Contribution of different biosynthetic pathways to species selectivity of aminoglycerophospholipids assembled into mitochondrial membranes of the yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, three pathways lead to the formation of cellular phosphatidylethanolamine (PtdEtn), namely the mitochondrial conversion of phosphatidylserine (PtdSer) to PtdEtn catalyzed by phosphatidylserine decarboxylase 1 (Psd1p), the equivalent reaction catalyzed by phosphatidylserine decarboxylase 2 (Psd2p) in the Golgi, and the CDP-ethanolamine branch of the so-called Kennedy pathway which is located to the microsomal fraction. To investigate the contributions of these three pathways to the cellular pattern of PtdEtn species (fatty acid composition) we subjected lipids of wild-type and yeast mutant strains with distinct defects in the respective pathways to mass spectrometric analysis. We also analyzed species of PtdSer and phosphatidylcholine (PtdCho) of these strains because formation of the three aminoglycerophospholipids is linked through their biosynthetic route. We demonstrate that all three pathways involved in PtdEtn synthesis exhibit a preference for the formation of C34:2 and C32:2 species resulting in a high degree of unsaturation in total cellular PtdEtn. In PtdSer, the ratio of unsaturated to saturated fatty acids is much lower than in PtdEtn, suggesting a high species selectivity of PtdSer decarboxylases. Finally, PtdCho is characterized by its higher ratio of C16 to C18 fatty acids compared to PtdSer and PtdEtn. In contrast to biosynthetic steps, import of all three aminoglycerophospholipids into mitochondria of wild-type and mutant cells is not highly specific with respect to species transported. Thus, the species pattern of aminoglycerophospholipids in mitochondria is mainly the result of enzyme specificities, but not of translocation processes involved. Our results support a model that suggests equilibrium transport of aminoglycerophospholipids between mitochondria and microsomes based on membrane contact between the two compartments.     

Synthetic lethal interaction of the mitochondrial phosphatidylethanolamine and cardiolipin biosynthetic pathways in Saccharomyces cerevisiae

Saccharomyces cerevisiae mitochondria contain enzymes required for synthesis of the phospholipids cardiolipin (CL) and phosphatidylethanolamine (PE), which are enriched in mitochondrial membranes. Previous studies indicated that PE may compensate for the lack of CL, and vice versa. These data suggest that PE and CL have overlapping functions and that the absence of both lipids may be lethal. To address this hypothesis, we determined whether the crd1delta mutant, which lacks CL, was viable in genetic backgrounds in which PE synthesis was genetically blocked. Deletion of the mitochondrial PE pathway gene PSD1 was synthetically lethal with the crd1delta mutant, whereas deletion of the Golgi and endoplasmic reticulum pathway genes PSD2 and DPL1 did not result in synthetic lethality. A 20-fold reduction in phosphatidylcholine did not affect the growth of crd1delta cells. Supplementation with ethanolamine, which led to increased PE synthesis, or with propanolamine, which led to synthesis of the novel phospholipid phosphatidylpropanolamine, failed to rescue the synthetic lethality of the crd1delta psd1delta cells. These results suggest that mitochondrial biosynthesis of PE is essential for the viability of yeast mutants lacking CL.     

The biogenesis of mitochondrial membranes in the yeast Saccharomyces cerevisiae.

Membrane lipids of yeast mitochondria have been enriched by growing yeast cells in minimal medium supplemented with specific unsaturated fatty acids as the sole lipid supplement. Using the activity of marker enzymes for the outer (kynurenine hydroxylase) and inner (cytochrome c oxidase and oligomycin-sensitive ATPase) mitochondrial membranes, Arrhenius plots have been constructed using both promitochondria and mitochondria obtained from O2-adapting cells in the presence of a second unsaturated fatty acid (i.e. linoleate (N2) to elaidic (O2)). Transition temperatures which reflect the unsaturated fatty acid enrichment of the new membranes reveal interesting features involved in the mechanism of the assembly of these two mitochondrial membranes. This approach was further enforced with both lipid depletion and mitochondrial protein inhibition studies. Kynurenine hydroxylase which does not require fatty acid for its continued synthesis during aerobiosis seems to be incorporated into the preformed linoleate-anaerobic outer membrane. The newly synthesized activities of inner mitochondrial membrane enzymes on the other hand, appear to integrate their activity into newly formed aerobic-elaidic-rich inner membrane. These latter enzymes show a distinct dependence on fatty acid supplement for their continued synthesis during their aerobic phase. This suggests that O2-dependent proteo-lipid precursors are formed before these enzymes are integrated into their membrane mosaic. Two separate models are proposed to explain these results, one for the lipid-rich outer mitochondrial membrane and another for the protein-rich inner mitochondrial membrane.     

