Nuclear fusion during yeast mating occurs by a three-step pathway

In Saccharomyces cerevisiae, mating culminates in nuclear fusion to produce a diploid zygote. Two models for nuclear fusion have been proposed: a one-step model in which the outer and inner nuclear membranes and the spindle pole bodies (SPBs) fuse simultaneously and a three-step model in which the three events occur separately. To differentiate between these models, we used electron tomography and time-lapse light microscopy of early stage wild-type zygotes. We observe two distinct SPBs in ~80% of zygotes that contain fused nuclei, whereas we only see fused or partially fused SPBs in zygotes in which the site of nuclear envelope (NE) fusion is already dilated. This demonstrates that SPB fusion occurs after NE fusion. Time-lapse microscopy of zygotes containing fluorescent protein tags that localize to either the NE lumen or the nucleoplasm demonstrates that outer membrane fusion precedes inner membrane fusion. We conclude that nuclear fusion occurs by a three-step pathway.     

Isolation and partial characterization of Borrelia burgdorferi inner and outer membranes by using isopycnic centrifugation.

In order to characterize the protein composition of the outer membrane of Borrelia burgdorferi, we have isolated inner and outer membranes by using discontinuous sucrose density step gradients. Outer and inner membrane fractions isolated by this method contained less than 1 and 2%, respectively, of the total lactate dehydrogenase activity (soluble marker) in cell lysate. More importantly, the purified outer membranes contained less than 4% contamination by the C subunit of F1/F0 ATPase (inner membrane marker). Very little flagellin protein was present in the outer membrane sample. This indicated that the outer membranes were relatively free of contamination by cytoplasmic, inner membrane or flagellar components. The outer membrane fractions (rho = 1.19 g/cm3) contained 0.15 mg (dry weight) of protein per mg. Inner membrane samples (rho = 1.12 g/cm3) contained 0.60 mg (dry weight) of protein per mg. Freeze-fracture electron microscopy revealed that the outer membrane vesicles contained about 1,700 intramembranous particles per micron 2 while inner membrane densities for inner and outer membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and nonequilibrium pH gel electrophoresis-SDS-PAGE analyses of inner and outer membrane samples revealed several proteins unique to the inner membrane and 20 proteins that localized specifically to the outer membrane. This analysis clearly shows that the inner and outer membranes isolated by this technique are unique structures.     

Outer and inner membrane preparations of extreme thermophile and their physico-chemical properties

Outer and inner (cytoplasmic) membranes were partially purified from the gram negative extremely thermophilic bacteria, Thermus thermophilus HB-8 by sucrose density gradient centrifugation. In spite of our efforts to separate them, the inner membrane fraction contained some outer membrane components as determined by enzyme assay and electrophoresis. When studied by 5DS spin labeling, the outer membranes showed a larger 2T11 value (lower fluidity) than the inner membranes, although the fatty acid compositions were similar. The inner membranes of the cells cultured at higher temperature showed a larger 2T11 value than the cells cultured at lower temperature. A similar phenomenon was observed with the TEMPO parameter of liposomal membranes. The upper break point (Th) of the inner membranes observed by spin labeling was slightly lower than the culture temperature of the cells, and the lower break point (T1) corresponded well to the lowest temperature limit of growth. The calorimetric heating curve of the inner membranes had a broader temperature range of transition than that of the liposomal membranes. The transition temperature observed by calorimetry seems to reflect the melting properties of the membrane lipids, while fatty acid spin probe probably reports the local environment of the membrane, which is more directly related to its biological function.     

Freeze-fracture electron microscopic analysis of ultrarapidly frozen envelope membranes on intact chloroplasts and after purification

Summary Freeze-fracture electron microscopy of ultrarapidly frozen intact pea chloroplasts has been used to characterize the supramolecular architecture of their outer and inner envelope membranes, to follow changes in these membranes caused by experimental treatments, and to identify the composition of purified envelope membrane subfractions. Examination of intact chloroplasts revealed that the two membranes exhibit dramatically different densities of intramembrane particles, with the inner membrane particle density approximately fourfold that of the outer. Analysis of purified envelope membrane subfractions indicates that the low bouyant density fraction (1.08 g/cm³) corresponds to the outer envelope membrane, whereas the relatively higher bouyant density fraction (1.13 g/cm³) is predominantly inner membrane. From qualitative and quantitative morphological data we conclude that the outer membrane subfraction is pure whereas the inner membrane subfraction is significantly contaminated by outer membrane. These results confirm conclusions reached from biochemical analysis of these membranes.During the course of the studies on intact chloroplasts, sites were observed where the outer and inner envelope membranes appear to adhere to each other (contact sites). Some of the contact sites observed on intact chloroplasts survived the envelope purification procedures as evidenced by their presence on a small number of vesicles in inner membrane preparations. The practical significance of these putative contact sites is discussed.     

