Altered distribution profiles of endoplasmic reticulum subfractions after incubation of Krebs II ascites cells with different concentrations of cytochalasin B

Information on the interaction between endoplasmic reticulum (ER) membranes and components of the skeletal network of the cell was gained by treating cells with the antimicrofilament agent cytochalasin B prior to cell disruption by nitrogen cavitation. Treatment of Krebs II ascites cells with cytochalasin B (5–10 μg ml^?1) resulted in an increased yield of three ER membrane subfractions — heavy rough (HR), light rough (LR) and smooth (S) membranes, as judged by ³H-choline incorporation in gradient fractions following discontinuous sucrose gradient centrifugation. The major increase was observed in the HR fraction. These results indicate that the actual yield of the respective ER membrane subfractions after cell disruption is dependent on the degree of direct and/or indirect interaction between individual ER membranes and actin containing filaments of the cytoskeleton in the intact cell.     

Ultrastructure and polysome content of the microsomal subfractions of mouse plasmacytoma cells

Summary The endoplasmic reticulum (ER) of MPC-11 cells released as vesicles upon cell disruption by nitrogen cavitation was separated from the bulk of mitochondria, lysosomes and plasma membranes by a low speed centrifugation. The ER membranes were fractionated on discontinuous sucrose gradients into heavy rough (HR), light rough (LR) and smooth (S) membranes. The morphology of subcellular fractions was studied by electron microscopy and the ER membranes were shown to be virtually free of contaminating organelles. The S fraction was easily distinguishable because of the lack of ribosomes but there were no apparent morphological differences between the HR and LR fractions. Of total activity in the microsomal subfractions, 70% of the UDPase and 67% of the 5′-nucleotidase activity was associated with the S fraction. Polysomes were present in the HR, LR and nuclear-associated ER fractions but not in the S fraction. The HR and LR fractions did not appear to be contaminated to any great extent with free polysomes. RNA/protein and RNA/phospholipid ratios of the HR fraction were higher than those of the LR fraction, indicating a greater density of ribosomes in the former fraction. These ratios were much lower in the S fraction reflecting the low ribosome content.     

Compartmentation of the rough endoplasmic reticulum

It has become evident during recent years that a wide variety of proteins are synthesized on membrane-bound polysomes, very many of which are not ultimately secreted from the cell. The majority of proteins appear to go through some form of post-translational modification before the final appearance of an 'active' product, and in some cases the polypeptide chain may be modified before the completed protein molecule is released from the ribosome. This then raises the question concerning the possibility of the organization of the rough endoplasmic reticulum into individual domains, or compartments, each of which may have the responsibility of performing definite and well defined functions. During recent years the behaviour of two subfractions of the rough endoplasmic reticulum in a variety of cell types and under a variety of conditions has been studied in order to gain insight into a possible compartmentation of this organelle. Throughout the studies disruption of cells has been performed by nitrogen cavitation. This technique was chosen in order to provide conditions of homogenization which were extremely reproducible since shearing forces, mechanical damage and the effects of local heating were eliminated. Endoplasmic reticulum (ER) membranes isolated from the post-mitochondrial supernatant have been separated into subfractions by centrifugation on discontinuous sucrose gradients. By virtue of their high density imparted by the association of ribosomes, rough ER (RER) membranes penetrate 1.4 M sucrose accumulating above either 2.0 M sucrose (light rough -LR membranes) or a cushion of 2.3 M sucrose (heavy rough -HR membranes). Smooth (S) membranes, which are virtually devoid of ribosomes, collect above 1.4 M sucrose. The HR, LR and S subfractions in MPC-11 cells differ in a number of respects: RNA/protein and RNA/phospholipid ratios, polysome profiles and marker enzymes. When cells were homogenized in buffer containing 25 mM KCl then all three ER subfractions were observed, however, when the buffer contained 100 mM KCl then only the LR and S subfractions were observed in gradients, radioactivity equivalent to that in the HR fraction was not recovered in the other two subfractions. Four times as many light chain immunoglobulin polypeptides were found associated with polysomes of HR membranes compared to LR membranes. The nuclear associated ER (NER), though very active in protein synthesis, was only 20% as active in the synthesis of light chain as the combined LR/HR fraction. Studies with MPC-11 cells showed that the relative amounts of the three ER subfractions were related to the phase of the cell cycle.(ABSTRACT TRUNCATED AT 400 WORDS)     