Benzodiazepine binding to mitochondrial membranes of the amoeba Acanthamoeba castellanii and the yeast Saccharomyces cerevisiae

Benzodiazepine binding sites were studied in mitochondria of unicellular eukaryotes, the amoeba Acathamoeba castellanii and the yeast Saccharomyces cerevisiae, and also in rat liver mitochondria as a control. For that purpose we applied Ro5-4864, a well-known ligand of the mitochondrial benzodiazepine receptor (MBR) present in mammalian mitochondria. The levels of specific [(3)H]Ro5-4864 binding, the dissociation constant (K(D)) and the number of [(3)H]Ro5-4864 binding sites (B(max)) determined for fractions of the studied mitochondria indicate the presence of specific [(3)H]Ro5-4864 binding sites in the outer membrane of yeast and amoeba mitochondria as well as in yeast mitoplasts. Thus, A. castellanii and S. cerevisiae mitochondria, like rat liver mitochondria, contain proteins able to bind specifically [(3)H]Ro5-4864. Labeling of amoeba, yeast and rat liver mitochondria with [(3)H]Ro5-4864 revealed proteins identified as the voltage dependent anion selective channel (VDAC) in the outer membrane and adenine nucleotide translocase (ANT) in the inner membrane. Therefore, the specific MBR ligand binding is not confined only to mammalian mitochondria and is more widespread within the eukaryotic world. However, it can not be excluded that MBR ligand binding sites are exploited efficiently only by higher multicellular eukaryotes. Nevertheless, the MBR ligand binding sites in mitochondria of lower eukaryotes can be applied as useful models in studies on mammalian MBR.     

Cardiolipin and mitochondrial phosphatidylethanolamine have overlapping functions in mitochondrial fusion in Saccharomyces cerevisiae

The two non-bilayer forming mitochondrial phospholipids cardiolipin (CL) and phosphatidylethanolamine (PE) play crucial roles in maintaining mitochondrial morphology. We have shown previously that CL and PE have overlapping functions, and the loss of both is synthetically lethal. Because the lack of CL does not lead to defects in the mitochondrial network in Saccharomyces cerevisiae, we hypothesized that PE may compensate for CL in the maintenance of mitochondrial tubular morphology and fusion. To test this hypothesis, we constructed a conditional mutant crd1Δpsd1Δ containing null alleles of CRD1 (CL synthase) and PSD1 (mitochondrial phosphatidylserine decarboxylase), in which the wild type CRD1 gene is expressed on a plasmid under control of the TET(OFF) promoter. In the presence of tetracycline, the mutant exhibited highly fragmented mitochondria, loss of mitochondrial DNA, and reduced membrane potential, characteristic of fusion mutants. Deletion of DNM1, required for mitochondrial fission, restored the tubular mitochondrial morphology. Loss of CL and mitochondrial PE led to reduced levels of small and large isoforms of the fusion protein Mgm1p, possibly accounting for the fusion defect. Taken together, these data demonstrate for the first time in vivo that CL and mitochondrial PE are required to maintain tubular mitochondrial morphology and have overlapping functions in mitochondrial fusion.     

Contribution of Hsc70 to barotolerance in the yeast Saccharomyces cerevisiae

The contribution of Hsc70 to barotolerance in logarithmic-phase cells of the HSC70 (ssb1 and ssb2) deletion mutant and in strains expressing the HSC70 gene on either a low- or a high-copy-number plasmid was studied. The deletion-mutant strain had higher thermotolerance and a slightly lower barotolerance than the control strain. The strain that expresses the HSC70 gene in high copy number had a higher barotolerance than the strain that expresses the gene in low copy number. These results suggest that Hsc70 contributes to barotolerance during exponentially growing conditions as does Hsp104 during heat-shock treatment.     

