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A CUG codon adapted two-hybrid system for the pathogenic fungus Candida albicans.

Stynen B, Van Dijck P, Tournu H - Nucleic Acids Res. (2010)

Bottom Line: Most notably, C. albicans does not follow the universal genetic code, by translating the CUG codon into serine instead of leucine.We further confirmed interactions between components of the filamentation/mating MAP kinase pathway, including the unsuspected interaction between the MAP kinases Cek2 and Cek1.We conclude that this system can be used to enhance our knowledge of protein-protein interactions in C. albicans.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Cell Biology, Department of Molecular Microbiology, Institute of Botany and Microbiology, Katholieke Universiteit Leuven, VIB, Kasteelpark Arenberg 31, B-3001 Leuven-Heverlee, Flanders, Belgium.

ABSTRACT
The genetics of the most common human pathogenic fungus Candida albicans has several unique characteristics. Most notably, C. albicans does not follow the universal genetic code, by translating the CUG codon into serine instead of leucine. Consequently, the use of Saccharomyces cerevisiae as a host for yeast two-hybrid experiments with C. albicans proteins is limited due to erroneous translation caused by the aberrant codon usage of C. albicans. To circumvent the need for heterologous expression and codon optimalization of C. albicans genes we constructed a two-hybrid system with C. albicans itself as the host with components that are compatible for use in this organism. The functionality of this two-hybrid system was shown by successful interaction assays with the protein pairs Kis1-Snf4 and Ino4-Ino2. We further confirmed interactions between components of the filamentation/mating MAP kinase pathway, including the unsuspected interaction between the MAP kinases Cek2 and Cek1. We conclude that this system can be used to enhance our knowledge of protein-protein interactions in C. albicans.

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Related in: MedlinePlus

(A) Two-hybrid interaction of C. albicans Ino4 with Ino2. Ino4 and Ino2 interact strongly, shown by growth of strain SC2H3 on SC–HIS after one day with Ino4 as bait and Ino2 as prey. This interaction is confirmed in the galactosidase assay where the activity measured (in Miller units) was 2.5-fold the background activity. (B) Two-hybrid interaction of C. albicans Kis1 with Snf4. Kis1 and Snf4, two proteins of the Snf1 complex, interact with each other as shown by growth of SC2H3 on SC–HIS and a high galactosidase activity. For the HIS1 reporter assay, cells were incubated for up to 2 days. (C) Co-immunoprecipitation of Kis1 and Snf4. The interaction between bait LexA–HA–Kis1 (73 kDa) and prey VP16–FLAG–Snf4 (47 kDa) is confirmed in a co-IP experiment. Total protein concentrations were equal for each sample. Lane 1: loading control of Kis1; lane 2: IP of Kis1 with anti-HA antibodies; lane 3: co-IP of Kis1 with anti-FLAG antibodies; lane 4: negative control of Kis1 immunoprecipitation without antibodies; lane 5: loading control of Snf4; lane 6: co-IP of Snf4 with anti-HA antibodies; lane 7: IP of Snf4 with anti-FLAG antibodies; lane 8: negative control of Snf4 immunoprecipitation without antibodies. The antibodies used for western blotting are indicated on the right side of each blot and the antibodies for immunoprecipitation are shown below the blot. IP-AB: antibody used for immunoprecipitation; LC: loading control; αFL: anti-FLAG antibody; αHA: anti-HA antibody.
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Figure 3: (A) Two-hybrid interaction of C. albicans Ino4 with Ino2. Ino4 and Ino2 interact strongly, shown by growth of strain SC2H3 on SC–HIS after one day with Ino4 as bait and Ino2 as prey. This interaction is confirmed in the galactosidase assay where the activity measured (in Miller units) was 2.5-fold the background activity. (B) Two-hybrid interaction of C. albicans Kis1 with Snf4. Kis1 and Snf4, two proteins of the Snf1 complex, interact with each other as shown by growth of SC2H3 on SC–HIS and a high galactosidase activity. For the HIS1 reporter assay, cells were incubated for up to 2 days. (C) Co-immunoprecipitation of Kis1 and Snf4. The interaction between bait LexA–HA–Kis1 (73 kDa) and prey VP16–FLAG–Snf4 (47 kDa) is confirmed in a co-IP experiment. Total protein concentrations were equal for each sample. Lane 1: loading control of Kis1; lane 2: IP of Kis1 with anti-HA antibodies; lane 3: co-IP of Kis1 with anti-FLAG antibodies; lane 4: negative control of Kis1 immunoprecipitation without antibodies; lane 5: loading control of Snf4; lane 6: co-IP of Snf4 with anti-HA antibodies; lane 7: IP of Snf4 with anti-FLAG antibodies; lane 8: negative control of Snf4 immunoprecipitation without antibodies. The antibodies used for western blotting are indicated on the right side of each blot and the antibodies for immunoprecipitation are shown below the blot. IP-AB: antibody used for immunoprecipitation; LC: loading control; αFL: anti-FLAG antibody; αHA: anti-HA antibody.