Light rough endoplasmic reticulum membranes re-appear before heavy rough membranes after feeding starved MPC-11 cells

In 48 hr starved MPC-11 cells smooth (S) endoplasmic reticulum (ER) membranes accounted for more than 80% of the total ER membrane fraction. After 2 1/2 hr feeding the amount of S membranes was reduced to below 55% while light rough (LR) membranes had increased from about 16% of the total to 42%. The percentage of heavy rough (HR) membranes only showed a minor increase during the incubation period. The results show that following an accumulation of S membranes in starved cells the LR membranes 're-appear' more rapidly than the HR membranes. This difference in behaviour provides further support to the concept that LR and HR membranes represent distinct domains of the ER system.     

Correlation of the effect of feeding upon the cell cycle distribution profiles of MPC-11 cells with the relative appearance of [3H]-choline labeled material in microsomal subfractions and other cell fractions

Summary Cultures of mouse plasmacytoma cells (MPC-11) grown within the range 6–23 × 10⁵ cells/ml showed considerable variation in cell cycle distribution profiles and also differences with regard to relative amounts of microsomal subfractions. The variability of appearance of heavy rough (HR) and light rough (LR) microsomal subfractions was not merely due to differences in nutritional state of the culture. Cultures containing a high S/G₂ + M cell cycle distribution ratio showed a high content of HR microsomal membranes; as the S/G₂ + M ratio decreased, so too decreased the amount of HR material whilst the amount of LR microsomal membranes increased. The results indicate that there is a direct correlation between phase of cell cycle and both amount and relative distribution of rough microsomal membranes, the smooth fraction (S), however, remains relatively unchanged.     

The importance of salt concentration during nitrogen cavitation of MPC-11 cells for the isolation of heavy rough endoplasmic reticulum membranes

When MPC-11 cells are disrupted by nitrogen cavitation in the presence of buffer containing 25-40 mM KCl then endoplasmic reticulum membranes can be separated into three subfractions by sucrose density gradient centrifugation: heavy rough (HR), light rough (LR) and smooth (S) membranes. An increase in the salt concentration of the buffer to 50 mM or above results in the occurrence of only the LR and S membranes in gradients. However, when cells equilibrated at high pressure in the bomb in 100 mM KCl buffer were expelled into a diluting buffer such that the final buffer concentration was reduced to 25 mM KCl upon cell disruption, then appreciable amounts of HR membranes are observed in sucrose gradients. The results would suggest that salt concentrations above 25-40 mM KCl stabilize the interaction between HR membranes and the cytoskeleton to such a degree that these membranes are pelleted at low speed together with the nuclei. The yields of LR and S membranes are apparently not affected to any significant degree by altered salt concentration.     

Differences in the association of ribosomes with heavy rough and light rough endoplasmic reticulum membranes of MPC-11 cells

The treatment of total endoplasmic reticulum membranes of mouse plasmacytoma cells with EDTA resulted in an abolition of the heavy rough (HR) subfraction, while there was a large increase in smooth (S) membranes. When HR and light rough (LR) endoplasmic reticulum membranes were treated individually with EDTA and re-centrifuged on discontinuous sucrose gradients it was observed that HR were converted into S membranes, i.e. membranes virtually devoid of ribosomes. LR membranes were not affected to the same extent but there was a shift to a somewhat lower density. A quantitation of ribosomes released by EDTA showed that 95% of 60 S and 72% of 40 S subunits were removed from HR membranes while for LR membranes the corresponding values were 8.5 and 22.6% respectively. Ratios of radioactivity to absorbance at 260 nm calculated for 40 S and 60 S subunits isolated from HR and LR membranes show that 60 S subunits from LR membranes, in contrast to those from HR membranes, equilibrate only slowly with the free pool of ribosomal subunits. The results indicate that the ribosomes associated with HR membranes are 'loosely bound' and those with LR membranes 'tightly bound'. When poly(A)-containing mRNA isolated from HR and LR membranes was translated in vitro and the products analysed for light-chain immunoglobulin content, it was found that the HR fraction was enriched in light-chain mRNA.     

