Proton magnetic resonance spectroscopy of promitochondrial membranes from yeast grown under different regimes of lipid supplementation

Summary Promitochondrial membranes, prepared fromSaccharomyces cerevisiae grown anaerobically under different conditions of lipid supplementation, have been examined by PMR spectroscopy. Promitochondria from cells cultured anaerobically in media containing both unsaturated fatty acid and sterol supplements, or containing unsaturated fatty acid alone, yield high resolution spectra similar to those which are characteristic of aerobic mitochondria. By contrast, promitochondrial membranes from cells grown only with sterol supplementation in order to deplete unsaturated fatty acid and total phospholipid content of the organelles, yielded PMR spectra which were very substantially broadened. These spectra are similar to those obtained with rat liver mitochondria.PMR spectra of promitochondria from each cell type dispersed in trifluoroacetic acid, or of extracted lipids or residual proteins similarly dispersed, were different only in detail. It appears, therefore, that in the native state membranes of unsaturated fatty acid-depleted promitochondria are structurally different from promitochondria of the other two cell types. The difference may be a consequence of altered lipid-to-protein ratios, and thus of changes in the extent of lipid domain formation in the membranes of these organelles.     

The biogenesis of mitochondria. II. The influence of medium composition on the cytology of anaerobically grown Saccharomyces cerevisiae

Yeast cells grown anaerobically have been shown to vary in their ultrastructure and absorption spectrum depending upon the composition of the growth medium. The changes observed in the anaerobically grown cells are governed by the availability of unsaturated fatty acids and ergosterol and a catabolite or glucose repression. All the cells contain nuclear and plasma membranes, but the extent of the occurrence of vacuolar and mitochondrial membranes varies greatly with the growth conditions. Cells grown anaerobically on the least nutritive medium, composed of 0.5% Difco yeast extract-5% glucose-inorganic salts (YE-G), appear to contain little vacuolar membrane and no clearly recognizable mitochondrial profiles. Cells grown anaerobically on the YE-G medium supplemented with Tween 80 and ergosterol contain clearly recognizable vacuolar membrane and some mitochondrial profiles, albeit rather poorly defined. Cells grown on YE-G medium supplemented only with Tween 80 are characterized by the presence of large amounts of cytoplasmic membrane in addition to vacuolar membrane and perhaps some primitive mitochondrial profiles. When galactose replaces glucose as the major carbon source in the medium, the mitochondrial profiles within the cytoplasm become more clearly recognizable and their number increases. In aerobically grown cells, the catabolite repression also operates to reduce the total number of mitochondrial profiles. The possibility is discussed that cells grown anaerobically on the YE-G medium may not contain mitochondrial membrane and, therefore, that such cells, on aeration, form mitochondrial membrane from nonmitochondrial sources. A wide variety of absorption compounds is observed in anaerobically grown cells which do not correspond to any of the classical aerobic yeast cytochromes. The number and relative proportions of these anaerobic compounds depend upon the composition of the growth medium, the most complex spectrum being found in cells grown in the absence of lipid supplements.     

The effect of altered membrane sterol composition on the temperature dependence of yeast mitochondrial ATPase

The sterol content of cells of $\text{Saccharomyces cerevisiae}$ was manipulated by growing the organism anaerobically in a medium containing excess supplements of unsaturated fatty acids and a range of supplements of ergosterol. Anaerobic mitochondrial precursor structures were isolated whose membrane lipids contain the same fatty acid composition but whose sterol content varies from 7 to 105 mg/g mitochondrial protein. Arrhenius plots of the mitochondrial ATPase activity of the different preparations show a discontinuity with Arrhenius activation energies of about +40 and +80 KJ/mole, respectively, above and below the transition temperature. However, the temperature of the transition is markedly dependent on sterol composition, and increases by up to 17° as the sterol content of the mitochondria is progressively decreased. These results support the concepts that membrane lipid composition influences the activity of membrane-bound enzymes, and that sterols promote the gel to liquid phase transition in biological membranes.     

