Asymmetry of the phospholipid bilayer of rat liver endoplasmic reticulum

The phospholipids of intact microsomal membranes were hydrolysed 50% by phospholipase C of Clostridium welchii, without loss of the secretory protein contents of the vesicle, which are therefore not permeable to the phospholipase. Phospholipids extracted from microsomes and dispersed by sonication were hydrolysed rapidly by phospholipase C-Cl. welchii with the exception of phosphatidylinositol. Assuming that only the phospholipids of the outside of the bilayer of the microsomal membrane are hydrolysed in intact vesicles, the composition of this leaflet was calculated as 84% phosphatidylcholine, 8% phosphatidylethanolamine, 9% sphingomyelin and 4% phosphatidylserine, and that of the inner leaflet 28% phosphatidylcholine, 37% phosphatidylethanolamine, 6% phosphatidylserine and 5% sphingomyelin. Microsomal vesicles were opened and their contents released in part by incubation with deoxycholate (0.098%) lysophosphatidylcholine (0.005%) or treatment with the French pressure cell. Under these conditions, hydrolysis of the phospholipids by phospholipase C-Cl. welchii was increased and this was mainly due to increased hydrolysis of those phospholipids assigned to the inner leaflet of the bilayer, phosphatidylethanolamine and phosphatidylserine. Phospholipase A₂ of bee venom and phospholipase C of Bacillus cereus caused rapid loss of vesicle contents and complete hydrolysis of the membrane phospholipids, with the exception of sphingomyelin which is not hydrolysed by the former enzyme.     

Cryoresistance of rat liver endoplasmic reticulum membranes]

The effects of freezing of microsomes in liquid nitrogen and those of storage of microsomal suspensions at 2-4 degrees C and -3 - -5 degrees C for 24 hrs, on the enzymatic activities and hydrophobicity of membranes were studied. The hydrophobicity was determined by fluorescence of bound 1,8-anilino-naphthalene sulfonate. Rapid freezing of the microsomal suspension in liquid nitrogen followed by rapid warming did not change the hydrophobicity of the membranes, the rate of enzymatic lipid peroxidation, the level of cytochrome P-450 and the activity of NADH- and NADPH-cytochrome c reductase. A considerable decrease in the rate of enzymatic lipid peroxidation and membrane hydrophobicity was observed in the microsomes stored for 24 hrs at 2-4 degrees C. The 24-hr storage at -3 - -5 degrees C with subsequent thawing resulted in a rapid aggregation of the microsomes.     

Polypeptide translocation across the endoplasmic reticulum membrane.

Many polypeptides have been postulated to play direct roles in secretory protein translocation based on genetic criteria, cross-linking, and antibody inhibition. Much of the excitement in the next few years will come from the resolution of current controversies. What is the nature of the ribosome receptor, and is it essential for translocation? Is BiP required for translocation in mammalian cells? Are all of the polypeptides of signal peptidase and oligosaccharyltransferase required for catalytic function, or do some of them mediate steps of protein translocation? One of the best ways to resolve these problems will be to determine the importance of each in reconstituted translocation reactions by fractionation or immunodepletion, or by analysis in a purified reaction. Another approach is to identify homologues of these molecules in S. cerevisiae and to assess their importance in in vivo translocation. Several mechanistic questions remain to be addressed as well. Does the protein translocation apparatus consist of protein, or lipid, or both? How are integral membrane proteins inserted? How is the translocon gated to admit only unfolded or partially folded secretory polypeptides and to exclude cytoplasmic molecules? The answers to these questions will illuminate a basic enigma in cell biology that has remained unanswered for many years.     

Outet mitochondrial membrane continuous with endoplasmic reticulum

Summary Continuity of outer mitochondrial membrane with tubular profiles of endoplasmic reticulum (ER) character is found in rat liver hepatocytes and in the ciliateTetrahymena pyriformis. Such membrane continuity is evident from thin sections through intact cells and isolated fractions as well as with negatively stained isolated mitochondria. The ER-profiles continuous with the outer mitochondrial membrane appear predominantly smooth, whereas in some cases they showed a few ribosomes associated with them. The observations are discussed as another indication of the close structural and chemical relationship between the outer mitochondrial membrane and the ER. In addition, it is hypothesized that this cisternal continuity provides a route for transfer of special proteins (and possibly lipid components, too) from the rough ER into the mitochondria.The authors thank Drs. H.Falk (this institute), E. C.Bracker, and D. J.Morré (Purdue University) for the permanent and stimulating discussions during the course of the work as well as for access to unpublished information. The work was partially supported by the Deutsche Forschungsgemeinschaft.     

