Lipid composition and turnover of rough and smooth microsomal membranes in rat liver

Subfractions of rat liver microsomes (rough, smooth I, and smooth II), isolated in a cation-containing sucrose gradient system, were analyzed. After removal of adsorbed and luminal protein, these subfractions had the same phospholipid/protein ratio, about 0.40. Both the classes and the relative amounts of phospholipids were similar in the three subfractions, but the relative amounts of neutral lipids (predominantly free cholesterol and triglycerides) were higher in smooth I and especially in smooth II than in rough microsomes. Various pieces of evidence indicate that the neutral lipids are tightly bound to the membranes. Glycerol-(3)H was incorporated into the phospholipids of the rough and smooth I microsomes significantly faster than into those of the smooth II membranes; (32)P incorporation followed a similar but less pronounced pattern. Acetate-(3)H was incorporated into the free cholesterol of smooth I microsomes only half as fast as into the other two subfractions. Injection of phenobarbital increased the cellular phospholipid and neutral lipid content in the rough and smooth I, but not in the smooth II microsomes. Consequently, the neutral lipid/phospholipid ratio of all three subfractions remained unchanged after phenobarbital treatment. It is concluded that the membranes of the rough and the two smooth microsomal subfractions from rat liver have a similar phospholipid composition, but are dissimilar in their neutral lipid content and in the incorporation rate of precursors into membrane lipids.     

Membranes of Tetrahymena. IV. Isolation and characterization of temperature-responsive smooth and rough microsomal subfractions.

Temperature-responsive microsomes of the ciliate protozoan Tetrahymena have been originally fractionated by step centrifugation on two-layered, Mg2+-containing sucrose gradients. Three fractions have been obtained, which are termed smooth I, smooth II and rough according to the appearance of the membrane vesicles upon electron-microscopy. Smooth I, smooth II, and rough microsomes exhibit RNA/protein ratios of 0.09, 0.20, and 0.34; their phospholipid/protein ratios and their neutral lipid/phospholipid ratios were 0.52, 0.43 and 0.25, and 0.17, 0.18 and 0.13, respectively. All three fractions contain equivalent, low succinic dehydrogenase and 5'-nucleotidase activities. Glucose-6-phosphatase and acid phosphatase are more concentrated in smooth I membranes than in rough membranes. The reverse is true for ATPase. The smooth II membranes occupy an intermediate position except that their ATPase activity is the lowest of the three fractions. The specific activities of these enzymes of the three microsomal fractions are compared to those of homogenates of whole cells. Thin-layer chromatography reveals a very similar polar and nonpolar lipid pattern of the three microsomal fractions. The major phospholipid compounds are phosphatidlethanolamine, glycerideaminoethylphosphonate and phosphatidylcholine, while diglycerides, an unknown NL-compound, and triglycerides are the major apolar lipids. Gas liquid chromatography shows that the fatty acids are mainly even-numbered ranging between C12 and C18. The smooth I, smooth II and rough membranes contain 65.2, 69.3 and 72.7% unsaturated fatty acids in their polar lipids, whereas only 52.7, 49.7 and 48.3% unsaturated acids are found in their apolar lipids, respectively. The fatty acids are more unevenly distributed among the individual polar lipids than in the apolar ones.     

Ethanolamine Base-Exchange Reaction in Rat Brain Microsomal Subfractions

Crude microsomal fractions have been subfractionated by differential ultracentrifugation into subfractions A, B, and C, corresponding to light smooth, heavy smooth, and rough microsomal membranes, respectively. The purity and the vesiculation of the membranes were checked biochemically. Subfraction C showed the highest ethanolamine base-exchange activity, both on phospholipid and protein bases. The other two subfractions had roughly similar activities. The kinetic behavior of the enzyme activity, although anomalous, was similar in the three subfractions. Treatment of the vesicles with Pronase or with mercury-dextran produced inactivation of the ethanolamine base-exchange reaction in the three subfractions. These findings suggest that the active site of base-exchange activity would be localized on the external leaflet of the vesicles. Treatment of the membranes with trinitrobenzenesulfonic acid (TNBS) has shown that the newly synthesized phosphatidylethanolamine (PE) belongs to a pool easily reacting with the probe, independent of the subfraction investigated. On the other hand, the distribution of the bulk membrane PE reacting with TNBS differs in the three subfractions examined. It is concluded that the newly synthesized PE and probably the active site of the enzyme are on the external leaflet of the membrane in all subfractions and that the ethanolamine base-exchange reaction has similar properties in all subfractions.     

