Isolation of the mitochondrial nucleoids from yeast Kluyveromyces lactis and analyses of the nucleoid proteins

Mitochondrial (mt) nucleoids were isolated from yeast Kluyveromyces lactis with morphological intactness. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) revealed more than 20 proteins that are associated with the mt-nucleoids. However, the protein profile of the mt-nucleoids of K. lactis was significantly different from that of the mt-nucleoid proteins from Saccharomyces cerevisiae. SDS-DNA PAGE, which detected an Abf2p, a major mitochondrial DNA-binding protein, among the mt-nucleoid proteins of S. cerevisiae on a gel, detected only a 17-kDa protein in the K. lactis mt-nucleoid proteins. The 17-kDa protein was purified as homogeneous from the mt-nucleoids by a combination of acid extraction, hydroxyapatite chromatography and DNA-cellulose chromatography. The 17-kDa protein introduced a negative supercoil into circular plasmid DNA in the presence of topoisomerase I, as does S. cerevisiae Abf2p, and it packed K. lactis mtDNA into nucleoid-like particles in vitro. These results, together with the determination of the N-terminal amino acid sequence, suggested that the 17-kDa protein is an Abf2p homologue of K. lactis and plays structural roles in compacting mtDNA in cooperation with other nucleoid proteins.     

Identification of an essential core element and stimulatory sequences in a Kluyveromyces lactis ARS element, KARS101

A Kluyveromyces lactis chromosomal sequence of 913 bp is sufficient for replication in Saccharomyces cerevisiae and K. lactis . This fragment contains a 12 bp sequence 5'-ATTTATTGTTTT-3' that is related to the S. cerevisiae ACS (ARS consensus sequence). This dodecamer was removed by site-directed mutagenesis and the effect on K. lactis and S. cerevisiae ARS (autonomous replicating sequence) activity was determined. The dodecamer is essential for S. cerevisiae ARS function but only contributes to K. lactis ARS activity; therefore, its role in K. lactis is unlikely to be the same as that of the essential S. cerevisiae ACS.
A 103 bp subclone was found to retain ARS activity in both yeasts, but the plasmid was very unstable in S. cerevisiae . Deletion and linker substitution mutagenesis of this fragment was undertaken to define the DNA sequence required for K. lactis ARS function and to test whether the sequence required for ARS activity in K. lactis and S. cerevisiae coincide. We found a 39 bp core region essential for K. lactis ARS function flanked by sequences that contribute to ARS efficiency. The instability of the plasmid in S. cerevisiae made a fine-structure analysis of the S. cerevisiae ARS element impossible. However, the sequences that promote high-frequency transformation in S. cerevisiae overlap the essential core of the K. lactis ARS element but have different end-points.     

sir2 mutants of Kluyveromyces lactis are hypersensitive to DNA-targeting drugs.

A Kluyveromyces lactis mutant, hypersensitive to the DNA-targeting drugs ethidium bromide (EtBr), berenil, and HOE15030, can be complemented by a wild-type gene with homology to SIR2 of Saccharomyces cerevisiae (ScSIR2). The deduced amino acid sequence of the K. lactis Sir2 protein has 53% identity with ScSir2 protein but is 108 residues longer. K. lactis sir2 mutants show decreased mating efficiency, deficiency in sporulation, an increase in recombination at the ribosomal DNA locus, and EtBr-induced death. Some functional equivalence between the Sir2 proteins of K. lactis and S. cerevisiae has been demonstrated by introduction of ScSIR2 into a sir2 mutant of K. lactis. Expression of ScSIR2 on a multicopy plasmid restores resistance to EtBr and complements sporulation deficiency. Similarly, mating efficiency of a sir2 mutant of S. cerevisiae is partially restored by K. lactis SIR2 on a multicopy plasmid. Although these observations suggest that there has been some conservation of Sir2 protein function, a striking difference is that sir2 mutants of S. cerevisiae, unlike their K. lactis counterparts, are not hypersensitive to DNA-targeting drugs.     

