Limits...
Functional study of the Hap4-like genes suggests that the key regulators of carbon metabolism HAP4 and oxidative stress response YAP1 in yeast diverged from a common ancestor.

Petryk N, Zhou YF, Sybirna K, Mucchielli MH, Guiard B, Bao WG, Stasyk OV, Stasyk OG, Krasovska OS, Budin K, Reymond N, Imbeaud S, Coudouel S, Delacroix H, Sibirny A, Bolotin-Fukuhara M - PLoS ONE (2014)

Bottom Line: Based mostly on global gene expression studies performed in the S. cerevisiae HAP4 disruption mutant (ScΔhap4), we show here that HpHap4-A is functionally equivalent to ScHap4, whereas HpHap4-B is not.Finally, a transcriptomic analysis performed in the ScΔyap1 strain overexpressing HpHAP4-B shows that HpHap4-B acts both on oxidative stress response and carbohydrate metabolism in a manner different from both ScYap1 and ScHap4.These data, placed in an evolutionary context, raise new questions concerning the evolution of the HAP4 transcriptional regulation function and suggest that Yap1 and Hap4 have diverged from a unique regulatory protein in the fungal ancestor.

View Article: PubMed Central - PubMed

Affiliation: Institut de Génétique et Microbiologie, IFR Génome 115, Université Paris-Sud and CNRS, Orsay, France; Institute of Cell Biology, National Academy of Sciences, Lviv, Ukraine; Centre de Génétique Moléculaire, CNRS, Gif sur Yvette, France.

ABSTRACT
The transcriptional regulator HAP4, induced by respiratory substrates, is involved in the balance between fermentation and respiration in S. cerevisiae. We identified putative orthologues of the Hap4 protein in all ascomycetes, based only on a conserved sixteen amino acid-long motif. In addition to this motif, some of these proteins contain a DNA-binding motif of the bZIP type, while being nonetheless globally highly divergent. The genome of the yeast Hansenula polymorpha contains two HAP4-like genes encoding the protein HpHap4-A which, like ScHap4, is devoid of a bZIP motif, and HpHap4-B which contains it. This species has been chosen for a detailed examination of their respective properties. Based mostly on global gene expression studies performed in the S. cerevisiae HAP4 disruption mutant (ScΔhap4), we show here that HpHap4-A is functionally equivalent to ScHap4, whereas HpHap4-B is not. Moreover HpHAP4-B is able to complement the H2O2 hypersensitivity of the ScYap1 deletant, YAP1 being, in S. cerevisiae, the main regulator of oxidative stress. Finally, a transcriptomic analysis performed in the ScΔyap1 strain overexpressing HpHAP4-B shows that HpHap4-B acts both on oxidative stress response and carbohydrate metabolism in a manner different from both ScYap1 and ScHap4. Deletion of these two genes in their natural host, H. polymorpha, confirms that HpHAP4-A participates in the control of the fermentation/respiration balance, while HpHAP4-B is involved in oxidative stress since its deletion leads to hypersensitivity to H2O2. These data, placed in an evolutionary context, raise new questions concerning the evolution of the HAP4 transcriptional regulation function and suggest that Yap1 and Hap4 have diverged from a unique regulatory protein in the fungal ancestor.

Show MeSH

Related in: MedlinePlus

Functional comparison between ScYAP1 and the HpHAP4-B genes.Part A: Comparison of the new bZIP type motif identified in HpHap4-B with the YAP family motif. Q234, Q239, A241 and F/Y242 are four basic regions (DNA-binding sites) characteristic residues of the YAP protein family which are rarely or never observed in other bZIP proteins [34]. Part B: Heterologous complementation of the growth deficiency of S. cerevisiae Δyap1 in the presence of H202 by the HpHAP4-B gene. The gene is expressed on a multicopy plasmid (pBFG1, see Methods) and the growth monitored on minimal medium (W0) containing 0.5 mM of H202. 1: S. cerevisiae Δyap1 strain (with or without H202). 2:Δyap1 strain with empty plasmid BFG1. 3: Δyap1 with BFG1 plasmid containing HpHap4-B. 4: Δyap1 with BFG1 plasmid containing HpHap4-B with deleted bZIP domain (see Methods). Tests 2, 3 and 4 were performed on medium containing H202. Strains were grown on minimal glucose medium supplemented with the necessary aminoacids at 28°C for 5 days.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4257542&req=5

