The submicrosomal localization of acyl-coenzyme A–cholesterol acyltransferase and its substrate, and of cholesteryl esters in rat liver

To determine the submicrosomal distribution of acyl-CoA–cholesterol acyltransferase and of cholesteryl esters, the microsomal fraction and the digitonin-treated microsomal preparation of rat liver were subjected to analytical centrifugation on sucrose density gradients. With untreated microsomal fractions the distribution profile and the median density of acyl-CoA–cholesterol acyltransferase were very similar to those of RNA. This is in contrast with hydroxymethylglutaryl-CoA reductase and cholesterol 7α-hydroxylase, which are confined to endoplasmic reticulum membranes with low ribosomal coating. In digitonin-treated microsomal preparations activity of acyl-CoA–cholesterol acyltransferase was not detectable. The labelling of untreated microsomal fractions with trace amounts of [¹⁴C]cholesterol followed by subfractionation of the labelled microsomal fraction showed that the specific radioactivity of cholesteryl esters obtained in vitro by the various subfractions was similar with all subfractions but different from the specific radioactivity of the 7α-hydroxycholesterol obtained in vitro by the same subfraction. These results demonstrate the existence of two pools of cholesterol confined to membranes from the endoplasmic reticulum, one acting as substrate for cholesterol 7α-hydroxylase and the other acting as substrate for acyl-CoA–cholesterol acyltransferase. The major part of cholesteryl esters present in both untreated and digitonin-treated microsomal fractions was distributed at densities similar to those of membranes from the smooth endoplasmic reticulum and at densities lower than those of smooth membranes from Golgi apparatus. The ratio of the concentrations of non-esterified to esterified cholesterol in the subfractions from both untreated and digitonin-treated microsomal fractions was highest at the maximum distribution of plasma membranes.     

The nuclear associated endoplasmic reticulum and membrane biogenesis in MPC-11 cells

The incorporation of 3H-glucosamine, 3H-choline and 14C-fucose into subcellular fractions of MPC-11 cells was studied. After a 20 min period of labelling with both 3H-glucosamine and 3H-choline, greatest incorporation was observed in nuclear-associated endoplasmic reticulum (NER). 14C-fucose, however, was incorporated to a greater extent in endoplasmic reticulum (ER) membranes. Pulse-chase experiments with 3H-glucosamine showed a loss of radioactivity from NER and a simultaneous increase in the ER fraction. In comparison to NER, ER membranes were poorly labeled with 3H-glucosamine after a 20 min pulse. Following a 2 h incubation there was a 12 fold increase in radioactivity in ER membranes in comparison to a 1.2 fold increase in NER. There were no individual differences between subfractions of ER membranes with respect to 3H-glucosamine content after the pulse, or following the 2 h incubation. The results indicate that the NER is a major, early site of the synthesis of 3H-glucosamine labeled membrane glycoproteins, and that these proteins migrate into other ER membranes early after their synthesis.     

Biosynthesis of high density lipoprotein by chicken liver: conjugation of nascent lipids with apoprotein A1

To study the assembly of newly synthesized lipids with apoprotein A1, we administered [2-3H]glycerol to young chickens and determined the hepatic intracellular sites of lipid synthesis and association of nascent lipids with apoprotein A1. [2-3H]glycerol was rapidly incorporated into hepatic lipids, reaching maximal levels at 5 min, and this preceded the appearance of lipid radioactivity in the plasma. The liver was fractionated into rough and smooth endoplasmic reticulum and Golgi cell fractions. The isolated cell fractions were further subfractionated into membrane and soluble (content) fractions by treatment with 0.1 M Na2CO3, pH 11.3. At various times, the lipid radioactivity was measured in each of the intracellular organelles, in immunoprecipitable apoprotein A1, and in materials that floated at buoyant densities similar to those of plasma lipoproteins. Maximal incorporation occurred at 1 min in the rough endoplasmic reticulum, at 3-5 min in the smooth endoplasmic reticulum, and at 5 min in the Golgi cell fractions. The majority (66-93%) of radioactive glycerol was incorporated into triglycerides with smaller (4-27%) amounts into phospholipids. About 80% of the lipid radioactivity in the endoplasmic reticulum and 70% of that in the Golgi cell fractions was in the membranes. The radioactive lipids in the content subfraction were distributed in various density classes with most nascent lipids floating at a density less than or equal to 1.063 g/ml. Apoprotein A1 from the Golgi apparatus, obtained by immunoprecipitation, contained sixfold more nascent lipids than did that from the endoplasmic reticulum. These data indicate that [2-3H]glycerol is quickly incorporated into lipids of the endoplasmic reticulum and the Golgi cell fractions, that most of the nascent lipids are conjugated with apoproteins A1 in the Golgi apparatus, and that very little association of nascent lipid to apoprotein A1 occurs in the endoplasmic reticulum.     

