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Specific mutations in alpha- and gamma-subunits of F1-ATPase affect mitochondrial genome integrity in the petite-negative yeast Kluyveromyces lactis. 总被引:1,自引:0,他引:1
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We have shown previously that mutations in nuclear genes, termed MGI, for mitochondrial genome integrity, can convert the petite-negative yeast Kluyveromyces lactis into a petite-positive form with the ability to produce mitochondrial genome deletion mutants (Chen and Clark-Walker, Genetics, 133, 517-525, 1993). Here we describe that two genes, MGI2 and MGI5, encode the alpha- and gamma-subunits of mitochondrial F1-ATPase. Specific mutations, Phe443-->Ser and Ala333-->Val in MGI2, and Thr275-->Ala in MGI5, confer on cells the ability to produce petite mutants spontaneously with deletions in mitochondrial (mt) DNA and the capacity to lose their mitochondrial genomes upon treatment with ethidium bromide. Structural integrity of the F1 complex seems to be needed for expression of the Mgi- phenotype as null mutations in MGI2 and MGI5 remove the ability to form mtDNA deletions. It is suggested that mgi mutations allow petites to survive because an aberrant F1 complex prevents collapse of the mitochondrial inner membrane potential that normally occurs on loss of mtDNA-encoded F0 subunits. 相似文献
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I Ferrero C Rossi M P Landini P P Puglisi 《Biochemical and biophysical research communications》1978,80(2):340-348
The effect of glucose in two different strains of the petite-negative yeast is studied. The results obtained show that one strain () is glucose repressible for Glutamate Dehydrogenase and β-Galactosidase, whereas the other one (CBS 2360) is almost completely insensitive. The effect of Erythromycin on expression of catabolite repression in CBS 2359 is also analyzed. The results show that the dependence of catabolite repression on mitochondrial protein synthesis reflect the degree of interaction between the nuclear and mitochondrial compartments. 相似文献
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Production of glucoamylase in pyruvate decarboxylase deletion mutants of the yeast Kluyveromyces lactis 总被引:1,自引:0,他引:1
Narita J Okano K Tateno T Tanino T Sewaki T Sung MH Fukuda H Kondo A 《Applied microbiology and biotechnology》2006,69(5):564-572
We have developed a novel Escherichia coli cell surface display system by employing PgsA as an anchoring motif. In our display system, C-terminal fusion to PgsA anchor
protein from Bacillus subtilis was used. The enzymes selected for display were α-amylase (AmyA) from Streptococcus bovis 148 and lipase B (CALB) from Candida antarctica. The molecular mass values of AmyA and CALB are approximately 77 and 34 kDa, respectively. The enzymes were displayed on
the surface as a fusion protein with a FLAG peptide tag at the C terminus. Both the PgsA-AmyA-FLAG and PgsA-CALB-FLAG fusion
proteins were shown to be displayed by immunofluorescence labeling using anti-FLAG antibody. The displayed enzymes were active
forms, and AmyA and CALB activities reached 990 U/g (dry cell weight) and 4.6 U/g (dry cell weight), respectively. AmyA-displaying
E. coli cells grew utilizing cornstarch as the sole carbon source, while CALB-displaying E. coli cells catalyzed enantioselective transesterification, indicating that they are effective whole-cell biocatalysts. Since a
target enzyme with a size of 77 kDa and an industrially useful lipase have been successfully displayed on the cell surface
of E. coli for the first time, PgsA protein is probably a useful anchoring motif to display various enzymes. 相似文献
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Analysis of the regions coding for transfer RNAs in Kluyveromyces lactis mitochondrial DNA. 总被引:5,自引:2,他引:3
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The major regions coding for the transfer RNA genes in the mitochondrial DNA of K. lactis were studied. Twenty one, out of a supposed twenty four tRNA genes were identified and localized with respect to other mitochondrial genes. Most of the tRNA genes were found in a cluster downstream of the large ribosomal RNA gene. The order of a few groups of genes is conserved with respect to S. cerevisiae and T. glabrata. The highly diverged intergenic sequences contained a large number of guanine-cytosine clusters which frequently formed long palindromic sequences. 相似文献
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Miyakawa I Sato H Maruyama Y Nakaoka T 《The Journal of General and Applied Microbiology》2003,49(2):85-93
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. 相似文献
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sir2 mutants of Kluyveromyces lactis are hypersensitive to DNA-targeting drugs. 总被引:4,自引:0,他引:4
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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. 相似文献
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G Parenti-Castelli M Degli Esposti E Bertoli G Lenaz 《Bollettino della Società italiana di biologia sperimentale》1979,55(19):1988-1992
Short-chain ubiquinone (UQ-3) abolishes oligomycin sensitivity of ATPase in submitochondrial particles and the effect is reversed by long-chain ubiquinone (UQ-7). Ubiquinone-3 also abolishes DCCD sensitivity of ATPase in submitochondrial particles but the effect is not reversed by long-chain ubiquinones. These data suggest that ubiquinone interferes with energy transfer process by interaction with mitochondrial ATPase. 相似文献