Purified outer membranes of Serpulina hyodysenteriae contain cholesterol.

We have isolated outer and inner membranes of Serpulina hyodysenteriae by using discontinuous sucrose density gradients. The outer and inner membrane fractions contained less than 1 and 2%, respectively, of the total NADH oxidase activity (soluble marker) in the cell lysate. Various membrane markers including lipooligosaccharide (LOS), the 16-kDa outer membrane lipoprotein (SmpA), and the C subunit of the F1F0 ATPase indicated that the lowest-density membrane fraction contained outer membranes while the high-density membrane fraction contained inner membranes and that both are essentially free of contamination by the periplasmic flagella, a major contaminant of membranes isolated by other techniques. The outer membrane fractions (rho = 1.10 g/cm3) contained 0.25 mg of protein/mg (dry weight), while the inner membrane samples (rho = 1.16 g/cm3) contained significantly more protein (0.55 mg of protein/mg [dry weight]). Lipid analysis revealed that the purified outer membranes contained cholesterol as a major component of the membrane lipids. Treatment of intact S. hyodysenteriae with different concentrations of digitonin, a steroid glycoside that interacts with cholesterol, indicated that the outer membrane could be selectively removed at concentrations as low as 0.125%.     

Physiological and biochemical analysis of the effects of alkaline phosphatase overproduction in Escherichia coli.

Overexpression of the Escherichia coli phoA gene, coding for alkaline phosphatase (PhoA), on multicopy plasmids caused a severe defect in the precursor processing (secretion) of PhoA, beta-lactamase, and the outer membrane protein OmpA. This secretion defect continued even after the repression of phoA expression, indicating that protein secretion was irreversibly impaired in cells. Among the secretory proteins, only OmpA gradually secreted posttranslationally. The inverted inner membrane vesicles prepared from cells with the secretion defect showed appreciably reduced translocation activity in vitro. But the membrane vesicles retained the ability to generate a proton motive force which, together with ATP, is essential as an energy source for the efficient secretion of proteins in E. coli. An appreciable amount of incompletely translocated PhoA molecules was detected in the inner membranes of cells with the secretion defect.     

Separation and Properties of the Inner and Outer Membranes of a Psychrophilic Bacterium, Vibrio sp. Strain ABE-1

The inner and outer membranes from a marine psychrophilic bacterium,Vibrio sp. ABE-1, were separated and purified by desalting,lysozyme treatment, and ultracentrifugation. Enzyme assay, electrophoresis,and chemical analysis showed that they had been highly purified.The isolated outer membrane had no 3-deoxy-D-mannooctulosonate(KDO) and chemical analysis revealed that it contained aboutthree times as much total lipid as the inner membrane. Consequently,the density of the inner membrane was higher than that of theouter membrane (1.19–1.23 gµcm³ for IM; 1.16–1.20g–cm³ for OM). The absolute majority of the lipids inthese membranes were phospholipids, more than half of whichwas identified as phosphatidylethanolamine. Virtually all cardiolipinwas found exclusively in the inner membrane, whereas an unidentifiedphospholipid containing hexosamine was only in the outer membrane.The ratios of unsaturated to total fatty acids in the innerand outer membranes were 76.0% and 69.6%, respectively. (Received February 23, 1984; Accepted August 8, 1984)     

Conjugative junctions in RP4-mediated mating of Escherichia coli

The physical association of bacteria during conjugation mediated by the IncPalpha plasmid RP4 was investigated. Escherichia coli mating aggregates prepared on semisolid medium were ultrarapidly frozen using copper block freezing, followed by freeze substitution, thin sectioning, and transmission electron microscopy. In matings where the donor bacteria contained conjugative plasmids, distinctive junctions were observed between the outer membranes of the aggregates of mating cells. An electron-dense layer linked the stiffly parallel outer membranes in the junction zone, but there were no cytoplasmic bridges nor apparent breaks in the cell walls or membranes. In control experiments where the donors lacked conjugative plasmids, junctions were not observed. Previous studies have shown that plasmid RP4 carries operons for both plasmid DNA processing (Tra1) and mating pair formation (Tra2). In matings where donor strains carried Tra2 only or Tra2 plus the pilin-processing protease TraF, junctions were found but they were shorter and more interrupted than the wild type. If the donor strain had the pilin gene knocked out (trbC), junctions were still found. Thus, it appears that the electron-dense layer between the outer membranes of the conjugating cells is not composed of pilin.     

