The role of the Hxk2 protein in glucose repression has been a matter of controversy for many years. Our data challenge the view that its role in glucose-repression signalling is directly related to its catalytic activity. This view was based on the inverse correlation between Hxk2 and invertase activity in a collection of random Hxk2 mutants [1
]. Although previous results have shown Hxk2 mutants with low catalytic activity that are still functional in glucose signalling [15
], a complete separation of the two activities never has been achieved. We have used the accumulated knowledge about the structure of the catalytic and regulatory domains of the Hxk2 protein to design a set of new mutations. We probed the role of these Hxk2 mutations in the following Hxk2 functions: catalytic activity, regulatory activity and interactions with proteins essential for these functions. By screening for suppression of the catalytic activity while maintaining the regulatory function and suppression of the regulatory function and maintaining the catalytic activity in the Hxk2 protein, we have isolated novel alleles of the HXK2
gene that have been long sought after. The results confirm what has been suspected for some time: that Hxk2 mediates catalytic and regulatory activities through different domains in the protein. The two separation-of-function mutants isolated, Hxk2wca
, are remarkable since they convert the Hxk2 from a bifunctional protein into a single-function protein with activity as a mediating factor in transcription (Hxk2wca
) or as an enzyme with hexose-phosphorylating activity (Hxk2wrf
The Hxk2wca mutant protein encodes an Hxk2 protein without the last eight C-terminal amino acids and a replacement of Ser304, which is located in the active centre of the enzyme, with phenylalanine. The Hxk2wrf mutant is an Hxk2 protein with an internal deletion between amino acids Lys6 and Met15. Since there is no overlap between the two domains controlling the Hxk2 catalytic and regulatory functions respectively, in the present study, we demonstrated that a correlation between catalytic activity and regulatory function in this protein does not exist.
In the glucose-repression signalling pathway, two major mediators are the proteins Mig1 and Hxk2. Under high-glucose conditions, nuclear Hxk2 interacts both with Mig1 and the Snf1 subunit of the Snf1 protein kinase complex to establish the repression state of several genes. Hxk2 interacts strongly with Mig1 in high-glucose-grown cells and previous findings implicate Ser311
of Mig1 as a critical residue for this interaction [9
]. The interaction between Snf1 and Hxk2 was detected both under high- and low-glucose conditions and the data suggest that both proteins interact constitutively [9
In low-glucose-grown cells, the Hxk2 interaction with Mig1 is abolished and a transient increase in interaction between Snf1 and Mig1 was detected [33
]. This interaction pattern potentially stimulates Mig1 phosphorylation by Snf1 kinase at Ser311
. Phosphorylation of Ser311
results in most Mig1 being exported from the nucleus to the cytoplasm, and, ultimately, the derepression of genes regulated by the Hxk2–Mig1 glucose-repression signalling pathway.
To analyse the mechanism by which the loss-of-function mutants operate through the regulatory machinery, we have determined protein–protein interactions with Mig1 and Snf1 proteins, and protein–DNA interactions with the Mig1-binding site of the SUC2
promoter. Since the Hxk2–Mig1 complex plays an essential role in glucose-repression signalling of several genes [7
], detailed knowledge of the interaction between Hxk2wca
mutant proteins with Mig1 is required to understand how these two mutants operate in the glucose-repression mechanism. One of the important observations described in the present study is that two-hybrid assays, co-immunoprecipitation and GST pull-down experiments with purified Mig1 protein demonstrated that Hxk2wrf
does not interact with Mig1 under both high- and low-glucose conditions. The lack of interaction between Hxk2wrf
and Mig1 implies a loss of regulatory function. However, two-hybrid assays, co-immunoprecipitation and GST pull-down experiments with purified Mig1 protein demonstrated that Hxk2wca
interacted with Mig1 under high- but not low-glucose conditions, a similar behaviour to the wild-type Hxk2.
Although the interaction between Snf1 and Hxk2wrf
was detected both under high- and low-glucose conditions and our data suggest that these proteins interact constitutively with Snf1, a loss of regulatory function was detected in the Hxk2wrf
mutant protein. Lack of the regulatory function of the Hxk2wrf
protein could be explained because the Hxk2 protein operates as a bridge between Snf1 and Mig1 and is required to inhibit Mig1 phosphorylation at Ser311
by Snf1 kinase under high-glucose conditions [9
]. Since Hxk2wrf
has lost its interaction capacity with Mig1 under high-glucose conditions, the Mig1 protein could be phosphorylated by Snf1, and both Mig1 and Hxk2 are exported from the nucleus to the cytoplasm by the Msn5 [34
] and Xpo1 (Crm1) exporters [35
] respectively. Thus, under high-glucose conditions, several genes controlled by the Mig1 repressor complex are expressed because the Hxk2wrf
protein has lost its regulatory function.
This hypothesis is supported by the following facts: (i) although it has been reported that Snf1 is inactive under high-glucose conditions [31
], it has also been described that the non-phosphorylated form of Snf1 may have a direct role in controlling the expression of the TRK1
genes of the Trk high-affinity potassium-uptake system [36
]; (ii) in the absence of Hxk2, the Snf1 protein kinase complex assumes an active conformation even in the presence of glucose [9
], because the Mig1 protein from both high- and low-glucose-grown cells has decreased electrophoretic mobility, indicating that the protein is phosphorylated [9
]; (iii) in the absence of Snf1 or both Hxk2 and Snf1, Mig1 has an increased mobility pattern, indicating that the protein is dephosphorylated [9
]; (iv) in Hxk2wrf
mutants, lacking a domain required for Mig1–Hxk2 interaction, Mig1 is phosphorylated under both high- and low-glucose conditions (A). Thus our data strongly suggest that the Hxk2 interaction with Mig1 under high-glucose conditions might prevent phosphorylation of Mig1 by Snf1.
In the present study, we have demonstrated that Hxk2wrf is unable to signal glucose repression in S. cerevisiae because it was not present in the Mig1 repressor complex of the SUC2 promoter. Furthermore, we have also demonstrated that Hxk2wca is able to signal glucose repression in S. cerevisiae because it is physically associated with the Mig1 protein repressor, and ChIP assays confirmed that both Hxk2 and Hxk2wca interacted with Mig1 in DNA fragments containing the MIG1 site of the SUC2 promoter. Therefore our results suggest that both Hxk2wca and Hxk2 use similar mechanisms to regulate glucose signalling in yeast.