Candidate osmosensors from Candida utilis and Kluyveromyces lactis: structural and functional homology to the Sho1p putative osmosensor from Saccharomyces cerevisiae

In Saccharomyces cerevisiae, increases in external osmolarity evoke osmostress-induced signalling via the HOG MAP kinase pathway. One of the upstream components of this signal transduction route is the putative osmosensor, Sho1p. With the aim to elucidate the molecular basis of osmosensing in budding yeast, we have cloned SHO1 homologues from Candida utilis and Kluyveromyces lactis which allowed determination of conserved domains of Sho1p. Results obtained from sequence comparisons, confirmed the importance of the transmembrane domains and the SH3 domain for Sho1p function. The K. lactis and S. cerevisiae Sho1p show the highest degree of homology, the isoform from C. utilis is a shorter protein. SHO1 from C. utilis, however, did complement the osmosensitivity of the sho1ssk2ssk22 strain by restoring HOG pathway function, since Hog1p dual phosphorylation after high osmotic challenge was restored in this strain after transformation with a plasmid bearing this SHO1 homologue.     

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.     

The KlPGS1 gene encoding phosphatidylglycerolphosphate synthase in Kluyveromyces lactis is essential and assigned to chromosome I

The phosphatidylglycerolphosphate synthase (CDP-diacylglycerol:sn-glycerol-3-phosphate 3-phosphatidyltransferase, EC 2.7.8.5) is an essential enzyme in biosynthesis of cardiolipin. In this work we report the isolation, heterological cloning, molecular characterization and physical mapping of the Saccharomyces cerevisiae PEL1/PGS1 homologue from Kluyveromyces lactis. The pel1 mutant strain of S. cerevisiae was used to isolate this homologue by screening a K. lactis genomic library. The novel cloned gene was named KlPGS1. Its coding region was found to consist of 1623 bp. The corresponding protein exhibits 55% amino acid identity to its S. cerevisiae counterpart. The presence of the mitochondrial presequence indicates its mitochondrial localization. Sporulation and ascus dissection of diploids heterozygous for single-copy disruption of KlPGS1 revealed that the KlPGS1 gene, is essential in K. lactis. Using a DIG-dUTP-labeled DNA probe-originated from the KlPGS1 gene and Southern hybridization of contour-clamped homogeneous electric field (CHEF)-separated K. lactis chromosomal DNA, the KlPGS1 gene was assigned to chromosome I. The nucleotide sequence data reported in this paper were submitted to GenBank and assigned the Accession No. AY176328.