pone-0112263-g004: Functional comparison between ScYAP1 and the HpHAP4-B genes.Part A: Comparison of the new bZIP type motif identified in HpHap4-B with the YAP family motif. Q234, Q239, A241 and F/Y242 are four basic regions (DNA-binding sites) characteristic residues of the YAP protein family which are rarely or never observed in other bZIP proteins [34]. Part B: Heterologous complementation of the growth deficiency of S. cerevisiae Δyap1 in the presence of H202 by the HpHAP4-B gene. The gene is expressed on a multicopy plasmid (pBFG1, see Methods) and the growth monitored on minimal medium (W0) containing 0.5 mM of H202. 1: S. cerevisiae Δyap1 strain (with or without H202). 2:Δyap1 strain with empty plasmid BFG1. 3: Δyap1 with BFG1 plasmid containing HpHap4-B. 4: Δyap1 with BFG1 plasmid containing HpHap4-B with deleted bZIP domain (see Methods). Tests 2, 3 and 4 were performed on medium containing H202. Strains were grown on minimal glucose medium supplemented with the necessary aminoacids at 28°C for 5 days.

Mentions: The additional bZIP-type DNA-binding motif observed in many proteins encoded by putative HAP4 orthologues, especially when the species harbouring them are phylogenetically distant from S. cerevisiae, is a well conserved motif of 25 amino acids which is similar to the DNA-binding motif of the S. cerevisiae YAP family of proteins (Fig 4A). It contains four characteristic amino acid residues, Q234, Q239, A241 and F/Y242 specific of the Yap family [34]. This sequence, called the "Basic Region" (abbreviated here as BR), is necessary for the ScYap1 transactivator to recognize its specific cis-DNA binding sequences (called ARE). The ZIP motif (leucine zipper), which is adjacent to the BR region, was not identified in these HAP4 orthologues; only putative coiled-coiled motifs quite distant to the basic motif could be detected [12].


Functional study of the Hap4-like genes suggests that the key regulators of carbon metabolism HAP4 and oxidative stress response YAP1 in yeast diverged from a common ancestor.

Petryk N, Zhou YF, Sybirna K, Mucchielli MH, Guiard B, Bao WG, Stasyk OV, Stasyk OG, Krasovska OS, Budin K, Reymond N, Imbeaud S, Coudouel S, Delacroix H, Sibirny A, Bolotin-Fukuhara M - PLoS ONE (2014)

Functional comparison between ScYAP1 and the HpHAP4-B genes.Part A: Comparison of the new bZIP type motif identified in HpHap4-B with the YAP family motif. Q234, Q239, A241 and F/Y242 are four basic regions (DNA-binding sites) characteristic residues of the YAP protein family which are rarely or never observed in other bZIP proteins [34]. Part B: Heterologous complementation of the growth deficiency of S. cerevisiae Δyap1 in the presence of H202 by the HpHAP4-B gene. The gene is expressed on a multicopy plasmid (pBFG1, see Methods) and the growth monitored on minimal medium (W0) containing 0.5 mM of H202. 1: S. cerevisiae Δyap1 strain (with or without H202). 2:Δyap1 strain with empty plasmid BFG1. 3: Δyap1 with BFG1 plasmid containing HpHap4-B. 4: Δyap1 with BFG1 plasmid containing HpHap4-B with deleted bZIP domain (see Methods). Tests 2, 3 and 4 were performed on medium containing H202. Strains were grown on minimal glucose medium supplemented with the necessary aminoacids at 28°C for 5 days.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4257542&req=5