Highly diverged homologs of Saccharomyces cerevisiae mitochondrial mRNA-specific translational activators have orthologous functions in other budding yeasts

Translation of mitochondrially coded mRNAs in Saccharomyces cerevisiae depends on membrane-bound mRNA-specific activator proteins, whose targets lie in the mRNA 5'-untranslated leaders (5'-UTLs). In at least some cases, the activators function to localize translation of hydrophobic proteins on the inner membrane and are rate limiting for gene expression. We searched unsuccessfully in divergent budding yeasts for orthologs of the COX2- and COX3-specific translational activator genes, PET111, PET54, PET122, and PET494, by direct complementation. However, by screening for complementation of mutations in genes adjacent to the PET genes in S. cerevisiae, we obtained chromosomal segments containing highly diverged homologs of PET111 and PET122 from Saccharomyces kluyveri and of PET111 from Kluyveromyces lactis. All three of these genes failed to function in S. cerevisiae. We also found that the 5'-UTLs of the COX2 and COX3 mRNAs of S. kluyveri and K. lactis have little similarity to each other or to those of S. cerevisiae. To determine whether the PET111 and PET122 homologs carry out orthologous functions, we deleted them from the S. kluyveri genome and deleted PET111 from the K. lactis genome. The pet111 mutations in both species prevented COX2 translation, and the S. kluyveri pet122 mutation prevented COX3 translation. Thus, while the sequences of these translational activator proteins and their 5'-UTL targets are highly diverged, their mRNA-specific functions are orthologous.     

Phenotypic expression of Kluyveromyces lactis killer toxin against Saccharomyces spp.

The secretion of killer toxins by some strains of yeasts is a phenomenon of significant industrial importance. The activity of a recently discovered Kluyveromyces lactis killer strain against a sensitive Saccharomyces cerevisiae strain was determined on peptone-yeast extract-nutrient agar plates containing as the carbon source glucose, fructose, galactose, maltose, or glycerol at pH 4.5 or 6.5. Enhanced activity (50 to 90% increase) was found at pH 6.5, particularly on the plates containing galactose, maltose, or glycerol, although production of the toxin in liquid medium was not significantly different with either glucose or galactose as the carbon source. Results indicated that the action of the K. lactis toxin was not mediated by catabolite repression in the sensitive strain. Sensitivities of different haploid and polyploid Saccharomyces yeasts to the two different killer yeasts S. cerevisiae (RNA-plasmid-coded toxin) and K. lactis (DNA-plasmid-coded toxin) were tested. Three industrial polyploid yeasts sensitive to the S. cerevisiae killer yeast were resistant to the K. lactis killer yeast. The S. cerevisiae killer strain itself, however, was sensitive to the K. lactis killer yeast.     

Phenotypic expression of Kluyveromyces lactis killer toxin against Saccharomyces spp

The secretion of killer toxins by some strains of yeasts is a phenomenon of significant industrial importance. The activity of a recently discovered Kluyveromyces lactis killer strain against a sensitive Saccharomyces cerevisiae strain was determined on peptone-yeast extract-nutrient agar plates containing as the carbon source glucose, fructose, galactose, maltose, or glycerol at pH 4.5 or 6.5. Enhanced activity (50 to 90% increase) was found at pH 6.5, particularly on the plates containing galactose, maltose, or glycerol, although production of the toxin in liquid medium was not significantly different with either glucose or galactose as the carbon source. Results indicated that the action of the K. lactis toxin was not mediated by catabolite repression in the sensitive strain. Sensitivities of different haploid and polyploid Saccharomyces yeasts to the two different killer yeasts S. cerevisiae (RNA-plasmid-coded toxin) and K. lactis (DNA-plasmid-coded toxin) were tested. Three industrial polyploid yeasts sensitive to the S. cerevisiae killer yeast were resistant to the K. lactis killer yeast. The S. cerevisiae killer strain itself, however, was sensitive to the K. lactis killer yeast.     

Why does Kluyveromyces lactis not grow under anaerobic conditions? Comparison of essential anaerobic genes of Saccharomyces cerevisiae with the Kluyveromyces lactis genome

Although some yeast species, e.g. Saccharomyces cerevisiae, can grow under anaerobic conditions, Kluyveromyces lactis cannot. In a systematic study, we have determined which S. cerevisiae genes are required for growth without oxygen. This has been done by using the yeast deletion library. Both aerobically essential and nonessential genes have been tested for their necessity for anaerobic growth. Upon comparison of the K. lactis genome with the genes found to be anaerobically important in S. cerevisiae, which yielded 20 genes that are missing in K. lactis, we hypothesize that lack of import of sterols might be one of the more important reasons that K. lactis cannot grow in the absence of oxygen.     