Mentions: To validate the functionality of the two-hybrid system, interactions between C. albicans proteins that were previously shown to occur were analyzed with the two-hybrid tool. We first examined the heterodimerization of the proteins Ino2 and Ino4. Ino2 is a transcription factor that, together with Ino4, putatively regulates ribosomal protein genes in C. albicans (8). The Ino2–Ino4 dimer binds ICRE (inositol/choline-responsive element)-related motif sequences. After modification of a CTG codon in the basic helix-loop-helix region of Ino4, Ino4 and Ino2 were previously shown to interact in a yeast two-hybrid assay (8). In the Candida background, we have shown that Ino2 could activate HIS1 and StlacZ in a one-hybrid assay (Figure 2A). We cloned full-length INO2 into the prey plasmid pC2HP and full-length INO4 in the bait plasmid pC2HB. The two-hybrid strain SC2H3 was transformed with pC2HB–Ino4 or with the empty control vector pC2HB on medium lacking leucine. A second transformation step on medium lacking arginine was performed with the prey plasmid pC2HP-Ino2 or with the empty control vector pC2HP. As shown in Figure 3A, only the combination pC2HB–Ino4 with pC2HP–Ino2 led to growth on selective medium, indicating interaction between the two proteins. For validation of the binding of Ino4 with Ino2, the second reporter assay, with StlacZ, was performed. As shown in Figure 3A, the values of a galactosidase assay were significantly higher for the interaction compared to the negative controls. For sustainable and strong interactions, growth was clearly visible on selective medium after 1 to 2 days of incubation. For interactions that may be either transient or weaker, one can expect growth on selective medium to occur within 5–7 days. In parallel, LacZ expression levels >100 U can be considered the read-out for a strong and constitutive interaction.Figure 3.


A CUG codon adapted two-hybrid system for the pathogenic fungus Candida albicans.

Stynen B, Van Dijck P, Tournu H - Nucleic Acids Res. (2010)