The association of phosphorylase kinase with membranes of rat liver smooth endoplasmic reticulum

Upon fractionation of a post mitochondrial supernatant from rat liver, phosphorylase kinase activity was largely recovered in the cytosol and the smooth endoplasmic reticulum (SER) fraction. The presence of phosphorylase kinase in SER vesicles was not due to an interaction of the enzyme with glycogen particles, since previous elimination of SER glycogen either by 48 h animal starvation or by treatment of the membrane fraction with -amylase did not significantly alter phosphorylase kinase activity content. Washing of the initial pellet of SER fraction (crude SER) by dilution and recentrifugation, released in the supernatant an amount of phosphorylase kinase activity, which is dependent on: i) the degree of dilution, ii) the number of washes, iii) the ionic strength of the washing solution and iii) the presence or absence of Ca²⁺. Crude SER-associated phosphorylase kinase was marginally affected by increased concentrations of antibody against rabbit skeletal muscle holoenzyme which nevertheless drastically inhibited cytosolic enzyme activity, while it showed a higher resistance to partial proteolysis and a different Western blotting profile with anti-phosphorylase kinase when compared with the soluble kinase. A small but significant fraction of SER phosphorylase kinase was strongly associated with the microsomal fraction being partly extractable only in presence of detergents. This membrane-bound enzyme form exhibited an alkaline pH optimum, in contrast to the neutral pH optima of both soluble and weakly associated phosphorylase kinase.Abbreviations SER smooth endoplasmic reticulum - RER rough endoplasmic reticulum - PMS post mitochondrial supernatant - MES 2-(N-morpholino) ethane sulfonic acid - PMSF phenylmethylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

The effects of inducers of the endoplasmic reticulum, peroxisomes and mitochondria on the amounts and synthesis of ubiquinone in rat liver subcellular membranes

Rats were treated with inducers of peroxisomes, mitochondria and the endoplasmic reticulum, as well as receiving diets and drug known to influence the mevalonate pathway. Treatment with clofibrate and 2-diethylhexylphthalate (DEHP) increased microsomal and mitochondrial ubiquinone contents, but a decrease was observed in lysosomes. In vivo labeling of this lipid with [3H]mevalonate was also elevated. The amount of cholesterol did not change upon exposure to these inducers of peroxisomes and mitochondria, but its rate of labeling was decreased. The concentration of dolichol increased only after treatment with DEHP and only in lysosomes. The inducers of the endoplasmic reticulum phenobarbital, 3-methylcholanthrene and N-nitrosodiethylamine enhanced the rate of ubiquinone synthesis and exposure to the latter two substances also elevated the amount of this lipid in microsomes. A cholesterol-rich diet increased the labeling of ubiquinone and decreased cholesterol labeling, while cholestyramine treatment had opposite effects on lipid labeling in both microsomes and mitochondria. The results demonstrate that the ubiquinone contents of the various membranes of hepatocytes change in a characteristic manner under the influence of inducers and dietary factors. Clearly, the level of ubiquinone and its biosynthesis are regulated separately from those of the other products of the mevalonate pathway, cholesterol and dolichol.     

Changes in phospholipid composition of the mitochondrial membrane after orthotopic liver transplantation

Changes in phospholipids and their fatty acid composition in liver mitochondria obtained from allogenic rats with orthotopic liver transplants were measured with and without immunosuppressive treatment. In untreated allogenic rats, mitochondrial phosphorylation activity was severely deteriorated at 8 days after transplantation. A significant change was also found in the amount of cardiolipin compared with other classes of phospholipids. Namely, cardiolipin decreased, and lysodiphosphatidylglycerol and phosphatidylglycerol increased concomitantly. Furthermore, the percentage of linoleic acid in cardiolipin decreased dramatically. Decrease in cardiolipin and changes in its fatty acid composition may be attributed to the deterioration of mitochondrial function upon acute rejection.     

