Sites for interaction between Gal80p and Gal1p in Kluyveromyces lactis: structural model of galactokinase based on homology to the GHMP protein family.

The induction of transcription of the galactose genes in yeast involves the galactose-dependent binding of ScGal3p (in Saccharomyces cerevisiae) or KlGal1p (in Kluyveromyces lactis) to Gal80p. This binding abrogates Gal80's inhibitory effect on the activation domain of Gal4p, which can then activate transcription. Here, we describe the isolation and characterization of new interaction mutants of K.lactis GAL1 and GAL80 using a two-hybrid screen. We present the first structural model for Gal1p to be based on the published crystal structures of other proteins belonging to the GHMP (galactokinase, homoserine kinase, mevalonate kinase and phosphomevalonate kinase) kinase family and our own X-ray diffraction data of Gal1p crystals at 3A resolution. The locations of the various mutations in the modelled Gal1p structure identify domains involved in the interaction with Gal80p and provide a structural explanation for the phenotype of constitutive GAL1 mutations.

Comparative genetic and physiological studies of the MAP kinase Mpk1p from Kluyveromyces lactis and Saccharomyces cerevisiae.

MAP kinases are essential components of signal transduction pathways in yeasts and higher eukaryotes. Here, we report on the isolation of the gene encoding the MAP kinase KlMpk1p by complementation of the respective Saccharomyces cerevisiae deletion mutant with a genomic library from Kluyveromyces lactis. Sequencing revealed the presence of an open reading frame capable of encoding a protein of 520 amino acid residues with a deduced molecular mass of 59.726 Da. The deduced protein sequence displayed a high degree of similarity to known MAP kinases from yeast to man, with an overall identity of 70 % to ScMpk1p. One-hybrid analysis demonstrated the presence of a cryptic transcriptional activation domain in the C-terminal part of the protein. Deletion of this sequence in ScMpk1p resulted in a reduced MAP kinase activity (measured by an indirect assay), an increased sensitivity towards caffeine and an increased resistance against Calcofluor white. Complete deletion mutants of Klmpk1 display an osmo-remedial phenotype on rich medium, but are capable of growth in the absence of osmotic stabilization on synthetic medium. As Scmpk1 deletion mutants, they are sensitive to cell surface destabilizing agents such as Calcofluor white and SDS, and growth is inhibited in the presence of 5 mM caffeine. Overexpression of KlMPK1 did not produce a growth defect in S. cerevisiae or in K. lactis.

Characterization of KLBCK1, encoding a MAP kinase kinase kinase of Kluyveromyces lactis.

The cellular integrity and response to hypoosmotic conditions in the yeast Saccharomyces cerevisiae are ensured by a MAP kinase signal transduction pathway mediated by the yeast homolog of mammalian protein kinase C. Bck1p functions as the MAP kinase kinase kinase of this pathway. Here we report on the cloning and analysis of the BCK1 homolog from the milk yeast Kluyveromyces lactis (KlBCK1). The deduced protein sequences display three highly conserved domains with the serine/threonine kinase domain containing 89 % identical amino acid residues. Interestingly, a region identified in KlBck1p as a putative SAM domain, mediating protein-protein interactions, is also conserved in ScBck1p. Yet, two-hybrid analyses indicate that this region may not be involved in dimerization of KlBck1p in contrast to its S. cerevisiae counterpart. Expression of KlBCK1 fully complements the defects in a Scbck1 null mutant and is capable of activating the pathway as indicated by a reporter system based on the transcription factor Rlm1p. However, deletion from the haploid K. lactis genome does not result in a loss of cellular integrity under a variety of conditions tested. Thus, despite the functional conservation in this component of the MAP kinase pathway in both yeast, cellular integrity in K. lactis may depend at least in part on different signalling mechanisms when compared with S. cerevisiae.