Three target genes for the transcriptional activator Cat8p of Kluyveromyces lactis: acetyl coenzyme A synthetase genes KlACS1 and KlACS2 and lactate permease gene KlJEN1

The aerobic yeast Kluyveromyces lactis and the predominantly fermentative Saccharomyces cerevisiae share many of the genes encoding the enzymes of carbon and energy metabolism. The physiological features that distinguish the two yeasts appear to result essentially from different organization of regulatory circuits, in particular glucose repression and gluconeogenesis. We have isolated the KlCAT8 gene (a homologue of S. cerevisiae CAT8, encoding a DNA binding protein) as a multicopy suppressor of a fog1 mutation. The Fog1 protein is a homologue of the Snf1 complex components Gal83p, Sip1p, and Sip2p of S. cerevisiae. While CAT8 controls the key enzymes of gluconeogenesis in S. cerevisiae, KlCAT8 of K. lactis does not (I. Georis, J. J. Krijger, K. D. Breunig, and J. Vandenhaute, Mol. Gen. Genet. 264:193-203, 2000). We therefore examined possible targets of KlCat8p. We found that the acetyl coenzyme A synthetase genes, KlACS1 and KlACS2, were specifically regulated by KlCAT8, but very differently from the S. cerevisiae counterparts. KlACS1 was induced by acetate and lactate, while KlACS2 was induced by ethanol, both under the control of KlCAT8. Also, KlJEN1, encoding the lactate-inducible and glucose-repressible lactate permease, was found under a tight control of KlCAT8.     

Role of Snf1p in regulation of intracellular sorting of the lactose and galactose transporter Lac12p in Kluyveromyces lactis

The protein kinase Snf1/AMPK plays a central role in carbon and energy homeostasis in yeasts and higher eukaryotes. To work out which aspects of the Snf1-controlled regulatory network are conserved in evolution, the Snf1 requirement in galactose metabolism was analyzed in the yeast Kluyveromyces lactis. Whereas galactose induction was only delayed, K. lactis snf1 mutants failed to accumulate the lactose/galactose H+ symporter Lac12p in the plasma membran,e as indicated by Lac12-green fluorescent protein fusions. In contrast to wild-type cells, the fusion protein was mostly intracellular in the mutant. Growth on galactose and galactose uptake could be restored by the KHT3 gene, which encodes a new transporter of the HXT subfamily of major facilitators These findings indicate a new role of Snf1p in regulation of sugar transport in K. lactis.     

The Snf1 kinase of the filamentous fungus Hypocrea jecorina phosphorylates regulation-relevant serine residues in the yeast carbon catabolite repressor Mig1 but not in the filamentous fungal counterpart Cre1

In Saccharomyces cerevisiae, the SNF1 gene product phosphorylates the carbon catabolite repressor protein Mig1 under conditions when glucose is limiting, thereby relieving the fungus from catabolite repression. We have investigated whether the corresponding counterpart of filamentous fungi-the Cre1 protein-is also phosphorylated by Snf1. To this end, snf1, an ortholog of SNF1, was isolated from the ascomycete Hypocrea jecorina. The gene encodes a protein with high similarity to Snf1 kinases from other eukaryotes in its N-terminal catalytic domain, but little similarity in the C-terminal half of the protein, albeit some short aa-areas were detected, however, which are conserved in filamentous fungi and in yeast. Expression of snf1 is independent of the carbon source. An overexpressed catalytic domain of H. jecorina Snf1 readily phosphorylated yeast Mig1, but not a Mig1 mutant form, in which all four identified Snf1 phosphorylation sites (Phi XRXXSXXX Phi) had been mutated. The enzyme did neither phosphorylate H. jecorina Cre1 nor histone H3, another substrate of Snf1 kinase in yeast. H. jecorina Snf1 also phosphorylated peptides comprising the strict Snf1 consensus, but notably did not phosphorylate peptides containing the regulatory serine residue in Cre1 (=Ser(241) in H. jecorina Cre1 and Ser(266) in Sclerotinia sclerotiorum CRE1). The use of cell-free extracts of H. jecorina as protein source for Snf1 showed phosphorylation of an unknown 36 kDa protein, which was present only in extracts from glucose-grown mycelia. We conclude that the Snf1 kinase from H. jecorina is not involved in the phosphorylation of Cre1.     