Biogenesis of mitochondria. The effects of altered membrane lipid composition on cation transport by mitochondria of Saccharomyces cerevisiae

1. The fatty acid composition of the membrane lipids of a fatty acid desaturase mutant of Saccharomyces cerevisiae was manipulated by growing the organism in a medium containing defined fatty acid supplements. 2. Mitochondria were obtained whose fatty acids contain between 20% and 80% unsaturated fatty acids. 3. Mitochondria with high proportions of unsaturated fatty acids in their lipids have coupled oxidative phosphorylation with normal P/O ratios, accumulate K(+) ions in the presence of valinomycin and an energy source, and eject protons in an energy-dependent fashion. 4. If the unsaturated fatty acid content of the mitochondrial fatty acids is lowered to 20%, the mitochondria simultaneously lose active cation transport and the ability to couple phosphorylation to respiration. 5. The loss of energy-linked reactions is accompanied by an increased passive permeability of the mitochondria to protons. 6. Free fatty acids uncouple oxidative phosphorylation in yeast mitochondria and the effect is reversed by bovine serum albumin. 7. The free fatty acid contents of yeast mitochondria are unaffected by depletion of unsaturated fatty acids, and free fatty acids are not responsible for the uncoupling of oxidative phosphorylation in organelles depleted in unsaturated fatty acids. 8. It is suggested that the loss of energy-linked reactions in yeast mitochondria that are depleted in unsaturated fatty acids is a consequence of the increased passive permeability to protons, and is caused by a change in the physical properties of the lipid phase of the inner mitochondrial membrane.     

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.     

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 pro-mitochondria 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.     

Development of mitochondrial membranes in anaerobically grown yeast cells.

Biochemical analyses of mitochondrial marker substances, especially cardiolipin and oligomycin-sensitive ATPase [EC 3.6.1.3], as well as electron microscopic observations were carried out to eludicate the process of mitochondrial development in annaerobic yeast cells. Cardiolipin was found to be localized in the mitochondria in anaerobic cells. Its cellular content was a little higher in the stationary phase than in the exponential phase in glucose-grown cells and increased further in galactose-grown cells. The lipid content of the mitochondrial preparation obtained from glucose-grown stationary cells was nearly as high as that from galactose-grown cells. It was also comparable to that of aerobic cells in the stationary phase, where mitochondria are fully developed. Both cellular and mitochondrial levels of oligomycin-sensitive ATPase activity were also found to rise markedly in galactose-grown anaerobic cells, although not in stationary phase cells grown anaerobically on glucose. These high levels of the mitochondrial markers indicate a developmental change in mitochondrial structure even in anaerobically grown cells, which lack mitochondrial cytochromes. In the process of aerobic adaptation, respiratory system formation was observed to occur much faster in galactose-grown cells than in glucose-grown cells, and not to be inhibited by chloramphenicol and high concentrations of glucose structure in anaerobic cells. The developmental change was also corroborated by electron microscopic observations, which revealed the occurrence of two types of mitochondria in anaerobic cells. One was found in glucose-repressed cells and was characterized by the presence of numerous electron-dense granules in the matrix. In contrast, the other type, found in glucose-derepressed cells, had an electron-lucent matrix. No crista membrane was seen in either type of mitochondria in anaerobic cells, although the infoldings of the inner membrane, which partition the matrix into two parts and therefore are called "septum membranes," appeared frequently in the stationary phase cells. On the basis of these results, the process of mitochondrial development in yeast cells is discussed.     

Effect of unsaturated fatty acids on the biosynthesis of glucose-repressed enzymes in yeast

In anaerobically glucose-grown yeast isocitrate lyase (EC 4.1.3.1.), malate synthase (EC 4.1.3.2.) and malate dehydrogenase (EC 1.1.1.37.) are repressed by glucose. 24 h cultures still contain 0.3–0.4% glucose in the medium, which is enough to completely repress these activities. Aeration of these cells, in buffer containing acetate, initiates the formation of the three enzymes. Within 16 h, the specific activities of these enzymes increase about 140, 120 and 70-fold, respectively. Glucose-6-phosphate dehydrogenase activity was not altered. When the yeast was grown anaerobically, but with a supplement of an unsaturated fatty acid in the medium, synthesis of the three enzymes was much faster and the specific activities after 16 h of derepression were considerably higher. A relationship exists between the number of double bonds in the unsaturated fatty acid molecule and its capability to stimulate enzyme synthesis: linolenic acid is more effective than linoleic acid, which, in turn, is much more effective than oleic acid. Increasing periods of aeration with glucose of anaerobically grown cells prior to derepression results in an increasing stimulation of enzyme synthesis on subsequent derepression. Anaerobic incubation of yeast in the presence of an unsaturated fatty acid in advance to derepression also increased the velocity of enzyme formation. It is suggested that during the aeration period with glucose and during anaerobic incubation with an unsaturated fatty acid a more active protein synthesizing apparatus was formed.     