Stability of the plasma membrane in Saccharomyces cerevisiae enriched with phosphatidylcholine or phosphatidylethanolamine.

Spheroplasts from Saccharomyces cerevisiae NCYC 366, enriched in phosphatidylethanolamine after growth in medium supplemented with 1 mM ethanolamine, were more resistant to osmotic lysis than were spheroplasts from cells grown in the presence of 1 mM choline and enriched in phosphatidylcholine.     

Roles of phosphatidylethanolamine and of its several biosynthetic pathways in Saccharomyces cerevisiae

Three different pathways lead to the synthesis of phosphatidylethanolamine (PtdEtn) in yeast, one of which is localized to the inner mitochondrial membrane. To study the contribution of each of these pathways, we constructed a series of deletion mutants in which different combinations of the pathways are blocked. Analysis of their growth phenotypes revealed that a minimal level of PtdEtn is essential for growth. On fermentable carbon sources such as glucose, endogenous ethanolaminephosphate provided by sphingolipid catabolism is sufficient to allow synthesis of the essential amount of PtdEtn through the cytidyldiphosphate (CDP)-ethanolamine pathway. On nonfermentable carbon sources, however, a higher level of PtdEtn is required for growth, and the amounts of PtdEtn produced through the CDP-ethanolamine pathway and by extramitochondrial phosphatidylserine decarboxylase 2 are not sufficient to maintain growth unless the action of the former pathway is enhanced by supplementing the growth medium with ethanolamine. Thus, in the absence of such supplementation, production of PtdEtn by mitochondrial phosphatidylserine decarboxylase 1 becomes essential. In psd1Delta strains or cho1Delta strains (defective in phosphatidylserine synthesis), which contain decreased amounts of PtdEtn, the growth rate on nonfermentable carbon sources correlates with the content of PtdEtn in mitochondria, suggesting that import of PtdEtn into this organelle becomes growth limiting. Although morphological and biochemical analysis revealed no obvious defects of PtdEtn-depleted mitochondria, the mutants exhibited an enhanced formation of respiration-deficient cells. Synthesis of glycosylphosphatidylinositol-anchored proteins is also impaired in PtdEtn-depleted cells, as demonstrated by delayed maturation of Gas1p. Carboxypeptidase Y and invertase, on the other hand, were processed with wild-type kinetics. Thus, PtdEtn depletion does not affect protein secretion in general, suggesting that high levels of nonbilayer-forming lipids such as PtdEtn are not essential for membrane vesicle fusion processes in vivo.     

Isolation and characterization of highly purified mitochondrial outer membranes of the yeast Saccharomyces cerevisiae (method).

Mitochondrial outer membrane vesicles (OMV) from the yeast Saccharomyces cerevisiae were prepared by osmotic swelling and mechanical disruption of mitochondria that had been isolated at pH 6.0 and purified by density gradient centrifugation. The OMV were obtained in a yield of 1% (protein/protein) with respect to the mitochondria. The OMV were shown to be essentially free of mitochondrial inner membrane protein markers, while contamination with endoplasmic reticulum was around 5% (protein-based). The very low phosphatidylserine synthase activity present in the OMV preparation indicated that contamination with mitochondria-associated membranes (MAM) was negligible. The resistance of the outer membrane protein Tom40 to digestion by trypsin demonstrated the sealed nature and right-side out orientation of the OMV. Analysis of the phospholipid composition revealed that the contents of phosphatidylcholine and phosphatidylinositol are higher and the content of phosphatidylethanolamine is lower in the mitochondrial outer membrane as compared to whole mitochondria. Cardiolipin is largely depleted in the OMV.     

Isolation and characterization of highly purified mitochondrial outer membranes of the yeast Saccharomyces cerevisiae (Method)

Mitochondrial outer membrane vesicles (OMV) from the yeast Saccharomyces cerevisiae were prepared by osmotic swelling and mechanical disruption of mitochondria that had been isolated at pH 6.0 and purified by density gradient centrifugation. The OMV were obtained in a yield of 1% (protein/protein) with respect to the mitochondria. The OMV were shown to be essentially free of mitochondrial inner membrane protein markers, while contamination with endoplasmic reticulum was around 5% (protein-based). The very low phosphatidylserine synthase activity present in the OMV preparation indicated that contamination with mitochondria-associated membranes (MAM) was negligible. The resistance of the outer membrane protein Tom40 to digestion by trypsin demonstrated the sealed nature and right-side out orientation of the OMV. Analysis of the phospholipid composition revealed that the contents of phosphatidylcholine and phosphatidylinositol are higher and the content of phosphatidylethanolamine is lower in the mitochondrial outer membrane as compared to whole mitochondria. Cardiolipin is largely depleted in the OMV.     