Distribution of the integral membrane protein NADH-cytochrome b5 reductase in rat liver cells, studied with a quantitative radioimmunoblotting assay.

The intracellular localization of the post-translationally inserted integral membrane protein, NADH-cytochrome b5 reductase, was investigated, using a quantitative radioimmunoblotting method to determine its concentration in rat liver subcellular fractions. Subcellular fractions enriched in rough or smooth microsomes, Golgi, lysosomes, plasma membrane and mitochondrial inner or outer membranes were characterized by marker enzyme analysis and electron microscopy. Reductase levels were determined both with the NADH-cytochrome c reductase activity assay, and by radioimmunoblotting, and the results of the two methods were compared. When measured as antigen, the reductase was relatively less concentrated in microsomal subfractions, and more concentrated in fractions containing outer mitochondrial membranes, lysosomes and plasma membrane than when measured as enzyme activity. Rough and smooth microsomes had 4-5-fold lower concentrations, on a phospholipid basis than did mitochondrial outer membranes. Fractions containing Golgi, lysosomes and plasma membrane had approximately 14-, approximately 16, and approximately 9-fold lower concentrations of antigen than did mitochondrial outer membranes, respectively, and much of the antigen in these fractions could be accounted for by cross-contamination. No enzyme activity or antigen was detected in mitochondrial inner membranes. Our results indicate that the enzyme activity data do not precisely reflect the true enzyme localization, and show an extremely uneven distribution of reductase among different cellular membranes.     

Segregation of the polypeptide translocation apparatus to regions of the endoplasmic reticulum containing ribophorins and ribosomes. I. Functional tests on rat liver microsomal subfractions

A preparation of rat liver microsomes containing 70% of the total cellular endoplasmic reticulum (ER) membranes was subfractionated by isopycnic density centrifugation. Twelve subfractions of different ribosome content ranging in density from 1.06 to 1.29 were obtained and analyzed with respect to marker enzymes, RNA, and protein content, as well as the capacity of these membranes to bind 80S ribosomes in vitro. After removal of native polysomes from these microsomal subfractions by puromycin in a buffer of high ionic strength their capacity to rebind 80S ribosomes approached levels found in the corresponding native membranes before ribosome stripping. This indicates that in vitro rebinding of ribosomes occurs to the same sites occupied in the cell by membrane-bound polysomes. Microsomes in the microsomal subfractions were also tested for their capacity to effect the translocation of nascent secretory proteins into the microsomal lumen utilizing a rabbit reticulocyte translation system programmed with mRNA coding for the precursor of human placental lactogen. Membranes from microsomes with the higher isopycnic density and a high ribosome content showed the highest translocation activity, whereas membranes derived from smooth microsomes had only a very low translocation activity. These results indicate the membranes of the rough and smooth portions of the endoplasmic reticulum are functionally differentiated so that sites for ribosome binding and the translocation of nascent polypeptides are segregated to the rough domain of the organelle.     

Distribution of protein-bound sugar residues in microsomal subfractions and Golgi membranes

Liver microsomal subfractions and Golgi membranes free from adsorbed and secretory proteins have a characteristic sugar composition. The ratio of mannose to galactose is largest in rough microsomes, smaller in smooth I microsomes, still smaller in smooth II microsomes, and smallest in Golgi membranes. There is about twice as much glucosamine in Golgi membranes and 3 times as much in smooth II microsomes as in the other microsomal subfractions. Golgi membranes are rich in sialic acid in comparison to rough microsomes and it is present at even higher levels in the two smooth microsomal subfractions. Increasing concentrations of deoxycholate preferentially remove protein-bound mannose and glucosamine, while releasing significantly less galactose. About half of the microsomal mannose and galactose can be liberated from the surface of intact microsomal vesicles by treatment with trypsin. When trypsin is added to permeable vesicles where the inside surface can be also attacked, an additional 20% of the total mannose but no additional galactose is liberated.     

Distribution of protein-bound sugar residues in microsomal subfractions and Golgi membranes.