Variation in microsomal subfractions obtained from normal animal tissues and transformed cells following cell disruption by nitrogen cavitation

Summary Microsomal membranes were obtained from MPC-11 cells, L-cells, Krebs II ascites cells and various normal animal tissues following cell disruption by nitrogen cavitation. Membrane preparations were applied to discontinuous sucrose gradients designed to separate three fractions — heavy rough (HR), light rough (LR) and smooth (S) microsomes. In each of the transformed cell lines all three fractions were found whilst in the normal tissues tested the HR fraction was absent. Of the normal tissues liver and pancreas were rich in both LR and S microsomes, the presence of large amounts of LR indicating a rich protein synthesizing activity on membrane-bound polysomes. Kidney also contained appreciable LR but much less than both liver and pancreas. Both heart and lung contained virtually only S microsomal material — a reflection of low protein synthetic activity on membrane-bound polysomes. Attempts to promote the appearance of the HR fraction in liver, kidney and pancreas by incubation in tissue culture medium, or, in the case of pancreas, by cholecystokinin/pancreozymin/secretin, stimulation bothin vivo andin vitro were unsuccessful.     

Distribution of microsomal triglyceride transfer protein within sub-endoplasmic reticulum regions in human hepatoma cells

Very low-density lipoprotein (VLDL) particles are formed in the endoplasmic reticulum (ER) through the association of lipids with apolipoprotein B (apoB). Microsomal triglyceride transfer protein (MTP), which transfers lipid molecules to nascent apoB, is essential for VLDL formation in ER. However, little is known of the distribution and interaction of MTP with apoB within ER. In this study, distribution patterns of apoB and MTP large subunit (lMTP) within ER were examined. Microsomes prepared from HuH-7 cells, a human hepatoma cell line, were further fractionated into rough ER (RER)-enriched subfractions (ER-I fraction) and smooth ER (SER)-enriched subfractions (ER-II fraction) by iodixanol density-gradient ultracentrifugation. ApoB was evenly distributed in the ER-I and the ER-II fractions, while 1.5 times more lMTP molecules were present in the ER-I fraction than in the ER-II fraction. lMTP and apoB were coprecipitated both in the ER-I and in the ER-II fractions by immunoprecipitation whenever anti-apoB or an anti-lMTP antibodies were used. ApoB-containing lipoprotein particles showed a lower density in the ER-II fraction than those in the ER-I fraction. From these results, it is suggested that MTP can function in both rough and smooth regions of ER in human hepatoma cells.     

Electrophoretic studies on liver endoplasmic reticulum membrane polypeptides and on their phosphorylation in vivo and in vitro

