Improved proteome analysis of Saccharomyces cerevisiae mitochondria by free-flow electrophoresis

The analysis of complex cellular proteomes by means of two-dimensional gel electrophoresis (2-DE) is significantly limited by the power of resolution of this technique. Although subcellular fractionation can be a fundamental first step to increase resolution, it frequently leads to preparations contaminated with other cellular structures. Here, we chose mitochondria of Saccharomyces cerevisiae to demonstrate that an integrated zone-electrophoretic purification step (ZE), with a free-flow electrophoresis device (FFE), can assist in overcoming this problem, while significantly improving their degree of purity. Whereas mitochondrial preparations isolated by means of differential centrifugation include a considerable degree of non-mitochondrial proteins (16%), this contamination could be effectually removed by the inclusion of a ZE-FFE purification step (2%). This higher degree of purity led to the identification of many more proteins from ZE-FFE purified mitochondrial protein extracts (n = 129), compared to mitochondrial protein extracts isolated by differential centrifugation (n = 80). Moreover, a marked decrease of degraded proteins was found in the ZE-FFE purified mitochondrial protein extracts. It is noteworthy that even at a low 2-DE resolution level, a four-fold higher number (17 versus 4) of presumably low abundance proteins could be identified in the ZE-FFE purified mitochondrial protein extracts. Therefore these results represent a feasible approach for an in-depth proteome analysis of mitochondria and possibly other organelles.     

Rapd analysis of wine Saccharomyces cerevisiae strains differentiated by pulsed field gel electrophoresis

Summary Enological yeast strains involved simultaneously during a fermentation can be identified through the analysis of their electrophoretic karyotype. The right assignment of yeasts to different strains has been checked by analysing the random amplified polymorphic DNA (RAPD), using a simple minipreparation protocol to obtain the template DNA for the polymerase chain reaction (PCR).     

Fermentation of D-xylose by free and immobilized Saccharomyces cerevisiae

With D-xylose (50 g l ) as sole carbon substrate, aerobic cultures of S. cerevisiae consumed significant amounts of sugar (26.4 g l ), producing 4.0 g xylitol l but no ethanol. In the presence of a mixture of glucose (35 g l ) and xylose (15 g l ), yeasts consumed 1.6 g xylose l that was converted nearly stoichiometrically to xylitol. Anaerobic conditions lessened xylose consumption and its conversion into xylitol. Traces of ethanol (0.4 g l ) were produced when xylose was the only carbon source, however. Agar-entrapped yeasts behaved as anaerobically-grown cultures but with higher specific rates of xylose consumption and xylitol production.     

In vivo analysis of the mechanisms for oxidation of cytosolic NADH by Saccharomyces cerevisiae mitochondria

During respiratory glucose dissimilation, eukaryotes produce cytosolic NADH via glycolysis. This NADH has to be reoxidized outside the mitochondria, because the mitochondrial inner membrane is impermeable to NADH. In Saccharomyces cerevisiae, this may involve external NADH dehydrogenases (Nde1p or Nde2p) and/or a glycerol-3-phosphate shuttle consisting of soluble (Gpd1p or Gpd2p) and membrane-bound (Gut2p) glycerol-3-phosphate dehydrogenases. This study addresses the physiological relevance of these mechanisms and the possible involvement of alternative routes for mitochondrial oxidation of cytosolic NADH. Aerobic, glucose-limited chemostat cultures of a gut2Delta mutant exhibited fully respiratory growth at low specific growth rates. Alcoholic fermentation set in at the same specific growth rate as in wild-type cultures (0.3 h(-1)). Apparently, the glycerol-3-phosphate shuttle is not essential for respiratory glucose dissimilation. An nde1Delta nde2Delta mutant already produced glycerol at specific growth rates of 0.10 h(-1) and above, indicating a requirement for external NADH dehydrogenase to sustain fully respiratory growth. An nde1Delta nde2Delta gut2Delta mutant produced even larger amounts of glycerol at specific growth rates ranging from 0.05 to 0.15 h(-1). Apparently, even at a low glycolytic flux, alternative mechanisms could not fully replace the external NADH dehydrogenases and glycerol-3-phosphate shuttle. However, at low dilution rates, the nde1Delta nde2Delta gut2Delta mutant did not produce ethanol. Since glycerol production could not account for all glycolytic NADH, another NADH-oxidizing system has to be present. Two alternative mechanisms for reoxidizing cytosolic NADH are discussed: (i) cytosolic production of ethanol followed by its intramitochondrial oxidation and (ii) a redox shuttle linking cytosolic NADH oxidation to the internal NADH dehydrogenase.     

Saccharomyces cerevisiae polymerase zeta functions in mitochondria

The MtArg8 reversion assay, which measures point mutation in mtDNA, indicates that in budding yeast Saccharomyces cerevisiae, DNA polymerase zeta and Rev1 proteins participate in the mitochondrial DNA mutagenesis. Supporting this evidence, both polymerase zeta and Rev1p were found to be localized in the mitochondria. This is the first report demonstrating that the DNA polymerase zeta and Rev1 proteins function in the mitochondria.     

Expression of bacterial endonucleases in Saccharomyces cerevisiae mitochondria

Expression vectors were created in which the 5' end of the Saccharomyces cerevisiae CDC9 gene, which encodes a mitochondrial targeting peptide, was cloned in-frame with the coding regions of the EcoR I, Hind III, and Pst I endonuclease genes. Expression of the EcoR I and Hind III fusion proteins inhibited growth of yeast on glycerol-containing media and resulted in the nearly quantitative restriction digestion of their mitochondrial DNA. In contrast, expression of Pst I, which does not recognize any sites within yeast mitochondrial DNA, had no effect on growth in glycerol-containing media, and did not affect the integrity of the mitochondrial genome.     