(A) Two-hybrid interaction of C. albicans Ino4 with Ino2. Ino4 and Ino2 interact strongly, shown by growth of strain SC2H3 on SC–HIS after one day with Ino4 as bait and Ino2 as prey. This interaction is confirmed in the galactosidase assay where the activity measured (in Miller units) was 2.5-fold the background activity. (B) Two-hybrid interaction of C. albicans Kis1 with Snf4. Kis1 and Snf4, two proteins of the Snf1 complex, interact with each other as shown by growth of SC2H3 on SC–HIS and a high galactosidase activity. For the HIS1 reporter assay, cells were incubated for up to 2 days. (C) Co-immunoprecipitation of Kis1 and Snf4. The interaction between bait LexA–HA–Kis1 (73 kDa) and prey VP16–FLAG–Snf4 (47 kDa) is confirmed in a co-IP experiment. Total protein concentrations were equal for each sample. Lane 1: loading control of Kis1; lane 2: IP of Kis1 with anti-HA antibodies; lane 3: co-IP of Kis1 with anti-FLAG antibodies; lane 4: negative control of Kis1 immunoprecipitation without antibodies; lane 5: loading control of Snf4; lane 6: co-IP of Snf4 with anti-HA antibodies; lane 7: IP of Snf4 with anti-FLAG antibodies; lane 8: negative control of Snf4 immunoprecipitation without antibodies. The antibodies used for western blotting are indicated on the right side of each blot and the antibodies for immunoprecipitation are shown below the blot. IP-AB: antibody used for immunoprecipitation; LC: loading control; αFL: anti-FLAG antibody; αHA: anti-HA antibody.
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Figure 3: (A) Two-hybrid interaction of C. albicans Ino4 with Ino2. Ino4 and Ino2 interact strongly, shown by growth of strain SC2H3 on SC–HIS after one day with Ino4 as bait and Ino2 as prey. This interaction is confirmed in the galactosidase assay where the activity measured (in Miller units) was 2.5-fold the background activity. (B) Two-hybrid interaction of C. albicans Kis1 with Snf4. Kis1 and Snf4, two proteins of the Snf1 complex, interact with each other as shown by growth of SC2H3 on SC–HIS and a high galactosidase activity. For the HIS1 reporter assay, cells were incubated for up to 2 days. (C) Co-immunoprecipitation of Kis1 and Snf4. The interaction between bait LexA–HA–Kis1 (73 kDa) and prey VP16–FLAG–Snf4 (47 kDa) is confirmed in a co-IP experiment. Total protein concentrations were equal for each sample. Lane 1: loading control of Kis1; lane 2: IP of Kis1 with anti-HA antibodies; lane 3: co-IP of Kis1 with anti-FLAG antibodies; lane 4: negative control of Kis1 immunoprecipitation without antibodies; lane 5: loading control of Snf4; lane 6: co-IP of Snf4 with anti-HA antibodies; lane 7: IP of Snf4 with anti-FLAG antibodies; lane 8: negative control of Snf4 immunoprecipitation without antibodies. The antibodies used for western blotting are indicated on the right side of each blot and the antibodies for immunoprecipitation are shown below the blot. IP-AB: antibody used for immunoprecipitation; LC: loading control; αFL: anti-FLAG antibody; αHA: anti-HA antibody.
Mentions: To validate the functionality of the two-hybrid system, interactions between C. albicans proteins that were previously shown to occur were analyzed with the two-hybrid tool. We first examined the heterodimerization of the proteins Ino2 and Ino4. Ino2 is a transcription factor that, together with Ino4, putatively regulates ribosomal protein genes in C. albicans (8). The Ino2–Ino4 dimer binds ICRE (inositol/choline-responsive element)-related motif sequences. After modification of a CTG codon in the basic helix-loop-helix region of Ino4, Ino4 and Ino2 were previously shown to interact in a yeast two-hybrid assay (8). In the Candida background, we have shown that Ino2 could activate HIS1 and StlacZ in a one-hybrid assay (Figure 2A). We cloned full-length INO2 into the prey plasmid pC2HP and full-length INO4 in the bait plasmid pC2HB. The two-hybrid strain SC2H3 was transformed with pC2HB–Ino4 or with the empty control vector pC2HB on medium lacking leucine. A second transformation step on medium lacking arginine was performed with the prey plasmid pC2HP-Ino2 or with the empty control vector pC2HP. As shown in Figure 3A, only the combination pC2HB–Ino4 with pC2HP–Ino2 led to growth on selective medium, indicating interaction between the two proteins. For validation of the binding of Ino4 with Ino2, the second reporter assay, with StlacZ, was performed. As shown in Figure 3A, the values of a galactosidase assay were significantly higher for the interaction compared to the negative controls. For sustainable and strong interactions, growth was clearly visible on selective medium after 1 to 2 days of incubation. For interactions that may be either transient or weaker, one can expect growth on selective medium to occur within 5–7 days. In parallel, LacZ expression levels >100 U can be considered the read-out for a strong and constitutive interaction.Figure 3.

Bottom Line: Most notably, C. albicans does not follow the universal genetic code, by translating the CUG codon into serine instead of leucine.We further confirmed interactions between components of the filamentation/mating MAP kinase pathway, including the unsuspected interaction between the MAP kinases Cek2 and Cek1.We conclude that this system can be used to enhance our knowledge of protein-protein interactions in C. albicans.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Cell Biology, Department of Molecular Microbiology, Institute of Botany and Microbiology, Katholieke Universiteit Leuven, VIB, Kasteelpark Arenberg 31, B-3001 Leuven-Heverlee, Flanders, Belgium.

ABSTRACT
The genetics of the most common human pathogenic fungus Candida albicans has several unique characteristics. Most notably, C. albicans does not follow the universal genetic code, by translating the CUG codon into serine instead of leucine. Consequently, the use of Saccharomyces cerevisiae as a host for yeast two-hybrid experiments with C. albicans proteins is limited due to erroneous translation caused by the aberrant codon usage of C. albicans. To circumvent the need for heterologous expression and codon optimalization of C. albicans genes we constructed a two-hybrid system with C. albicans itself as the host with components that are compatible for use in this organism. The functionality of this two-hybrid system was shown by successful interaction assays with the protein pairs Kis1-Snf4 and Ino4-Ino2. We further confirmed interactions between components of the filamentation/mating MAP kinase pathway, including the unsuspected interaction between the MAP kinases Cek2 and Cek1. We conclude that this system can be used to enhance our knowledge of protein-protein interactions in C. albicans.

Show MeSH
Related in: MedlinePlus