Utilization of endogenous diacylglycerol for the synthesis of triacylglycerol, phosphatidylcholine and phosphatidylethanolamine by lipid particles from baker's yeast (Saccharomyces cerevisiae).

The activity of the enzymes diacylglycerol acyltransferase (EC 2.3.1.20), cholinephosphotransferase (EC 2.7.8.2) and ethanolaminephosphotransferase (EC 2.7.8.1) have been measured in a lipid particle preparation from baker's yeast (Saccharomyces cerevisiae) with endogenous 1,2-diacylglycerol as substrate. For all three enzymes the rate of diacylglycerol utilization was established with respect to substrate and Mg2+ concentration. Neither of the enzyme activities was stimulated significantly by addition of diacylglycerols. The conversion of diacylglycerol into triacylglycerol in the presence of CDP-choline and CDPethanolamine, and the synthesis of phospholipids in the presence of acyl-CoA either added or generated in situ were studied. Neither CDPcholine nor CDPethanolamine had an effect on triacylglycerol synthesis. Exogenous acyl-CoA had no effect on either choline- or ethanolaminephosphotransferase activity. However, when the necessary substrates for formation of acyl-CoAs in situ (ATP, CoA, Mg2+ and free fatty acids) were added a decrease in both cholinephosphotransferase and ethanolaminephosphotransferase activity was observed. This inhibition was shown to be due to ATP and might explained as a result of chelation of the Mg2+, a necessary activator of both the choline- and the ethanolaminephosphotransferase.     

Purification and characteristics of a mitochondrial endonuclease from the yeast Saccharomyces cerevisiae

Saccharomyces cerevisiae contains a membrane-bound mitochondrial nuclease. The enzyme was purified nearly 500-fold from sphaeroplasts of the organism by differential centrifugation, differential solubilization, heparin-agarose chromatography, and gel filtration. A final specific activity of 98 mumol min-1 (mg of protein)-1 was obtained. The enzyme required further purification to achieve homogeneity. Two peaks of activity were obtained after gel filtration with apparent molecular weights of 140000 and 57000. Otherwise, these two components have nearly identical characteristics. Without detergent the enzyme is insoluble and has very low activity. Zwittergent 3-14 or Triton X-100 in the presence of KCl could be used to solubilize and activate the enzyme. A number of other detergents were much less effective in solubilizing or activating the nuclease. The enzyme requires Mg2+ for activity, and this can be replaced to some degree by Mn2+ but not by Ca2+ or Zn2+. It is most active at pH 6.5-7.0 and degrades the substrate to small oligonucleotides with 5'-phosphate ends. The relative rates of hydrolysis were 100 for poly(A), 31 for ssDNA, 19 for RNA, 2.1 for dsDNA, and less than or equal to 0.2 for poly(C). Under the assay conditions used the enzyme appears to constitute about 90% of the total nuclease activity of the cell. The enzyme is unstable, especially at neutral and alkaline pH.     

Inorganic polyphosphates of different fractions in the mutant yeast Saccharomyces cerevisiae with impaired mitochondrial ATP synthesis

Impaired synthetase function of the mitochondrial ATPase induced by mutation in the ATP22 gene results in decreased accumulation of inorganic polyphosphates in the stationary growth phase of the yeast Saccharomyces cerevisiae grown on glucose. The content of polyphosphates in the mutant strain in this phase is 2.5 times lower than in the parent strain. This difference is most pronounced for the acid-soluble polyP1 fraction and the alkali-soluble polyP3 fraction. Polyphosphate chain length in mutant cells is less than in the parent cells in both the acid-soluble polyP1 and in the salt-soluble polyP2 fractions. The mutation had no effect on polyphosphates content in the mitochondria.     