The effects of unsaturated fatty acid depletion on the proton permeability and energetic functions of yeast mitochondria

1. The fatty acid composition of the ole-1 and ole-1 petite mutants of Saccharomyces cerevisiae was manipulated by growing the organism in the presence of defined supplements of Tween 80 or by allowing cells that had first been grown in the presence of Tween 80 to deplete their unsaturated fatty acids by sequent growth in the absence of Tween 80. 2. The transition temperature of Arrhenius plots of mitochondrial ATPase (adenosine triphosphatase) increases as the unsaturated fatty acid content is lowered. 3. Cells require larger amounts of unsaturated fatty acids to grow on ethanol at lower temperatures. 4. Cells that stop growing owing to unsaturated fatty acid depletion at low temperatures are induced to grow further by raising the temperature and this results in a further depletion of unsaturated acids. This is due to a higher rate, but not a greater efficiency, of mitochondrial ATP synthesis. 5. Arrhenius plots of the passive permeability of mitochondria to protons between 4 and 37 degrees C are linear. The rate and the Arrhenius activation energy of proton entry increase greatly as the unsaturated fatty acid content is lowered. 6. Unsaturated fatty acid depletion has the same effects on the proton permeability of ole-1 petite mitochondria, indicating that the mitochondrially synthesized subunits of the ATPase are not involved in the enhanced rates of proton entry. 7. The adenylate energy charge of depleted ole-1 cells is greatly decreased by growth on ethanol medium. 8. The adenylate energy charge of isolated mitochondria is also lowered by unsaturated fatty acid depletion. 9. The results confirm that unsaturated fatty acid depletion uncouples oxidative phosphorylation in yeast both in vivo and in vitro, and is a consequence of changes in the lipid part of the membrane.     

Morphologic, biochemical and functional properties of the mitochondria of the rabbit gastric mucosa]

Purified fractions of the rabbit gastric mucosa and liver mitochondria were isolated. Electrone microscopy and functional studies of mitochondria showed the most intact mitochondria of gastric mucosa to be localized in gradient fractions of p=1.16-1.18, and those of the liver in fractions with p=1.19-1.20. The gastric mitochondria differed from the liver mitochondria by specific localization in oxyntic cells, abundance of crists, small matrix volume, presence of high respiratory control at lesser values of pH, high respiratory activity, great amount of cytochromes of electron transport chain in inner mitochondria membrane, two-fold excess of cytochroma a in relation to b and c, absence of b5 and by presence of great amount of phosphathydilaethanolamine and unsaturated fatty acids.     

Development of Respiration and Mitochondria in Mucor genevensis After Anaerobic Growth: Absence of Glucose Repression

Respiration and mitochondria in Mucor genevensis, a facultatively anaerobic dimorphic mold, have been studied in aerobically and anaerobically grown cells and in anaerobically grown cells adapting to aerobic conditions. Respiration in hyphae continues at a high level during aerobic growth but drops rapidly on exhaustion of glucose. In anaerobically grown yeastlike cells, containing no recognizable aerobic cytochromes, a small cyanide-insensitive respiration occurs. Mitochondria with well defined cristae are visible in negative contrast after KMnO(4) fixation of stringently anaerobic cells containing low amounts of fatty acid of which 10% or less are unsaturated. On aeration of anaerobically grown cells, respiratory capacity and cytochromes develop rapidly, even in the presence of 10% glucose, indicating that glucose does not repress development of respiration. However, mycelium formation by adapting yeastlike cells is repressed by high glucose concentration. In adapting cells, apparent changes in mitochondrial ultrastructure appear to be more related to changes in fixation properties of cells than to changes in the structure of mitochondria.     

Mammalian MTHFD2L encodes a mitochondrial methylenetetrahydrofolate dehydrogenase isozyme expressed in adult tissues

Previous studies in our laboratory showed that isolated, intact adult rat liver mitochondria are able to oxidize the 3-carbon of serine and the N-methyl carbon of sarcosine to formate without the addition of any other cofactors or substrates. Conversion of these 1-carbon units to formate requires several folate-interconverting enzymes in mitochondria. The enzyme(s) responsible for conversion of 5,10-methylene-tetrahydrofolate (CH(2)-THF) to 10-formyl-THF in adult mammalian mitochondria are currently unknown. A new mitochondrial CH(2)-THF dehydrogenase isozyme, encoded by the MTHFD2L gene, has now been identified. The recombinant protein exhibits robust NADP(+)-dependent CH(2)-THF dehydrogenase activity when expressed in yeast. The enzyme is localized to mitochondria when expressed in CHO cells and behaves as a peripheral membrane protein, tightly associated with the matrix side of the mitochondrial inner membrane. The MTHFD2L gene is subject to alternative splicing and is expressed in adult tissues in humans and rodents. This CH(2)-THF dehydrogenase isozyme thus fills the remaining gap in the pathway from CH(2)-THF to formate in adult mammalian mitochondria.     