The differentiation of rat liver endoplasmic reticulum membranes: Apo–cytochrome P450 as a membrane protein

The proteins of washed microsomal membranes from adult rat liver were solubilized by 2% SDS and electrophoresed on polyacrylamide gels. Confirming earlier reports, a large Coomassie-Blue staining band in the ～50,000 MW region was identified as cytochrome P₄₅₀ by four criteria: similar electrophoretic mobility to a purified cytochrome P₄₅₀ preparation, an increase in this band after in vivo phenobarbital administration, a decrease in this band after in vivo allylisopropylacetamide administration, and direct specific binding of added purified heme to this region of a washed, unfixed gel. Although cyt P₄₅₀ is not spectrally evident until just at the time of birth of the rats, a large band in this region was detectable in gels of microsomal membrane protein at all times, from three days before birth onward; this band also bound added heme after membrane proteins from fetal rat liver microsomes were electrophoresed on the gels. The conclusion was that apo-cyt P₄₅₀ is present in microsomal membranes at these times during differentiation, and that, regarding this protein, during differentiation heme is bound to the apo-protein already there, concomitant with a synthesis of more cyt P₄₅₀ molecules. The process of differentiation of this membrane type is also discussed.     

Functional changes of endoplasmic reticulum membranes associated with liver regeneration

The fluidity and lipid composition of microsomal membranes have been studied at the earliest stage of liver regeneration in the rat (16 h after partial hepatectomy). The physical properties of the membranes have been measured by electron paramagnetic resonance analysis of freedom of motion of lipid and protein analogue probes. The fluidity of the hydrophobic core and of the microenvironment surrounding membrane proteins appeared to be modified, while no modifications were detectable in the fluidity at the surface or in bulk biochemical composition. The kinetic parameters of two enzymes of the endoplasmic reticulum (3-hydroxy-3-methyl glutaryl coenzyme A reductase and glucose-6-phosphatase) which are differentially localized within the membrane bilayer, were also measured. The temperature dependence of both enzymes was modified in the proliferating system, but these modifications were not consistent with the changes detectable in their specific activity. A model to explain the changes that occur in this proliferating membrane system is presented.     

Interactions of liver endoplasmic reticulum membranes and polysomesin vitro

Summary The interactions of various preparations of endoplasmic reticulum membranes and polysomes have been studied by means of a sandwich sucrose gradient that clearly isolates free ribosomes, smooth endoplasmic reticulum (S.E.R.) and rough endoplasmic reticulum (R.E.R.) from the microsomal fraction of rat liver homogenates. Reconstructed rough membranes separate well from the native R.E.R. but occupy the same position along the gradients as the S.E.R. and the rough membranes, stripped of their ribosomes by means of LiCl. Native R.E.R. and S.E.R. do not bind any added labeled polysomes at 0°C; previous treatment with LiCl does not modify the behavior of S.E>R. The presence of cell sap during the binding reaction does not increase polysome fixation by stripped-rough membranes but protects in some way the polysomes and preserves all their original functional capacity of amino acid incorporation into protein.     

Polypeptide and phospholipid composition of the Rickettsia prowazekii membrane and its immunogenic properties

The composition of the cell membrane in R. prowazekii strain Breinl has been studied by the methods of electrophoresis in 7.5% polyacrylamide gel with 0.1% sodium dodecyl sulfate, iodination with Na125I in the presence of chloramine T or lactoperoxidase and the thin-layer chromatography of the common lipid fraction. Of six major polypeptides contained in whole rickettsial particles (3, 16, 26, 27, 28, 29), five polypeptides (3, 26, 27, 28, 29) making up 54% of all polypeptides of purified rickettsiae have been detected in membrane preparations obtained by either treatment. The molecular weights of these membrane polypeptides are, respectively, 133600, 34000, 29600, 21500 and 12400 daltons. The main membrane phospholipids are phosphatidylethanolamine (68.4%), phosphatidylglycerol (17.2%), phosphatidylcholine (5.1%) and cardiolipin (2.1%). The presence of cholesterol has also been established. The preparations of R. prowazekii membranes and individual membrane polypeptides are immunogenic and induce the formation of specific antibodies in white mice. The preparations of both membranes and surface polypeptide 3 (glycoprotein) have been found to possess a certain protective activity: the effect of the protection of white mice inoculated with R. prowazekii culture has proved to be 64%.     