Two distinct pathways for trehalose assimilation in the yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae can synthesize trehalose and also use this disaccharide as a carbon source for growth. However, the molecular mechanism by which extracellular trehalose can be transported to the vacuole and degraded by the acid trehalase Ath1p is not clear. By using an adaptation of the assay of invertase on whole cells with NaF, we showed that more than 90% of the activity of Ath1p is extracellular, splitting of the disaccharide into glucose. We also found that Agt1p-mediated trehalose transport and the hydrolysis of the disaccharide by the cytosolic neutral trehalase Nth1p are coupled and represent a second, independent pathway, although there are several constraints on this alternative route. First, the AGT1/MAL11 gene is controlled by the MAL system, and Agt1p was active in neither non-maltose-fermenting nor maltose-inducible strains. Second, Agt1p rapidly lost activity during growth on trehalose, by a mechanism similar to the sugar-induced inactivation of the maltose permease. Finally, both pathways are highly pH sensitive and effective growth on trehalose occurred only when the medium was buffered at around pH 5.0. The catabolism of trehalose was purely oxidative, and since levels of Ath1p limit the glucose flux in the cells, batch cultures on trehalose may provide a useful alternative to glucose-limited chemostat cultures for investigation of metabolic responses in yeast.     

Synthetic lethal interaction of the mitochondrial phosphatidylethanolamine biosynthetic machinery with the prohibitin complex of Saccharomyces cerevisiae

The majority of mitochondrial phosphatidylethanolamine (PtdEtn), a phospholipid essential for aerobic growth of yeast cells, is synthesized by phosphatidylserine decarboxylase 1 (Psd1p) in the inner mitochondrial membrane (IMM). To identify components that become essential when the level of mitochondrial PtdEtn is decreased, we screened for mutants that are synthetically lethal with a temperature-sensitive (ts) allele of PSD1. This screen unveiled mutations in PHB1 and PHB2 encoding the two subunits of the prohibitin complex, which is located to the IMM and required for the stability of mitochondrially encoded proteins. Deletion of PHB1 and PHB2 resulted in an increase of mitochondrial PtdEtn at 30 degrees C. On glucose media, phb1Delta psd1Delta and phb2Delta psd1Delta double mutants were rescued only for a limited number of generations by exogenous ethanolamine, indicating that a decrease of the PtdEtn level is detrimental for prohibitin mutants. Similar to phb mutants, deletion of PSD1 destabilizes polypeptides encoded by the mitochondrial genome. In a phb1Delta phb2Delta psd1(ts) strain the destabilizing effect is dramatically enhanced. In addition, the mitochondrial genome is lost in this triple mutant, and nuclear-encoded proteins of the IMM are assembled at a very low rate. At the nonpermissive temperature mitochondria of phb1Delta phb2Delta psd1(ts) were fragmented and aggregated. In conclusion, destabilizing effects triggered by low levels of mitochondrial PtdEtn seem to account for synthetic lethality of psd1Delta with phb mutants.     

A mitochondrial pyruvate dehydrogenase bypass in the yeast Saccharomyces cerevisiae.

Spheroplasts of the yeast Saccharomyces cerevisiae oxidize pyruvate at a high respiratory rate, whereas isolated mitochondria do not unless malate is added. We show that a cytosolic factor, pyruvate decarboxylase, is required for the non-malate-dependent oxidation of pyruvate by mitochondria. In pyruvate decarboxylase-negative mutants, the oxidation of pyruvate by permeabilized spheroplasts was abolished. In contrast, deletion of the gene (PDA1) encoding the E1alpha subunit of the pyruvate dehydrogenase did not affect the spheroplast respiratory rate on pyruvate but abolished the malate-dependent respiration of isolated mitochondria. Mutants disrupted for the mitochondrial acetaldehyde dehydrogenase gene (ALD7) did not oxidize pyruvate unless malate was added. We therefore propose the existence of a mitochondrial pyruvate dehydrogenase bypass different from the cytosolic one, where pyruvate is decarboxylated to acetaldehyde in the cytosol by pyruvate decarboxylase and then oxidized by mitochondrial acetaldehyde dehydrogenase. This pathway can compensate PDA1 gene deletion for lactate or respiratory glucose growth. However, the codisruption of PDA1 and ALD7 genes prevented the growth on lactate, indicating that each of these pathways contributes to the oxidative metabolism of pyruvate.