Ultrastructural aspects and amino acid composition of the purified inner and outer membranes of human liver mitochondria as compared to rat liver mitochondria.

1. The mitochondria isolated from human or rat liver were fractionated into submitochondrial particles and purified inner and outer membrane. According to different marker enzymes the inner membranes were enriched about 5-6-fold and the outer membranes about 12-14-fold. The electron microscopical appearance of the membranes was that expected on the basis of enzymic characterization. 2. A comparison of the average amino acid composition of the membrane proteins from the two types of mitochondria has been made. In the case of submitochondrial particles there were statistically significant differences between the human and rat hydrolysates for only five amino acids. Analysing the purified mitochondrial membranes there were significant differences between the two species for nine amino acids in the case of outer membranes and for 12 amino acids in the case of inner membranes. 3. With one exception all amino acids that were increased or decreased in the outer membrane exhibited a similar trend in the inner membrane of human compared with rat liver mitochondria. It appears that liver mitochondrial membranes have a species-dependent pattern of amino acid composition of their proteins.     

Lethality of the covalent linkage between mislocalized major outer membrane lipoprotein and the peptidoglycan of Escherichia coli.

The major outer membrane lipoprotein (Lpp) of Escherichia coli possesses serine at position 2, which is thought to function as the outer membrane sorting signal, and lysine at the C terminus, through which Lpp covalently associates with peptidoglycan. Arginine (R) is present before the C-terminal lysine in the wild-type Lpp (LppSK). By replacing serine (S) at position 2 with aspartate (D), the putative inner membrane sorting signal, and by deleting lysine (K) at the C terminus, Lpp mutants with a different residue at either position 2 (LppDK) or the C terminus (LppSR) or both (LppDR) were constructed. Expression of LppSR and LppDR little affected the growth of E. coli. In contrast, the number of viable cells immediately decreased when LppDK was expressed. Prolonged expression of LppDK inhibited separation of the inner and outer membranes by sucrose density gradient centrifugation, whereas short-term expression did not. Pulse-labeled LppDK and LppDR were localized in the inner membrane, indicating that the amino acid residue at position 2 functions as a sorting signal for the membrane localization of Lpp. LppDK accumulated in the inner membrane covalently associated with the peptidoglycan and thus prevented the separation of the two membranes. Globomycin, an inhibitor of lipoprotein-specific signal peptidase II, was lethal for E. coli only when Lpp possessed the C-terminal lysine. Taken together, these results indicate that the inner membrane accumulation of Lpp per se is not lethal for E. coli. Instead, a covalent linkage between the inner membrane Lpp having the C-terminal lysine and the peptidoglycan is lethal for E. coli, presumably due to the disruption of the cell surface integrity.     

Effect of plasmid RP1 on phase changes in inner and outer membranes and lipopolysaccharide from Acinetobacter calcoaceticus: a Fourier transform infrared study

The successful transfer of the resistance plasmid RP1 into the Gram-negative bacterium Acinetobacter calcoaceticus resulted in increased resistance of this microorganism to the antibiotics kanamycin and tetracycline. Microorganisms harboring the RP1 plasmid showed altered fatty acid composition in the lipopolysaccharide fraction and increased outer membrane permeability compared to organisms without the plasmid. Thermotropic gel to liquid crystal lipid phase changes were detected in both inner and outer membranes and purified lipopolysaccharide by Fourier transform infrared spectroscopy. The phase transition temperatures observed in the outer membranes and isolated lipopolysaccharide of the plasmid-containing cells were significantly higher than those of the plasmid-free organisms, while little difference was observed for the inner membranes. The plasmid-induced decrease in outer membrane fluidity may play a mediating role in the mechanisms of antibiotic resistance and susceptibility to host immune cells in Gram-negative microorganisms.     