Dynamic analysis of the KlGAL regulatory system in Kluyveromyces lactis: a comparative study with Saccharomyces cerevisiae

The GAL regulatory system is highly conserved in yeast species of Saccharomyces cerevisiae and Kluyveromyces lactis. While the GAL system is a well studied system in S. cerevisiae, the dynamic behavior of the KlGAL system in K. lactis has not been characterized. Here, we have characterized the GAL system in yeast K. lactis by developing a dynamic model and comparing its performance to its not-so-distant cousin S. cerevisiae. The present analysis demonstrates the significance of the autoregulatory feedbacks due to KlGal4p, KlGal80p, KlGal1p and Lac12p on the dynamic performance of the KlGAL switch. The model predicts the experimentally observed absence of bistability in the wild type strain of K. lactis, unlike the short term memory of preculturing conditions observed in S. cerevisiae. The performance of the GAL switch is distinct for the two yeast species although they share similarities in the molecular components. The analysis suggests that the whole genome duplication of S. cerevisiae, which resulted in a dedicated inducer protein, Gal3p, may be responsible for the high sensitivity of the system to galactose concentrations. On the other hand, K. lactis uses a bifunctional protein as an inducer in addition to its galactokinase activity, which restricts its regulatory role and hence higher galactose levels in the medium are needed to trigger the GAL system. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11693-011-9082-7) contains supplementary material, which is available to authorized users.     

Isolation and characterization of the genes encoding delta(8)-sphingolipid desaturase from Saccharomyces kluyveri and Kluyveromyces lactis

Saccharomyces kluyveri IFO 1685 and Kluyveromyces lactis IFO 1090 synthesize cerebroside containing 9-methyl- trans-4, trans-8-sphingadienine as a sphingoid base. From the genome of the two strains, the regions encompassing Delta(8)-sphingolipid desaturase were amplified and sequenced. The nucleotide sequences of these regions revealed single open reading frames of 1707 bp for S. kluyveri and 1722 bp for K. lactis, encoding polypeptides of 568 and 573 amino acids with molecular weights of 66.5 and 67.1 kDa, respectively. Conversion of 4-hydroxysphinganine to 4-hydroxy- trans-8-sphingenine in the cells of Saccharomyces cerevisiae was observed by the expressed gene from K. lactis and not by that from S. kluyveri. These findings may be explained by the difference in substrate specificity for the sphingoid base moiety between Delta(8)-sphingolipid desaturases of S. kluyveri and K. lactis.     

Analysis of the deubiquitinating enzymes of the yeast Saccharomyces cerevisiae

Attachment of proteins to ubiquitin is reversed by specialized proteases called deubiquitinating enzymes (Dubs), which are also essential for ubiquitin precursor processing. In the genome of Saccharomyces cerevisiae, 17 potential DUB genes can be discerned. We have now constructed strains deleted for each of these genes. Surprisingly, given the essential nature of the ubiquitin system, none of the mutants is lethal or strongly growth defective under standard conditions, although a number have detectable abnormalities. Including results from this study, 14 of the 17 Dubs have now been shown to have ubiquitin-cleaving activity. The most extensively characterized yeast Dub is Doa4, which is required for both ubiquitin homeostasis and proteasome-dependent proteolysis. To help determine what distinguishes Doa4 functionally from other Dubs, we have cloned a DOA4 ortholog from the yeast Kluyveromyces lactis. The K. lactis protein is 42% identical to Doa4, but unexpectedly the K. lactis gene is slightly closer in nucleotide sequence to UBP5, which cannot substitute for DOA4 even in high dosage. The data suggest that the DOA4 locus underwent a duplication after the divergence of K. lactis and S. cerevisiae. This information will facilitate fine-structure analysis of the Doa4 protein to help delineate its key functional elements.     

Identification of the sequences required for chromosomal replicator function in Kluyveromyces lactis

The analysis of replication intermediates of a Kluyveromyces lactis chromosomal autonomous replicating sequence (ARS), KARS101, has shown that it is active as a chromosomal replicator. KARS101 contains a 50 bp sequence conserved in two other K. lactis ARS elements. The deletion of the conserved sequence in KARS101 completely abolished replicator activity, in both the plasmids and the chromosome. Gel shift assays indicated that this sequence binds proteins present in K. lactis nuclear extracts, and a 40 bp sequence, previously defined as the core essential for K. lactis ARS function, is required for efficient binding. Reminiscent of the origin replication complex (ORC), the binding appears to be ATP dependent. A similar pattern of protection of the core was seen with in vitro footprinting. KARS101 also functions as an ARS sequence in Saccharomyces cerevisiae. A comparative study using S. cerevisiae nuclear extracts revealed that the sequence required for binding is a dodecanucleotide related to the S. cerevisiae ARS consensus sequence and essential for S. cerevisiae ARS activity.