Liver microsomal subfractions and Golgi membranes free from adsorbed and secretory proteins have a characteristic sugar composition. The ratio of mannose to galactose is largest in rough microsomes, smaller in smooth I microsomes, still smaller in smooth II microsomes, and smallest in Golgi membranes. There is about twice as much glucosamine in Golgi membranes and 3 times as much in smooth II microsomes as in the other microsomal subfractions. Golgi membranes are rich in sialic acid in comparison to rough microsomes and it is present at even higher levels in the two smooth microsomal subfractions. Increasing concentrations of deoxycholate preferentially remove protein-bound mannose and glucosamine, while releasing significantly less galactose. About half of the microsomal mannose and galactose can be liberated from the surface of intact microsomal vesicles by treatment with trypsin. When trypsin is added to permeable vesicles where the inside surface can be also attacked, an additional 20% of the total mannose but no additional galactose is liberated.     

Study of electrophoretic mobility of cellular membranes isolated from rat liver

Plasma membranes as well as mitochondrial and microsomal subfractions were subjected to zone electrophoresis. Treatment with neuraminidase, phospholipase A or C does not influence the movement of plasma membranes and smooth microsomes. Trypsin increases mobility of plasma membranes and smooth by about 20%, and further treatment with phospholipase C decreases mobility of plasma membranes, total smooth and smooth I microsomes, which, however, is not the case with smooth II microsomes. Low concentrations of trypsin also solubilize enzyme proteins of smooth microsomes from phenobarbital-treated rat liver, but electrophoretic mobility is not increased, indicating structural differences in induced membranes. The mobility of the outer and inner mitochondrial membranes is significantly higher than that of submitochondrial particles. For microsomes the negative surface charge density occurs in the decreasing order of: ribosomes — rough — smooth I — smooth II. A 10 mM CsCl gradient decreases the mobility of rough microsomes by 40% and of ribosomes by 20% but has no effect on total smooth microsomes. On the other hand, 5 mM MgCl₂ decreased the mobility of all three fractions. EDTA-treated rough and EDTA-treated smooth microsomes have the same electrophoretic mobilities. However, the mobilities of non-treated rough and smooth microsomes differ significantly from each other.     

Association of sialic acid with microsomal membrane structures in rat liver

The amount of sialic acid on phospholipid basis increases from rough, through smooth II and smooth I microsomes, to Golgi membranes, all of them free from most of the adsorbed and luminal protein. The incorporation rate of glucosamine-³H into sialic acid also follows a similar order. Deoxycholate removes phospholipid and sialic acid to an identical extent, and a significant part of the latter remains after trypsin and neuraminidase treatment. The sialic acid/phospholipid ratio decreases in phenobarbital-induced smooth but not in rough membranes, while the incorporation rate of glycosamine-³H into sialic acid decreases in both subfractions.     

Phosphatidylcholine synthesis for incorporation into membranes or for secretion as plasma lipoproteins by Golgi membranes of rat liver

Phosphatidylcholine, the major phospholipid of very low density lipoproteins, is packaged with triglyceride in the Golgi cisternae. CTP-phosphocholine cytidyltransferase and CDP-choline phosphotransferase activities of Golgi subfractions were higher than those of rough or smooth microsomes measured under the same conditions, indicating that phosphatidylcholine synthesis can occur in Golgi membranes. Consistent with this, the specific activity of phosphatidylcholine of Golgi membranes rose more rapidly than that of rough and smooth microsomes after injection of [14C]choline in vivo. The specific activity of the Golgi content phosphatidylcholine (non-membrane fraction) remained low. The S-adenosylmethionine phosphatidylethanolamine methyltransferase activity of Golgi subfractions was also higher than that of rough or smooth microsomes. After injection of [3H]methyl-labeled methionine in vivo, the specific activity of phosphatidylcholine of the Golgi membranes rose in parallel with that of the rough and smooth microsomes. The specific activity of the Golgi content phosphatidylcholine rose above that of the Golgi membranes and exhibited a different pattern, suggesting that this pathway may selectively label phosphatidylcholine which is secreted as lipoproteins. These observations indicate that the Golgi membranes have the enzymes necessary for synthesis of phosphatidylcholine, and incorporation of lipid precursors indicates that synthesis of phosphatidylcholine by Golgi membranes occurs in vivo.     