The effects of altered membrane sterol composition on oxidative phosphorylation in a haem mutant of Saccharomyces cerevisiae

1. The sterol, unsaturated fatty acid and cytochrome contents of cells of a delta-aminolaevulinate synthase mutant of Saccharomyces cerevisiae are manipulated by growing the organism in media containing defined supplements of delta-aminolaevulinate and other porphyrin intermediates. 2. If unsaturated fatty acids are added to the growth medium as Tween 80, sterol content and respiratory cytochromes alone are manipulated. 3. In the presence of delta-aminolaevulinate (10-50mg/1) cells exhibit moderate to high respiratory activity, but growth yields are low, indicating a loss of oxidative phosphorylation. This is associated with the depletion of membrane lipids, either unsaturated fatty acids and sterols together or sterols alone. 4. Sterol depletion leads to the loss of coupled mitochondrial oxidative phosphorylation in vitro. 5. The lesion in oxidative phosphorylation is associated with an increase in the passive permeability of sterol-depleted mitochondria to protons. 6. Arrhenius plots of mitochondrial permeability to protons indicate that the activation energy for proton entry increases as the sterol content of the membranes decreases. 7. Studies on a cytoplasmic petite mutant isolated from strain ole-3, which lacks a functional membrane-bound protein-translocating adenosine triphosphatase, indicate that proton permeability of the petite mitochondria varies as a function of sterol composition in the same way as that of ole-3 grande mitochondria. This indicates that sterols alone are probably directly responsible for the increased proton entry, owing to a reorganization of the lipid in the membrane. 8. Supplemented ole-3 cells with a normal lipid composition and normal or higher than normal respiratory activities have a growth efficiency only 65% of that of the wild-type, indicating that a further lesion in energy metabolism may be present.     

MEMBRANOUS STRUCTURES IN YEASTS

1. Most yeast cells carrying out active respiration have spherical or ellipsoidal mitochondria, with plate-like cristae. 2. Cytoplasmic petite strains of Saccharomyces cerevisiae have aberrant mitochondria, often containing whorled membranes. Mutants with deficiencies in the tricarboxylic acid cycle have mitochondria which appear normal when the cells are grown in low levels of glucose. 3. Cells of normal and petite S. cerevisiae grown strictly anaerobically show no recognizable mitochondrial profiles. 4. Carbon substrates which can only be respired promote the development of well-defined mitochondria. In certain facultatively anaerobic yeasts respiration is suppressed by glucose and the mitochondria under these conditions are large, pleomorphic and few in number. Other fermentable carbohydrates do not give this repression. 5. A number of antibacterial antibiotics, which inhibit mitochondrial protein synthesis, cause a disorganization of the mitochondrial cristae. 6. In yeast cells adapting from anaerobic to aerobic conditions mitochondria appear to develop from proliferations of the endoplasmic reticulum, which become progressively more organized. 7. Vacuoles often contain granular material, but in S. cerevisiae the vacuole, which has been described as a lysosome, frequently contains myelin-like lipid inclusions. The material in these inclusions is apparently derived from spherosomes. 8. Endoplasmic reticulum, orientated parallel to the plasmalemma, may be associated with fermentative ability in certain facultatively anaerobic yeasts. Endoplasmic reticulum is also actively involved in the budding process. 9. Normally the yeast-cell plasmalemma shows only minor convolutions, but in chloramphenicol-grown Rhodotorula glutinis the plasmalemma produces vesicular structures termed ‘paramural bodies’. 10. The yeast nuclear membrane has about 200 pores occupying 6–8 % of the total surface area. The nuclear membrane remains intact during mitotic division in yeasts until the daughter nuclei separate.     

Identification and localization of enzymes of the fumarate reductase and nitrate respiration systems of escherichia coli by crossed immunoelectrophoresis

Crossed immunoelectrophoresis was used to analyze the components of membrane vesicles of anaerobically grown Escherichia coli. The number of precipitation lines in the crossed immunoelectrophoresis patterns of membrane vesicles isolated from E. coli grown anaerobically on glucose plus nitrate and on glycerol plus fumarate were 83 and 70, respectively. Zymogram staining techniques were used to identify immunoprecipitates corresponding to nitrate reductase, formate dehydrogenase, fumarate reductase, and glycerol-3-phosphate dehydrogenase in crossed immunoelectrophoresis reference patterns. The identification of fumarate reductase by its succinate oxidizing activity was confirmed with purified enzyme and with mutants lacking or overproducing this enzyme. In addition, precipitation lines were found for hydrogenase, cytochrome oxidase, the membrane-bound ATPase, and the dehydrogenases for succinate, malate, dihydroorotate, D-lactate, 6-phosphogluconate, and NADH. Adsorption experiments with intact and solubilized membrane vesicles showed that fumarate reductase, hydrogenase, glycerol-3-phosphate dehydrogenase, nitrate reductase, and ATPase are located at the inner surface of the cytoplasmic membrane; on the other hand, the results suggest that formate dehydrogenase is a transmembrane protein.     