pone-0112263-g004: Functional comparison between ScYAP1 and the HpHAP4-B genes.Part A: Comparison of the new bZIP type motif identified in HpHap4-B with the YAP family motif. Q234, Q239, A241 and F/Y242 are four basic regions (DNA-binding sites) characteristic residues of the YAP protein family which are rarely or never observed in other bZIP proteins [34]. Part B: Heterologous complementation of the growth deficiency of S. cerevisiae Δyap1 in the presence of H202 by the HpHAP4-B gene. The gene is expressed on a multicopy plasmid (pBFG1, see Methods) and the growth monitored on minimal medium (W0) containing 0.5 mM of H202. 1: S. cerevisiae Δyap1 strain (with or without H202). 2:Δyap1 strain with empty plasmid BFG1. 3: Δyap1 with BFG1 plasmid containing HpHap4-B. 4: Δyap1 with BFG1 plasmid containing HpHap4-B with deleted bZIP domain (see Methods). Tests 2, 3 and 4 were performed on medium containing H202. Strains were grown on minimal glucose medium supplemented with the necessary aminoacids at 28°C for 5 days.
Mentions: The additional bZIP-type DNA-binding motif observed in many proteins encoded by putative HAP4 orthologues, especially when the species harbouring them are phylogenetically distant from S. cerevisiae, is a well conserved motif of 25 amino acids which is similar to the DNA-binding motif of the S. cerevisiae YAP family of proteins (Fig 4A). It contains four characteristic amino acid residues, Q234, Q239, A241 and F/Y242 specific of the Yap family [34]. This sequence, called the "Basic Region" (abbreviated here as BR), is necessary for the ScYap1 transactivator to recognize its specific cis-DNA binding sequences (called ARE). The ZIP motif (leucine zipper), which is adjacent to the BR region, was not identified in these HAP4 orthologues; only putative coiled-coiled motifs quite distant to the basic motif could be detected [12].

Bottom Line: Based mostly on global gene expression studies performed in the S. cerevisiae HAP4 disruption mutant (ScΔhap4), we show here that HpHap4-A is functionally equivalent to ScHap4, whereas HpHap4-B is not.Finally, a transcriptomic analysis performed in the ScΔyap1 strain overexpressing HpHAP4-B shows that HpHap4-B acts both on oxidative stress response and carbohydrate metabolism in a manner different from both ScYap1 and ScHap4.These data, placed in an evolutionary context, raise new questions concerning the evolution of the HAP4 transcriptional regulation function and suggest that Yap1 and Hap4 have diverged from a unique regulatory protein in the fungal ancestor.

View Article: PubMed Central - PubMed

Affiliation: Institut de Génétique et Microbiologie, IFR Génome 115, Université Paris-Sud and CNRS, Orsay, France; Institute of Cell Biology, National Academy of Sciences, Lviv, Ukraine; Centre de Génétique Moléculaire, CNRS, Gif sur Yvette, France.

ABSTRACT
The transcriptional regulator HAP4, induced by respiratory substrates, is involved in the balance between fermentation and respiration in S. cerevisiae. We identified putative orthologues of the Hap4 protein in all ascomycetes, based only on a conserved sixteen amino acid-long motif. In addition to this motif, some of these proteins contain a DNA-binding motif of the bZIP type, while being nonetheless globally highly divergent. The genome of the yeast Hansenula polymorpha contains two HAP4-like genes encoding the protein HpHap4-A which, like ScHap4, is devoid of a bZIP motif, and HpHap4-B which contains it. This species has been chosen for a detailed examination of their respective properties. Based mostly on global gene expression studies performed in the S. cerevisiae HAP4 disruption mutant (ScΔhap4), we show here that HpHap4-A is functionally equivalent to ScHap4, whereas HpHap4-B is not. Moreover HpHAP4-B is able to complement the H2O2 hypersensitivity of the ScYap1 deletant, YAP1 being, in S. cerevisiae, the main regulator of oxidative stress. Finally, a transcriptomic analysis performed in the ScΔyap1 strain overexpressing HpHAP4-B shows that HpHap4-B acts both on oxidative stress response and carbohydrate metabolism in a manner different from both ScYap1 and ScHap4. Deletion of these two genes in their natural host, H. polymorpha, confirms that HpHAP4-A participates in the control of the fermentation/respiration balance, while HpHAP4-B is involved in oxidative stress since its deletion leads to hypersensitivity to H2O2. These data, placed in an evolutionary context, raise new questions concerning the evolution of the HAP4 transcriptional regulation function and suggest that Yap1 and Hap4 have diverged from a unique regulatory protein in the fungal ancestor.

Show MeSH
Related in: MedlinePlus