Phosphorylation of Yeast Hexokinase 2 Regulates Its Nucleocytoplasmic Shuttling

Nucleocytoplasmic shuttling of Hxk2 induced by glucose levels has been reported recently. Here we present evidence that indicates that Hxk2 nucleocytoplasmic traffic is regulated by phosphorylation and dephosphorylation at serine 14. Moreover, we identified the protein kinase Snf1 and the protein phosphatase Glc7-Reg1 as novel regulatory partners for the nucleocytoplasmic shuttling of Hxk2. Functional studies revealed that, in contrast to the wild-type protein, the dephosphorylation-mimicking mutant of Hxk2 retains its nuclear localization in low glucose conditions, and the phosphomimetic mutant of Hxk2 retains its cytoplasmic localization in high glucose conditions. Interaction experiments of Hxk2 with Kap60 and Xpo1 indicated that nuclear import of the S14D mutant of Hxk2 is severely decreased but that the export is significantly enhanced. Conversely, nuclear import of the S14A mutant of Hxk2 was significantly enhanced, although the export was severely decreased. The interaction of Hxk2 with Kap60 and Xpo1 was found to occur in the dephosphorylated and phosphorylated states of the protein, respectively. In addition, we found that Hxk2 is a substrate for Snf1. Mutational analysis indicated that serine 14 is a major in vitro and in vivo phosphorylation site for Snf1. We also provide evidence that dephosphorylation of Hxk2 at serine 14 is a protein phosphatase Glc7-Reg1-dependent process. Taken together, this study establishes a functional link between Hxk2, Reg1, and Snf1 signaling, which involves the regulation of Hxk2 nucleocytoplasmic shuttling by phosphorylation-dephosphorylation of serine 14.     

Glucose represses the lactose-galactose regulon in Kluyveromyces lactis through a SNF1 and MIG1- dependent pathway that modulates galactokinase (GAL1) gene expression.

Expression of the lactose-galactose regulon in Kluyveromyces lactis is induced by lactose or galactose and repressed by glucose. Some components of the induction and glucose repression pathways have been identified but many remain unknown. We examined the role of the SNF1 (KlSNF1) and MIG1 (KlMIG1) genes in the induction and repression pathways. Our data show that full induction of the regulon requires SNF1; partial induction occurs in a Klsnf1 -deleted strain, indicating that a KlSNF1 -independent pathway(s) also regulates induction. MIG1 is required for full glucose repression of the regulon, but there must be a KlMIG1 -independent repression pathway also. The KlMig1 protein appears to act downstream of the KlSnf1 protein in the glucose repression pathway. Most importantly, the KlSnf1-KIMig repression pathway operates by modulating KlGAL1 expression. Regulating KlGAL1 expression in this manner enables the cell to switch the regulon off in the presence of glucose. Overall, our data show that, while the Snf1 and Mig1 proteins play similar roles in regulating the galactose regulon in Saccharomyces cerevisiae and K.lactis , the way in which these proteins are integrated into the regulatory circuits are unique to each regulon, as is the degree to which each regulon is controlled by the two proteins.     

Phosphorylation positively regulates DNA binding of the carbon catabolite repressor Cre1 of Hypocrea jecorina (Trichoderma reesei)

Cre1 of the ascomycete Hypocrea jecorina is a Cys(2)His(2) zinc finger DNA-binding protein functioning as regulator for carbon catabolite repression. It represents the functional equivalent of yeast Mig1, known to be negatively regulated by the Snf1-kinase at the nuclear import level. We demonstrate that Cre1 is also a phosphoprotein, and identify Ser(241) within an acidic protein region as phosphorylation target. In contrast to Mig1 phosphorylation is required for DNA binding of Cre1. A S241E mutation mimics phosphorylation, whereas a S241A mutant protein shows phosphorylation-independent DNA binding activity, suggesting that phosphorylation is required to release Cre1 from an inactive conformation involving unphosphorylated Ser(241). Retransformation of a H. jecorina cre1-non functional mutant with Cre1-S241A leads to permanent carbon catabolite repression in cellobiohydrolase I expression. Contrary to Mig1, the amino acid sequence surrounding Ser(241) (HSNDEDD) suggests that phosphorylation may occur by a casein kinase II-like protein. This is supported by a mutation of E244V leading to loss of phosphorylation, loss of DNA binding, and gain of carbon catabolite derepression. Our results imply that the regulation of carbon catabolite repression at the level of DNA binding strongly differs between Saccharomyces cerevisiae and H. jecorina.