Identification and characterization of the TolC protein, an outer membrane protein from Escherichia coli

We used the cloned tolC gene to identify, locate, and purify its gene product. Strains carrying pPR13 or pPR42 overproduced a cell envelope protein (molecular weight, 52,000). A protein of the same molecular weight was identified in radioactively labeled minicells carrying pPR13; this protein was absent in pPR11-carrying minicells. This protein was the tolC gene product, since pPR11 differed from pPR13 in having a Tn10 insertion in the tolC gene. The protein seen in cell envelopes of whole cells (TolC protein) was found to exist in an aggregated state in the outer membrane; under conditions in which OmpC and OmpF were peptidoglycan associated, TolC protein was not likewise associated. Using these properties, we purified the TolC protein and determined the sequence of twelve amino acids from the amino-terminal end. The location of the TolC protein in the outer membrane was consistent with the proposed function for the tolC gene product as a processing protein in the outer membrane.     

Mitochondrial creatine kinase mediates contact formation between mitochondrial membranes

Purified mitochondrial creatine kinase (Mi-CK) (EC 2.7.3.2) from chicken heart was shown to interact simultaneously with purified inner and outer mitochondrial membranes, thereby creating an intermembrane chondrial membranes, thereby creating an intermembrane were purified from rat liver and thus were fully devoid of Mi-CK. Intermembrane contact formation was demonstrated by measuring the binding of inner membrane vesicles to outer membranes spread at the air-water interface. Mi-CK also mediated intermembrane adhesion when membranes formed with total lipid extracts of both membranes were used, pointing to the role of lipids as potential membrane anchors of Mi-CK in the mitochondrial intermembrane space. Other enzymes of the intermembrane space that (like Mi-CK) are also cationic, as well as cytosolic isoenzymes of creatine kinase, failed to induce contact formation. Thus, of the proteins tested, membrane contact formation was specific for Mi-CK. The two oligomeric forms of Mi-CK (octamer and dimer) differed in their ability to mediate intermembrane adhesion, the octamer being more potent. Highly basic peptides, i.e. poly-L-lysines, were shown to strongly interact with membranes formed with lipid extracts of mitochondrial membranes: they both induced intermembrane binding and fusion. Interestingly, the extent of contact formation mediated by poly-L-lysines was lower than that of octameric Mi-CK. The implications of these findings on the function and localization of Mi-CK and on the structure of the mitochondrial intermembrane compartment are discussed.     

Separate fusion of outer and inner mitochondrial membranes

Mitochondria are enveloped by two closely apposed boundary membranes with different properties and functions. It is known that they undergo fusion and fission, but it has remained unclear whether outer and inner membranes fuse simultaneously, coordinately or separately. We set up assays for the study of inner and outer membrane fusion in living human cells. Inner membrane fusion was more sensitive than outer membrane fusion to inhibition of glycolysis. Fusion of the inner membrane, but not of the outer membrane, was abolished by dissipation of the inner membrane potential with K+ (valinomycin) or H+ ionophores (cccp). In addition, outer and inner membrane fusion proceeded separately in the absence of any drug. The separate fusion of outer and inner membranes and the different requirements of these fusion reactions point to the existence of fusion machineries that can function separately.     

Membrane Association and Multimerization of Secreton Component PulC

The PulC component of the Klebsiella oxytoca pullulanase secretion machinery (the secreton) was found by subcellular fractionation to be associated with both the cytoplasmic (inner) and outer membranes. Association with the outer membrane was independent of other secreton components, including the outer membrane protein PulD (secretin). The association of PulC with the inner membrane is mediated by the signal anchor sequence located close to its N terminus. These results suggest that PulC forms a bridge between the two membranes that is disrupted when bacteria are broken open for fractionation. Neither the signal anchor sequence nor the cytoplasmic N-terminal region that precedes it was found to be required for PulC function, indicating that PulC does not undergo sequence-specific interactions with other cytoplasmic membrane proteins. Cross-linking of whole cells resulted in the formation of a ca. 110-kDa band that reacted with PulC-specific serum and whose detection depended on the presence of PulD. However, antibodies against PulD failed to react with this band, suggesting that it could be a homo-PulC trimer whose formation requires PulD. The data are discussed in terms of the possible role of PulC in energy transduction for exoprotein secretion.     

Detection and subcellular localization of mature protein H, involved in excretion of cloacin DF13

Abstract To investigate whether the pCloDF13 encoded protein H is synthesized as a precursor with a signal sequence and to detect the mature protein H, radioactive labeling experiments with Escherichia coli minicells harboring different pCloDF13 derivatives were carried out. Analysis of the labeled polypeptides on special sodium dodecyl sulfate (SDS)-polyacrylamide gels revealed that the polypeptide encoded by the pCloDF13 gene H is a precursor with an apparent M _r of about 5000. The mature polypeptide had an apparent M _r of about 2800. Localization studies revealed that the mature protein H is predominantly located in the membrane fraction, both in the cytoplasmic and in the outer membranes of the minicells.     