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With the recent development of powerful molecular genetic tools, Kluyveromyces lactis has become an excellent alternative yeast model organism for studying the relationships between genetics and physiology. In particular, comparative yeast research has been providing insights into the strikingly different physiological strategies that are reflected by dominance of respiration over fermentation in K. lactis versus Saccharomyces cerevisiae. Other than S. cerevisiae, whose physiology is exceptionally affected by the so-called glucose effect, K. lactis is adapted to aerobiosis and its respiratory system does not underlie glucose repression. As a consequence, K. lactis has been successfully established in biomass-directed industrial applications and large-scale expression of biotechnically relevant gene products. In addition, K. lactis maintains species-specific phenomena such as the "DNA-killer system, " analyses of which are promising to extend our knowledge about microbial competition and the fundamentals of plasmid biology. 相似文献
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A fragment of mitochondrial DNA containing the Kluyveromyces lactis gene for valine-tRNA (tRNAVAL) was isolated as a multicopy suppressor of a respiratory-deficient mutant of this yeast. The mutant produced a truncated Cox14p because of a +1 frameshift mutation in COX14, a nuclear gene encoding a protein imported into mitochondria which is necessary for respiration (Fiori et al. 2000 Yeast 16: 307-314). We report here that the mitochondrial tRNAVAL gene, when transformed into K. lactis cells, is transcribed outside mitochondria and suppresses the frameshift mutation in COX14 restoring the correct reading frame during translation of its mRNA. In fact, using histidine tagging, the existence of a suppressed Cox14p of normal length was demonstrated in mutants expressing the mt-tRNAVAL from the nucleus. Suppression could occur through a non-canonical four base pairing between the tRNAVAL and the mutated mRNA or through slippage of ribosomes during translation. This is a new case of informational suppression in that the suppression of a chromosomal mutation is not caused by a second mutation but to a mislocalization/expression of a mt-tRNA. 相似文献
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Secretion of proteins is the most common approach to protein expression in Kluyveromyces lactis. A proteomic analysis was performed on spent fermentation medium following bioreactor propagation of a wild-type industrial strain to identify proteins naturally secreted by K. lactis cells. Multidimensional separations were conducted and RP online ESI-MS/MS analysis identified 81 secreted proteins. In addition, an in silico analysis predicted 178 K. lactis proteins to be secreted via the general secretory pathway (GSP). These two datasets were compared and approximately 70% of the K. lactis proteins detected in the culture medium possessed a GSP sequence. The detected proteins included those involved with cell wall structure and synthesis, carbohydrate metabolism, and proteolysis, a result that may have significant bearing on heterologous protein expression. Additionally, both the experimental and in silico datasets were compared to similar, previously published datasets for Candida albicans. With the methodology presented here, we provide the deepest penetration into a yeast secretome yet reported. 相似文献
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A comparative molecular genetic study of 37 Kluyveromyces strains of different origin has made it possible to find molecular markers that can differentiate between the dairy yeast Kluyveromyces lactis var. lactis and the genetically close wild Kl. lactis strains from the European "krassilnikovii" population, which are unable to ferment lactose. A restriction fragment length polymorphism analysis of the IGS2 region of the strains' rDNA reveals two different AluI profiles, one of which corresponds to Kl. lactis var. lactis while the other corresponds to yeasts from the "krassilnikovii" population. The AluI restriction profile of the IGS2 region of the rDNA also makes it possible to differentiate between the physiologically similar species Kl. marxianus and Kl. lactis. The origin of clinical Kl. lactis var. lactis isolates is discussed. 相似文献
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An enzyme activity in Kluyveromyces lactis that catalyzes the transfer of N-acetylglucosamine from uridine diphosphate N-acetylglucosamine to alpha Man(1 leads to 3) alpha Man ( 1 leads to 2) alpha Man (1 leads to 2)Man to yield alpha Man(1 leads to 3) [alpha GlcNAc(1 leads to 2)] alpha Man(1 leads to 2) alpha Man (1 leads to 2)Man, a mannoprotein side-chain unit, has been solubilized by Triton X-100 and purified 18000-fold by a combination of ion-exchange chromatography, gel filtration, hydrophobic chromatography, and adsorption to a lectin column. The enzyme activity from a K. lactis mutant (mnn2-2) that made mannoprotein lacking N-acetylglucosamine in its side chains, but that possessed a normal level of transferase activity in cell extracts, was purified and compared with the enzyme from the wild-type strain. Both transferase activities are integral membrane proteins found in particles associated with endoplasmic reticulum. The two purified enzymes had the same apparent size, heat stability, Mn2+ requirement, and Km for donor and acceptor and a similar Vmax. Wild-type and mutant cells had similar pool sizes of sugar nucleotide donor, and they incorporated labeled N-acetylglucosamine into chitin at similar rates. No evidence was obtained for an inactive enzyme precursor in mutant cells that was activated upon breaking the cells, nor did the mutant cells contain a transferase inhibitor or a hexosaminidase that could remove the sugar from the mannoprotein during processing and secretion. The mnn2-2 locus appears to be allelic with a second mutant, mnn2-1, that has the same phenotype but that lacks transferase activity in cell extracts. This suggests that the two mutations affect the structural gene for the transferase, and we conclude that the mnn2-2 mutant could contain an altered enzyme that fails to function because it is improperly localized or oriented in the membrane. 相似文献