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to examine the polypeptide patterns of rat liver rough and smooth endoplasmic reticulum (ER) membrane fractions stripped of ribosomes. Approximately 67 polypeptides were resolved from the rough ER membrane fraction. The polypeptide pattern of the smooth ER membrane fraction was similar to that of the rough ER membrane fraction, but exhibited substantially lower amounts of some seven polypeptides. Three of these polypeptides, of apparent molecular weights 63,000, 65,000, and 87,000, were of particular interest, as they could not be ascribed to contamination of stripped rough ER membrane fractions by residual ribosomal polypeptides. Conditions of treatment with low concentrations of trypsin were established that markedly diminished the capacity of the stripped rough ER membrane fraction to bind ribosomes in vitro and that also effected a partial detachment of ribosomes from nonstripped rough ER membranes; the results of electrophoretic analyses of rough ER membrane fractions treated in these manners are described. Comparison of the polypeptide patterns of guinea pig, mouse, and rabbit liver ER membrane fractions with rat liver ER membrane fractions revealed considerable variations in the distribution of the polypeptides of 63,000, 65,000, and 87,000 molecular weight among the ER membrane fractions of these species. The combined results of these studies indicate that the polypeptide of 87,000 molecular weight, although particularly sensitive to attack by trypsin, is not involved in the binding of ribosomes to the rough ER membrane fraction. Studies by others (cf. Kreibich, G., Grebenau, R., Mok, W., Pereyra, B., Rodriguez-Boulan, E., and Sabatini, D. D. (1977) Fed. Proc. 36, 656) have implicated the polypeptides of 63,000 and 65,000 molecular weight in this process. The patterns of phosphorylated polypeptides of rough and smooth ER membrane fractions of rat and mouse liver were also examined, using labeling in vivo with sodium [32p]phosphate or in vitro with [gamma-32P]ATP. Approximately 25 phosphorylated components were resolved by electrophoresis in the ER membrane fractions of both species. Evidence is presented that suggests that the great majority of these components are phosphopolypeptides. Differences were noted in the patterns of phosphorylation produced by in vivo and in vitro labeling; minor differences were also observed between the patterns of phosphorylation of the rough and smooth ER membrane fractions in either situation. The overall results afford an indirect approach toward evaluating the possible involvement of specific rough ER membrane polypeptides in ribosome-binding and reveal that liver ER membranes contain a substantially greater number of phosphorylated polypeptides thatn previously reported.     

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.     

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.     

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.     

Characterization of the ribosomal binding site in rat liver rough microsomes: Ribophorins I and II,two integral membrane proteins related to ribosome binding

Rat liver rough endoplasmic reticulum membranes (ER) contain two characteristic transmembrane glycoproteins which have been designated ribophorins I and II and are absent from smooth ER membranes. These proteins (MW 65,000 and 63,000 respectively) are related to the binding sites for ribosomes, as suggested by the following findings: (i) The ribophorin content of the rough ER membranes corresponds stoichiometrically to the number of bound ribosomes; (ii) ribophorins are quantitatively recovered with the bound polysomes after most other ER membrane proteins are dissolved with the nonionic detegent Kyro EOB; (iii) in intact rough microsomes ribophorins can be crosslinked chemically to the ribosomes and therefore are in close proximity to them. Treatment of rough microsomes with a low Triton X-100 concentration leads to the lateral displacement of ribosomes on the microsomal surface and to the formation of aggregates of bound ribosomes in areas of membranes which frequently invaginate into the microsomal lumen. Subfractionation of Triton-treated microsomes containing invaginations led to the recovery of smooth and “rough-inverted” vesicles. Ribophorins were present only in the latter fraction, indicating that both proteins are displaced together with the ribosome-binding capacity of rough and smooth microsomal membranes reconstituted after solubilization with detergents sugest that ribophorins are necessary for in vitro ribosome binding. Ribophorin-like proteins were found in rough microsomes obtained from secretory tissues of several animal species. The two proteins present in rat lacrimal gland microsomes have the same mobility as hepatocyte ribophorins and cross-react with antisera against them.     

A model for transport of a viral membrane protein through the early secretory pathway: minimal sequence and endoplasmic reticulum lateral mobility requirements