Study of electrophoretic mobility of cellular membranes isolated from rat liver.

Plasma membranes as well as mitochondrial and microsomal subfractions were subjected to zone electrophoresis. Treatment with neuraminidase, phospholipase A or C does not influence the movement of plasma membranes and smooth microsomes. Trypsin increases mobility of plasma membranes and smooth by about 20%, and further treatment with phospholipase C decreases mobility of plasma membranes, total smooth and smooth I microsomes, which, however, is not the case with smooth II microsomes. Low concentrations of trypsin also solubilize enzyme proteins of smooth microsomes from phenobarbital-treated rat liver, but electrophoretic mobility is not increased, indicating structural differences in induced membranes. The mobility of the outer and inner mitochondrial membranes is significantly higher than that of submitochondrial particles. For microsomes the negative surface charge density occurs in the decreasing order of: ribosomes--rough--smooth I--smooth II. A 10 mM CsCl gradient decreases the mobility of rough microsomes by 40% and of ribosomes by 20% but has no effect on total smooth micromes. On the other hand, 5mM MgCl2 decreased the mobility of all three fractions. EDTA-treated rough and EDTA-treated smooth microsomes have the same electrophoretic mobilities. However, the mobilities of non-treated rough and smooth microsomes differ significantly from each other.     

Recovery of ribophorins and ribosomes in "inverted rough" vesicles derived from rat liver rough microsomes

Treatment of rat liver rough microsomes (3.5 mg of protein/ml) with sublytical concentrations (0.08%) of the neutral detergent Triton X-100 caused a lateral displacement of bound ribosomes and the formation of ribosomal aggregates on the microsomal surface. At slightly higher detergent concentrations (0.12-0.16%) membrane areas bearing ribosomal aggregates invaginated into the microsomal lumen and separated from the rest of the membrane. Two distinct classes of vesicles could be isolated by density gradient centrifugation from microsomes treated with 0.16% Triton X-100: one with ribosomes bound to the inner membrane surfaces ("inverted rough" vesicles) and another with no ribosomes attached to the membranes. Analysis of the fractions showed that approximately 30% of the phospholipids and 20-30% of the total membrane protein were released from the membranes by this treatment. Labeling with avidin-ferritin conjugates demonstrated that concanavalin A binding sites, which in native rough microsomes are found in the luminal face of the membranes, were present on the outer surface of the inverted rough vesicles. Freeze-fracture electron microscopy showed that both fracture faces had similar concentrations of intramembrane particles. SDS PAGE analysis of the two vesicle subfractions demonstrated that, of all the integral microsomal membrane proteins, only ribophorins I and II were found exclusively in the inverted rough vesicles bearing ribosomes. These observations are consistent with the proposal that ribophorins are associated with the ribosomal binding sites characteristic of rough microsomal membranes.     

STUDIES ON THE BIOGENESIS OF SMOOTH ENDOPLASMIC RETICULUM MEMBRANES IN HEPATOCYTES OF PHENOBARBITAL-TREATED RATS : II. The Site of Phospholipid Synthesis in the Initial Phase of Membrane Proliferation

The specific activity of the acyltransferases of smooth microsomes of rat liver rose threefold by 12 h after injection of phenobarbital, while the activity of the acyltransferases of the rough microsomes rose slightly to peak at 3–4 h, and subsequently fell. The latter rise was abolished by treatment of the animal with actinomycin D or puromycin, while that of the smooth microsomes was unaffected. Incorporation of [¹⁴C]glycerol into phospholipid of smooth microsomes was elevated 100% by phenobarbital, while that of the rough microsomes was elevated 15%, and this could be accounted for by exchange between the microsomal phospholipids. The phospholipid/protein ratio of the smooth microsomes rose 1.5 times 3–4 h after injection of phenobarbital, while that of the rough microsomes fell slightly. The specific activity of NADPH cytochrome c reductase and NADPH diaphorase rose first in the rough microsomes, and subsequently in the smooth microsomes at a time coinciding with the return of the phospholipid/protein ratio to the control level. The rise in phospholipid/protein ratio was unaffected by actinomycin D or puromycin. These results indicate that the proliferating smooth membranes are the site of phospholipid synthesis, and that the phospholipid/protein ratio of these membranes may change independently.     