Biogenesis of microbial transport systems: evidnce for coupled incorporation of newly synthesized lipids and proteins into membrane

The biogenesis of the independent β-galactoside and β-glueoside transport systems of Escherichia coli K12 has been studied using an unsaturated fatty acid auxotroph. The response of transport rate to temperature was determined for cells grown with different fatty acid supplements. A change in the slope of an Arrhenius plot for transport rate was obtained at transition temperatures unique for each of the five fatty acid supplements tested. Both of the transport systems studied here had identical transition temperatures when the cells were grown with the same fatty acid supplement, indicating that the transport temperature characteristics are determined primarily by the properties of the lipid phase of the membrane.     

Effects of unsaturated fatty acid deprivation on neutral lipid synthesis in Saccharomyces cerevisiae

The effects of unsaturated fatty acid deprivation on lipid synthesis in Saccharomyces cerevisiae strain GL7 were determined by following the incorporation of [14C]acetate. Compared to yeast cells grown with oleic acid, unsaturated fatty acid-deprived cells contained 200 times as much 14C label in squalene, with correspondingly less label in 2,3-oxidosqualene and 2,3;22,23-dioxidosqualene. Cells deprived of either methionine or cholesterol did not accumulate squalene, demonstrating that the effect of unsaturated fatty acid starvation on squalene oxidation was not due to an inhibition of cell growth. Cells deprived of olefinic supplements displayed additional changes in lipid metabolism: (i) an increase in 14C-labeled diacylglycerides, (ii) a decrease in 14C-labeled triacylglycerides, and (iii) increased levels of 14C-labeled decanoic and dodecanoic fatty acids. The changes in squalene oxidation and acylglyceride metabolism in unsaturated fatty acid-deprived cells were readily reversed by adding oleic acid. Pulse-chase studies demonstrated that the [14C]squalene and 14C-labeled diacylglycerides which accumulated during starvation were further metabolized when cells were resupplemented with oleic acid. These results demonstrate that unsaturated fatty acids are essential for normal lipid metabolism in yeasts.     

On the rate-limiting step in the transfer of long-chain acyl groups across the inner membrane of brown adipose tissue mitochondria

Brown adipose tissue mitochondria predominantly oxidize fatty acids in order to generate heat for non-shivering thermogenesis, and have an unusually high capacity for net transfer of long-chain fatty acyl groups from the outer to the inner (matrix) compartment. The activities of the "outer" and "inner" carnitine long-chain acyltransferases have been estimated in isolated mitochondria of cold-acclimated guinea pits by the continuous spectrophotometric recording of the redox level of flavoproteins in the acyl-CoA dehydrogenase pathway. This redox level is determined by the intramitochondrial content of acyl-CoA under the selected experimental conditions. The apparent initial rate of the "inner" acyltransferase (palmitoyl-L-carnitine added) is three order of magnitudes higher than the "outer" acyltransferase (palmitoyl-CoA added), and this difference is not influenced by the substrate concentration, pH and reaction temperature. Thus, the "outer" acyltransferase reaction is rate limiting in the transfer of long-chain acyl groups across the inner membrane of these mitochondria and catalyzes a non-equilibrium reaction in the intact organelle. Estimates of the absolute rate of the "outer" long-chain acyltransferase indicate that it exceeds that of rat liver mitochondria by a factor of 20.     

Lipidomics of intact mitochondria by MALDI-TOF/MS

A simple and fast method of lipid analysis of isolated intact mitochondria by means of MALDI-TOF mass spectrometry is described. Mitochondria isolated from bovine heart and yeast have been employed to set up and validate the new method of lipid analysis. The mitochondrial suspension is directly applied over the target and, after drying, covered by a thin layer of the 9-aminoacridine matrix solution. The lipid profiles acquired with this procedure contain all peaks previously obtained by analyzing the lipid extracts of isolated mitochondria by TLC and/or mass spectrometry. The novel procedure allows the quick, simple, precise, and accurate analysis of membrane lipids, utilizing only a tiny amount of isolated organelle; it has also been tested with intact membranes of the bacterium Paracoccus denitrificans for its evolutionary link to present-day mitochondria. The method is of general validity for the lipid analysis of other cell fractions and isolated organelles.