TY - JOUR

T1 - Key role of Ser562/661 in Snf1-dependent regulation of Cat8p in Saccharomyces cerevisiae and Kluyveromyces lactis

AU - Charbon, Godefroid

AU - Breunig, Karin D

AU - Wattiez, Ruddy

AU - Vandenhaute, Jean

AU - Noël-Georis, Isabelle

PY - 2004/5

Y1 - 2004/5

N2 - Utilization of nonfermentable carbon sources by Kluyveromyces lactis and Saccharomyces cerevisiae requires the Snf1p kinase and the Cat8p transcriptional activator, which binds to carbon source-responsive elements of target genes. We demonstrate that KlSnf1p and KlCat8p from K. lactis interact in a two-hybrid system and that the interaction is stronger with a kinase-dead mutant form of KlSnf1p. Of two putative phosphorylation sites in the KlCat8p sequence, serine 661 was identified as a key residue governing KlCat8p regulation. Serine 661 is located in the middle homology region, a regulatory domain conserved among zinc cluster transcription factors, and is part of an Snf1p consensus phosphorylation site. Single mutations at this site are sufficient to completely change the carbon source regulation of the KlCat8p transactivation activity observed. A serine-to-glutamate mutant form mimicking constitutive phosphorylation results in a nearly constitutively active form of KlCat8p, while a serine-to-alanine mutation has the reverse effect. Furthermore, it is shown that KlCat8p phosphorylation depends on KlSNF1. The Snf1-Cat8 connection is evolutionarily conserved: mutation of corresponding serine 562 of ScCat8p gave similar results in S. cerevisiae. The enhanced capacity of ScCat8S562E to suppress the phenotype caused by snf1 strengthens the hypothesis of direct phosphorylation of Cat8p by Snf1p. Unlike that of S. cerevisiae ScCAT8, KlCAT8 transcription is not carbon source regulated, illustrating the prominent role of posttranscriptional regulation of Cat8p in K. lactis.

AB - Utilization of nonfermentable carbon sources by Kluyveromyces lactis and Saccharomyces cerevisiae requires the Snf1p kinase and the Cat8p transcriptional activator, which binds to carbon source-responsive elements of target genes. We demonstrate that KlSnf1p and KlCat8p from K. lactis interact in a two-hybrid system and that the interaction is stronger with a kinase-dead mutant form of KlSnf1p. Of two putative phosphorylation sites in the KlCat8p sequence, serine 661 was identified as a key residue governing KlCat8p regulation. Serine 661 is located in the middle homology region, a regulatory domain conserved among zinc cluster transcription factors, and is part of an Snf1p consensus phosphorylation site. Single mutations at this site are sufficient to completely change the carbon source regulation of the KlCat8p transactivation activity observed. A serine-to-glutamate mutant form mimicking constitutive phosphorylation results in a nearly constitutively active form of KlCat8p, while a serine-to-alanine mutation has the reverse effect. Furthermore, it is shown that KlCat8p phosphorylation depends on KlSNF1. The Snf1-Cat8 connection is evolutionarily conserved: mutation of corresponding serine 562 of ScCat8p gave similar results in S. cerevisiae. The enhanced capacity of ScCat8S562E to suppress the phenotype caused by snf1 strengthens the hypothesis of direct phosphorylation of Cat8p by Snf1p. Unlike that of S. cerevisiae ScCAT8, KlCAT8 transcription is not carbon source regulated, illustrating the prominent role of posttranscriptional regulation of Cat8p in K. lactis.

KW - Base Sequence

KW - Carbon/metabolism

KW - DNA, Fungal/genetics

KW - Fungal Proteins/chemistry

KW - Genes, Fungal

KW - Kluyveromyces/genetics

KW - Models, Biological

KW - Mutagenesis, Site-Directed

KW - Phosphorylation

KW - Protein Structure, Tertiary

KW - Restriction Mapping

KW - Saccharomyces cerevisiae/genetics

KW - Saccharomyces cerevisiae Proteins/chemistry

KW - Serine/chemistry

KW - Species Specificity

KW - Trans-Activators/chemistry

KW - Transcriptional Activation

KW - Two-Hybrid System Techniques

M3 - Journal article

C2 - 15121831

VL - 24

SP - 4083

EP - 4091

JO - Molecular and Cellular Biology

JF - Molecular and Cellular Biology

SN - 0270-7306

IS - 10

ER -