Analytical study of microsomes and isolated subcellular membranes from rat liver VIII. Subfractionation of preparations enriched with plasma membranes, outer mitochondrial membranes, or Golgi complex membranes

Preparations enriched with plasmalemmal, outer mitochondrial, or Golgi complex membranes from rat liver were subfractionated by isopycnic centrifugation, without or after treatment with digitonin, to establish the subcellular distribution of a variety of enzymes. The typical plasmalemmal enzymes 5'-nucleotidase, alkaline phosphodiesterase I, and alkaline phosphatase were markedly shifted by digitonin toward higher densities in all three preparations. Three glycosyltransferases, highly purified in the Golgi fraction, were moderately shifted by digitonin in both this Golgi complex preparation and the microsomal fraction. The outer mitochondrial membrane marker, monoamine oxidase, was not affected by digitonin in the outer mitochondrial membrane marker, monoamine oxidase, was not affected by digitonin in the out mitochondrial membrane preparation, in agreement wit its behavior in microsomes. With the exception of NADH cytochrome c reductase (which was concentrated in the outer mitochondrial membrane preparation), typical microsomal enzymes (glucose-6-phosphatase, esterase, and NADPH cytochrome c reductase) displayed low specific activities in the three preparations; except for part of the glucose-6-phosphatase activity in the plasma membrane preparation, their density distributions were insensitive to digitonin, as they were in microsomes. The influence of digitonin on equilibrium densities was correlated with its morphological effects. Digitonin induced pseudofenestrations in plasma membranes. In Golgi and outer mitochondrial membrane preparations, a few similarly altered membranes were detected in subfractions enriched with 5'-nucleotidase and alkaline phosphodiesterase I. The alterations of Golgi membranes were less obvious and seemingly restricted to some elements in the Golgi preparation. No morphological modification was detected in digitonin-treated outer mitochondrial membranes. These results indicate that each enzyme is associated with the same membrane entity in all membrane preparations and support the view that there is little overlap in the enzymatic equipment of the various types of cytomembranes.     

Intermembrane transfer of squalene promoted by supernatant protein factor

Supernatant protein factor (SPF), a protein that stimulates squalene epoxidation, mediates the transfer of squalene between two separable microsomal populations (Kojima, Y., E. J. Friedlander, and K. Bloch, 1981. J. Biol Chem. 256: 7235-7239). We now show that SPF also promotes the transfer of squalene associated with mitochondria or with plasma membranes to total microsomes or rough or smooth microsomal subfractions. Both rough and smooth microsomes have squalene epoxidase activity that is stimulated by SPF.     

Subfractionation of rat liver plasma membrane. Uneven distribution of plasma membrane-bound enzymes on the liver cell surface.

Plasma membranes were isolated from rat liver mainly under isotonic conditions. As marker enzymes for the plasma membrane, 5'-nucleotidase and (Na+ + K+)-ATPase were used. The yield of plasma membrane was 0.6-0.9 mg protein per g wet weight of liver. The recovery of 5'-nucleotidase and (Na+ +K+)-ATPase activity was 18 and 48% of the total activity of the whole-liver homogenate, respectively. Judged from the activity of glucose-6-phosphatase and succinate dehydrogenase in the plasma membrane, and from the electron microscopic observation of it, the contamination by microsomes and mitochondria was very low. A further homogenization of the plasma membrane yielded two fractions, the light and heavy fractions, in a discontinuous sucrose gradient centrifugation. The light fraction showed higher specific activities of 5'-nucleotidase, alkaline phosphatase, (Na+ +K+)-ATPase and Mg2+-ATPase, whereas the heavy one showed a higher specific activity of adenylate cyclase. Ligation of the bile duct for 48 h decreased the specific activities of (Na2+ +K+)-ATPase and Mg2+-ATPase in the light fraction, whereas it had no significant influence on the activities of these enzymes in the heavy fraction. The specific activity of alkaline phosphate was elevated in both fractions by the obstruction of the bile flow. Electron microscopy on sections of the plasma membrane subfractions showed that the light fraction consisted of vesicles of various sizes and that the heavy fractions contained membrane sheets and paired membrane strips connected by junctional complexes, as well as vesicles. The origin of these two fractions is discussed and it is suggested that the light fraction was derived from the bile front of the liver cell surface and the heavy one contained the blood front and the lateral surface of it.     

