 (1) 190-199; DOI: 10.1128/EC.3.1.190-199.2004

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Candida albicans Lacking the Gene Encoding the Regulatory Subunit of Protein Kinase A Displays a Defect in Hyphal Formation and an Altered Localization of the Catalytic Subunit
Alejandro Cassola, Marc Parrot, Susana Silberstein, Beatrice B. Magee, Susana Passeron, Luc Giasson, María L. Cantore
Eukaryotic Cell Feb 2004, 3 (1) 190-199; DOI: 10.1128/EC.3.1.190-199.2004
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KEYWORDS

Candida albicans
Cyclic AMP-Dependent Protein Kinases

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