Resolution of microbial mixtures by free flow electrophoresis

Summary The resolution of bacterial mixtures by free flow electrophoresis (FFE) was not affected by the position of the microbes on the growth curve and approximately 70% of the individual cells applied were recovered as viable cells. The dependence of bacterial electrophoretic mobility on the pH, salt concentration, and viscosity of the electrolyte was determined. Suspending media and running electrolyte were developed which allowed collection of samples of>99% purity within two minutes of introduction of a mixture of Escherichia coli and Staphylococcus aureus. Most bacterial strains migrated in a single band, although some migrated in more than one band. Escherichia coli was resolved from each of 10 different species. The considerable variation in mobility found in 21 different E. coli strains, however, appears to preclude use of FFE as a method of species identification.     

Fatty acid synthesis in mitochondria from Saccharomyces cerevisiae

The ability of purified mitochondria isolated from S. cerevisiae to synthesize fatty acids and especially very long chain fatty acids (VLCFA) has been investigated. The VLCFA synthesis requires malonyl-CoA as the C2 unit donor and NADPH as the reducing agent. Moreover the yeast mitochondrial elongase is able to accept either exogenous long chain fatty acyl-CoAs as substrates or elongate endogenous substrates. In the latter case, ATP is required for full activity. Besides this important VLCFA formation, the mitochondria from S. cerevisiae were also able to synthesize C16 and C18.     

Localization of glucoamylase genes of Saccharomyces cerevisiae by pulsed field gel electrophoresis

The chromosomal locations of four glucoamylase-specifying genes in the yeastSaccharomyces cerevisiae have been determined. Chromosomes were separated by pulsed field gel electrophoresis and blots were probed with radiolabelledSTA2 and marker DNA from specific yeast chromosomes. The three genes encoding extracellular glucoamylases,STA1 (DEX2), STA2 (DEX1) andSTA3 (DEX3) are located on chromosomes IV, II and XIV, respectively.SGA, specifying the sporulation-specific glucoamylase, was positioned on chromosome IX.     

Role of mitochondria in ethanol tolerance of Saccharomyces cerevisiae

The presence of active mitochondria and oxidative metabolism is shown to be essential to maintain low inhibition levels by ethanol of the growth rate (), fermentation rate (v) or respiration rate () of Saccharomyces cerevisiae wild type strain S288C. Cells which have respiratory metabolism show K _i (ethanol inhibition constant) values for , v and , higher (K _i>1 M) than those of petite mutants or grande strains grown in anaerobiosis (K _i=0.7 M). In addition, the relationship between or v and ethanol concentration is linear in cells with respiratory metabolism and exponential in cells lacking respiration. When functional mitochondria are transferred to petite mutants, the resulting strain shows K _i values similar to those of the grande strain and the inhibition of and v by increasing ethanol concentrations becomes linear.     

Fusion of mitochondria with protoplasts in Saccharomyces cerevisiae.

Summary Protoplasts prepared from a neutral petite haploid BO60AF-1 (a ade2 arg4 leu2 trp C ^O E ^O O ^{O O O}) were mixed with mitochondria isolated from an oligomycin resistant respiring haploid AN^ROR 12D (a his4 leu2 thr4 C ^S E ^S O _II ^{R + +}) and treated with 30% polythylene glycol and CaCl₂. When the treated protoplasts were spread and incubated on selective agar plates, oligomycin resistant respiration-sufficient colonies appeared with low frequency. All of these colonies carried the mitochondrial genotype of C ^S E ^S O _II ^{R + +} and showed the same mating type and nutritional requirements as did BO60AF-1, thus evidencing the mitochondrial transfer into protoplasts. Recombination and transmission of the mitochondrial drug resistance markers were studied in crosses involving the strains issued from mitochondria accepted protoplasts.     

Proteomic analysis of Saccharomyces cerevisiae

Nowadays, proteomics is recognized as one of the fastest growing tools in many areas of research. This is especially true for the study of Saccharomyces cerevisiae, as it is considered to be a model organism for eukaryotic cells. Proteomic analysis provides an insight into global protein expressions from identification to quantitation, from localization to function, and from individual to network systems. Moreover, many methods for identification and quantitation of proteins based on tandem mass spectrometry workflows have recently been developed and widely applied in S. cerevisiae. The current methods and issues in the proteomic analysis of S. cerevisiae are reviewed here.     

Proteomic analysis of Saccharomyces cerevisiae

Nowadays, proteomics is recognized as one of the fastest growing tools in many areas of research. This is especially true for the study of Saccharomyces cerevisiae, as it is considered to be a model organism for eukaryotic cells. Proteomic analysis provides an insight into global protein expressions from identification to quantitation, from localization to function, and from individual to network systems. Moreover, many methods for identification and quantitation of proteins based on tandem mass spectrometry workflows have recently been developed and widely applied in S. cerevisiae. The current methods and issues in the proteomic analysis of S. cerevisiae are reviewed here.     

Saccharomyces cerevisiae mitochondria lack a bacterial-type sec machinery.

The bacterial Sec genes encode a generalized protein export machinery. Although the mitochondria present in eukaryotic cells are derived from bacterial ancestors, a comprehensive search of the complete genomic sequence for the eukaryotic yeast Saccharomyces cerevisiae did not reveal any close homologs of the bacterial Sec genes, strongly suggesting that yeast mitochondria lack a generalized bacterial-type export system. This finding has implications for the sorting of imported mitochondrial proteins to the intermembrane space compartment, and also for the insertion of mitochondrially encoded proteins into the inner membrane.