Viral movement proteins exploit host endomembranes and the cytoskeleton to move within the cell via routes that, in some cases, are dependent on the secretory pathway. For example, melon necrotic spot virus p7B, a type II transmembrane protein, leaves the endoplasmic reticulum (ER) through the COPII‐dependent Golgi pathway to reach the plasmodesmata. Here we investigated the sequence requirements and putative mechanisms governing p7B transport through the early secretory pathway. Deletion of either the cytoplasmic N–terminal region (CR) or the luminal C–terminal region (LR) led to ER retention, suggesting that they are both essential for ER export. Through alanine‐scanning mutagenesis, we identified residues in the CR and LR that are critical for both ER export and for viral cell‐to‐cell movement. Within the CR, alanine substitution of aspartic and proline residues in the DSSP β–turn motif (D₇AP₁₀A) led to movement of discrete structures along the cortical ER in an actin‐dependent manner. In contrast, alanine substitution of a lysine residue in the LR (K₄₉A) resulted in a homogenous ER distribution of the movement protein and inhibition of ER–Golgi traffic. Moreover, the ability of p7B to recruit Sar1 to the ER membrane is lost in the D₇AP₁₀A mutant, but enhanced in the K₄₉A mutant. In addition, fluorescence recovery after photobleaching revealed that K₄₉A but not D₇AP₁₀A dramatically diminished protein lateral mobility. From these data, we propose a model whereby the LR directs actin‐dependent mobility toward the cortical ER, where the cytoplasmic DSSP β–turn favors assembly of COPII vesicles for export of p7B from the ER.     

Chilling followed by incubation at 37 degrees causes a flow of 3H-choline labeled phospholipids from nuclear-associated endoplasmic reticulum to other endomembranes of L-cells

The exposure of L-cells to a period of 15 min incubation in ice followed by a return to normal culture conditions caused distinct alterations in the distribution pattern of 3H-choline incorporation in phospholipids in subcellular fractions. The amount of radioactivity appearing in the nuclei and nuclear-associated endoplasmic reticulum decreased while that in the mitochondrial and endoplasmic reticulum membrane fractions increased, suggesting a precursor-product relationship. These changes appeared to occur in a linear fashion. Furthermore, the increase in radioactivity in individual endoplasmic reticulum subfractions differed in that label increased fivefold in light rough membranes but only about twofold in the HR and S subfractions.     

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.     

Characterization of functional domains of the lymphocyte plasma membrane

Highly purified plasma membranes of calf thymocytes were fractionated by means of affinity chromatography on concanavalin A-Sepharose into two subfractions; one (fraction 1) eluted freely from the affinity column, the second (fraction 2) adhered specifically to concanavalin A-Sepharose. Previous analysis showed that both subfractions were right-side-out (Resch, K., Schneider, S. and Szamel, M. (1981) Anal. Biochem. 117, 282-292). The ratio of cholesterol to phospholipid was nearly identical in plasma membrane and both subfractions. When isolated plasma membranes were labelled with tritiated NaBH4, both subfractions exhibited identical specific radioactivities. After enzymatic radioiodination of thymocytes, the relative distribution of labelled proteins and externally exposed phospholipids was very similar in isolated plasma membranes and in both membrane subfractions, indicating the plasma membrane nature of the subfractions separated by affinity chromatography on concanavalin A-Sepharose. This finding was further substantiated by the nearly identical specific activities of some membrane-bound enzymes, Mg2+-ATPase, alkaline phosphatase and gamma-glutamyl transpeptidase. The specific activities of (Na+ + K+)-ATPase and of lysolecithin acyltransferase were several-fold enriched in fraction 2 compared to fraction 1, especially after rechromatography of fraction 1 on concanavalin A-Sepharose. Unseparated membrane vesicles contained two types of binding site for concanavalin A. In contrast, isolated subfractions showed a linear Scatchard plot; fraction 2 exhibited fewer binding sites for concanavalin A: the association constant was, however, 3.5-times higher than that measured in fraction 1. When plasma membranes isolated from concanavalin A-stimulated lymphocytes were separated by affinity chromatography, the yield of the two subfractions was similar to that of membranes from unstimulated lymphocytes. Upon stimulation with concanavalin A, Mg2+-ATPase, gamma-glutamyl transpeptidase and alkaline phosphatase were suppressed in their activities in both membrane subfractions. In contrast, the specific activities of (Na+ + K+)-ATPase and lysolecithin acyltransferase were enhanced preferentially in the adherent fraction (fraction 2). The data suggest the existence of domains in the plasma membrane of lymphocytes which are formed by a spatial and functional coupling of receptors with high affinity for concanavalin A, and certain membrane-bound enzymes, implicated in the initiation of lymphocyte activation.     

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.