Studies on the latency of UDP-galactose-asialo-mucin galactosyltransferase activity in microsomal and Golgi subfractions from rat liver

The activity of UDPgalactose-asialo-mucin galactosyltransferase (EC 2.4.1.74) in microsomal and Golig subfractions was stimulated 2.4-fold after disruption of the membrane permeability barrier by hypotonic incubation. In the presence of Triton X-100, galactose transfer to asialo-mucin was increased 12-fold in rough microsomes and 5-fold in smooth microsomes both with and without hypotonic incubation; while in the Golgi subfractions no stimulation by detergent was observed. These experiments indicate differences in enzyme-lipid or enzyme-protein interactions in microsomes and Golgi membranes. Furthermore, these results strongly support the conclusion that the UDP-galactose-asialo-mucin galactosyltransferase activity in microsomal fractions is not due to contamination by Golgi vesicles but represents an enzyme activity endogenous to the endoplasmic reticulum.     

Segregation of the polypeptide translocation apparatus to regions of the endoplasmic reticulum containing ribophorins and ribosomes. II. Rat liver microsomal subfractions contain equimolar amounts of ribophorins and ribosomes

Ribophorins I and II, two transmembrane glycoproteins characteristic of the rough endoplasmic reticulum (ER) are thought to be part of the translocation apparatus for proteins made on membrane bound polysomes. To study the stoichiometry between ribophorins and membrane-bound ribosomes we have determined the RNA and ribophorin content in rat liver microsomes or in microsomal subfractions of different density (i.e., ribosome content). The specificity of antibodies against the ribophorins was demonstrated by Western blot analysis of rat liver rough microsomes separated by 2-dimensional gel electrophoresis. The ribophorin content of microsomal subfractions was determined by indirect immunoprecipitation and for ribophorin I by a radioimmune assay. In the latter assay a molar ratio of ribophorin I/ribosomes approaching one was calculated for total microsomes as well as in the gradient subfractions. We therefore suggest that ribophorins mediate the binding of ribosomes to endoplasmic reticulum membranes or play a role in co-translational process which depend on this binding, such as the insertion of nascent polypeptides into the membrane or their transfer into the cisternal lumen.     

COMPOSITION OF CELLULAR MEMBRANES IN THE PANCREAS OF THE GUINEA PIG : III. Enzymatic Activities

A comparative study of the enzymic activities of membrane fractions derived from guinea pig pancreatic homogenates has yielded the following results: Rough microsomal membranes (derived from the rough ER) have the reductase activities of the two microsomal electron transport systems but lack enzyme activities of Golgi-type (TPPase) and plasmalemmal-type (5'-nucleotidase, β-leucyl naphthylamidase, Mg-ATPase). Smooth microsomal membranes (derived primarily from the Golgi complex), zymogen granule membranes, and plasmalemmal fractions possess overlapping enzyme activities of plasmalemmal type, in different relative concentrations for each fraction. In addition, the smooth microsomal membranes exhibit TPPase and ADPase activity and share with rough microsomes the reductase activities of the two electron transport chains. Taken together with recent data on the lipid composition of the same fractions (2), these results indicate that the membranes of the pancreatic exocrine cell are chemically and functionally distinct, and hence do not mix with one another during the transport of secretory products.     

SUBCELLUAR SITES FOR SYNTHESIS OF CHONDROMUCOPROTEIN OF CARTILAGE

Microsomes from embryonic cartilage have been subfractionated to yield smooth microsomes and rough microsomes. The in vitro enzymic activities involved in chondroitin sulfate biosynthesis have been assayed in these subfractions. The results demonstrate that all of the activities necessary for linkage to protein as well as for completion of the polysaccharide chain are present in both the rough and smooth fractions. Only in the case of the polymerization of N-acetylgalactosamine and glucuronic acid could enzyme assays be done independent of endogenous acceptor. This enzyme(s) was equally distributed between the rough and smooth fractions. The activities for the addition of xylose and galactose to protein were highest in the rough fraction while that for sulfation was highest in the smooth fraction. These findings suggest that polysaccharide chain-initiation occurs in the rough endoplasmic reticulum and that chain completion occurs in the smooth reticulum. This pattern is consistent with modern theories of synthesis, transfer, and export of extracellular macromolecules.