Purification of a hexokinase-binding protein from the outer mitochondrial membrane.

Brain hexokinase (ATP:D-hexose-6-phosphotransferase, EC 2.7.1.1) binds selectively to the outer membrane of rat liver mitochondria but not to inner mitochondrial or microsomal membranes nor to the plasma membrane of human erythrocytes. A protein having subunit molecular weight of 31,000, determined by sodium dodecyl sulfate-gel electrophoresis, has been highly purified from the outer mitochondrial membrane by repetitive solubilization with octyl-beta-D-glucopyranoside followed by reconstitution into membranous vesicles when the detergent is removed by dialysis. When incorporated into lipid vesicles, the protein confers the ability to bind brain hexokinase in a Glc-6-P-sensitive manner as is seen with the intact outer mitochondrial membrane. Hexokinase binding ability and the 31,000 subunit molecular weight protein co-sediment during sucrose density gradient centrifugation. Both hexokinase binding ability and the 31,000 subunit molecular weight protein are resistant to protease treatment of the intact outer mitochondrial membrane while other membrane proteins are extensively degraded. It is concluded that this protein, designated the hexokinase-binding protein (HBP), is an integral membrane protein responsible for the selective binding of hexokinase by the outer mitochondrial membrane.     

Characterization of the F plasmid mating aggregation gene traN and of a new F transfer region locus trbE.

The product of the F plasmid transfer gene, traN, is thought to be required for the formation of stable mating aggregates during F-directed conjugation. By testing chimeric plasmids that express F transfer region segments for complementation of F lac traN mutant transfer, we mapped traN to the F transfer region between trbC and traF. Both protein and DNA sequence analysis determined the traN product to be a large, 66,000-Mr, polypeptide that undergoes signal sequence processing. The mature polypeptide was associated with outer membrane protein fractions, and a protease accessivity test confirmed that at least one portion of TraN is exposed on the cell surface. Our DNA sequence analysis also revealed that another gene, trbE, is located between traN and traF. The product of trbE was identified and shown to be a small, integral, inner membrane protein. The mating efficiency and pilus-specific phage susceptibility of a trbE::kan insertion mutant suggested that trbE is not essential for F transfer from Escherichia coli K-12 under standard mating conditions.     

Organization of K88ac-encoded polypeptides in the Escherichia coli cell envelope: use of minicells and outer membrane protein mutants for studying assembly of pili

Escherichia coli K-12 minicells, harboring recombinant plasmids encoding polypeptides involved in the expression of K88ac adhesion pili on the bacterial cell surface, were labeled with [35S]methionine and fractionated by a variety of techniques. A 70,000-dalton polypeptide, the product of the K88ac adhesion cistron adhA, was primarily located in the outer membrane of minicells, although it was less clearly associated with this membrane than the classical outer membrane proteins OmpA and matrix protein. Two polypeptides of molecular weights 26,000 and 17,000 (the products of adhB and adhC, respectively) were located in significant amounts in the periplasmic space. The 29,000-dalton polypeptide was shown to be processed in E. coli minicells. The 23.500-dalton K88ac pilus subunit (the product of adhD) was detected in both inner and outer membrane fractions. E. coli mutants defective in the synthesis of murein lipoprotein or the major outer membrane polypeptide OmpA were found to express normal amounts of K88ac antigen on the cell surface, whereas expression of the K88ac antigen was greatly reduced in perA mutants. The possible functions of the adh cistron products are discussed.     

Import of chemically synthesized signal peptides into rat liver mitochondria

The signal peptides of pre-aldehyde dehydrogenase (22-mer) and pre-ornithine transcarbamylase (27-mer) were chemically synthesized and their imports into rat liver mitochondria were studied. Both signal peptides were imported rapidly (within 2 min) in the absence of a membrane potential, exogenous ATP, or rabbit reticulocyte lysate. Signal peptides also were imported into mitochondria treated with a low concentration of trypsin which removed the outer membrane proteins. It was concluded that the chemically synthesized signal peptide could be imported differently than the precursor proteins. The imported signal peptide were found to be associated with both outer and inner membranes. Pulse-chase experiments showed that the import was unidirectional and that the signal peptides associated with inner membranes increased during the chase time. The signal peptides inhibited import of precursor proteins to different extents. Association of signal peptides with inner membrane near or at translocator sites might result in inhibition of precursor import.