Lipid metabolism and structure-activity features of the endoplasmic reticulum membrane in regenerating rat liver]

The relationship between the neutral lipid and phospholipid metabolism and some structure-function peculiarities of regenerating rat liver endoplasmic reticulum membranes (13 hours after surgery, i.e., corresponding to the G1-period of the cell cycle) was studied. There was an increase in the degree of the endoplasmic reticulum membrane development and the nonesterified fatty acid (NFA) and triglyceride (TG) content in regenerating rat liver microsomes. The relative specific radioactivity of neutral lipid and phospholipid fractions in regenerating rat liver microsomes was lower than in control animals, presumably due to the high rate of the microsomal lipid exchange in the regenerating liver with other cell organelles. The changes in the lipid content and rate of their metabolism in the regenerating rat liver were associated with the increase in the membrane microviscosity and the decrease in the activity of the membrane-bound enzyme (glucose-6-phosphatase). The differences in the time-dependent changes in the synthesis and metabolism of lipids in the NFA and TG fractions may be regarded as an endogenous factor determining the structure-function peculiarities of endoplasmic reticulum membranes.     

Association of isolated bovine kidney cortex peroxisomes with endoplasmic reticulum

Close lateral membrane associations of peroxisomes with endoplasmic reticulum are a common feature in bovine kidney cortex epithelial cells. Isolated highly purified peroxisome preparations from this tissue showed a remarkable and persistent copurification of peroxisomal marker enzymes with small amounts (5%) of the microsomal reference enzymes esterase and glucose-6-phosphatase. Contamination with mitochondrial and lysosomal markers was negligible. Ultrastructural examination of such preparations revealed a peculiar association of vesicles or short tubular segments with the peroxisomal membrane. Short electron dense crossbridges seemed to maintain their structural association. The cytochemical localization of glucose-6-phosphatase in peroxisome-associated membrane structures confirmed their derivation from endoplasmic reticulum. The metabolic significance of such structural peroxisome-endoplasmic reticulum associations is discussed.     

Biosynthesis of glycerolipid precursors in rat liver peroxisomes and their transport and conversion to phosphatidate in the endoplasmic reticulum.

The transport of glycerolipid intermediates, viz. palmitoyl dihydroxyacetone phosphate (DHAP) and lysophosphatidate from peroxisomes and their conversion to phosphatidate in endoplasmic reticulum (microsomes) were studied in cell-free systems. The lipids were biosynthesized from [32P]DHAP, palmitoyl-CoA, and freshly made rat liver peroxisomes and microsomes in the presence or absence of Mg2+, NADPH, and bovine serum albumin (BSA). After incubation, the soluble fraction and the membranes were separated, and the distribution of radioactive lipids in these fractions were determined. The results showed that palmitoyl-DHAP and lysophosphatidate were recovered in the supernatant when BSA was present or when BSA was absent, but Mg2+ was removed after incubation by chelation with EDTA (or ATP). At low optimum palmitoyl-CoA concentration or when palmitoyl-CoA was generated in peroxisomes, and in the absence of BSA, the biosynthesized keto ether and ester lipids and lysophosphatidate were similarly present in the supernatant. Phosphatidate, however, was always localized in the membranes. Further fractionation showed that phosphatidate was associated with the microsomes. The critical micellar concentrations of palmitoyl-DHAP and 1-palmitoyl-rac-glycerol 3-phosphate, under the incubation conditions used, were determined to be 58 and 70 microM, respectively. These results suggest that at physiological concentrations the biosynthesized lysolipids are water soluble, and therefore, a carrier protein is unnecessary for their transport. These lipids freely diffuse from peroxisomes to endoplasmic reticulum where they are converted to membrane-bound phosphatidate.     

Antioxidants as cytochrome stabilizers in endoplasmic reticulum membranes of rat liver in vivo]

It has been shown that synthetic antioxidant 4-methyl-2, 6-ditrebuthylphenole (ionole) inhibits free radical oxidation of phospholipids in the membranes of endoplasmic reticulum, increases the life time of cytochrome P-448 after its induction with 20 methylcholantrene.