The localization of cholinephosphotransferase in the outer membrane of guinea-pig lung mitochondria

The activity of cholinephosphotransferase was measured in the subcellular fractions of guinea-pig lung. The specific activity of the enzyme was highest in a fraction, intermediate in density between mitochondria and microsomes. Similar subcellular distribution patterns were observed for both cholinephosphotransferase and rotenone-insensitive NADH-cytochrome c reductase, an enzyme associated with the outer membrane of mitochondria and endoplasmic reticulum, suggesting that cholinephosphotransferase may be localized in both of these organelles. The distribution of cholinephosphotransferase activity in the subfractions of mitochondria and the intermediate fractions recovered by linear density gradient paralleled that of the mitochondrial outer membrane marker enzyme, monoamine oxidase. RNA content of a subfraction enriched in cholinephosphotransferase and monoamine oxidase was not typical to that of either rough or smooth endoplasmic reticulum. The results of this study suggest that in guinea-pig lung, cholinephosphotransferase is localized in both the outer membrane of mitochondria, and the endoplasmic reticulum.     

Gonadotropin and prostaglandins binding sites in rough endoplasmic reticulum and Golgi fractions of bovine corpora lutea.

Highly purified rough endoplasmic reticulum and three subfractions of golgi were prepared from 105,000g pellet of the homogenate by centrifugation in floatation and sedimentation discontinuous sucrose gradients. Highly purified plasma membranes were also prepared from 9,000g pellet of the same homogenates for assessment under the same experimental conditions. Although 5′-nucleotidase, a marker for plasma membranes, was markedly enriched in plasma membranes, very little or none of this enzyme activity was found in other fractions. Very little or no NADH cytochrome c reductase activity, a marker for rough endoplasmic reticulum, was found in fractions other than rough endoplasmic reticulum. Galactosyl transferase, a marker for golgi, was found and enriched in all the fractions; however, enrichment in golgi fractions was higher than in other fractions. Very little or no lysosomal marker activity, i.e., acid phosphatase, was found in rough endoplasmic reticulum or golgi fractions as compared to lysosomes. These marker enzyme data suggested that rough endoplasmic reticulum and golgi fractions were relatively pure with little or no cross contamination with other organelles. The [¹²⁵I]human choriogonadotropin ([¹²⁵I]hCG), [³H]prostaglandin (PG)E₁, and [³H]PGF_2a specifically bound to rough endoplasmic reticulum and golgi fractions in addition to plasma membranes. The enrichments of binding in the former two fractions, in some cases, were as high as plasma membranes itself. The specific binding of some of the ligands was found to be partially latent in rough endoplasmic reticulum and golgi fractions but not in plasma membranes. Marker enzyme data, ratio between bindings and marker enzyme activities (an index of organelle contamination), and partial latency of binding suggest that rough endoplasmic reticulum and golgi fractions intrinsically contain gonadotropin and PGs binding sites.     

Effect of digitonin on rat myometrium subcellular membrane fractions

Isopycnic centrifugation experiments using sucrose density gradients showed that in digitonin-treated microsomes the distribution of the plasma membrane (PM) marker 5'-nucleotidase was shifted to higher densities. The treatment also caused similar but less pronounced changes in the distribution of protein, the putative endoplasmic reticulum (ER) marker NADPH-dependent cytochrome c reductase, and the inner mitochondrial marker cytochrome c oxidase. Similar experiments using more purified membrane fractions showed that the digitonin treatment led to a comparable increase in the densities of the fractions N1 and N2 previously described as subfractions of plasma membrane and to considerably less increase in the density of the fraction N3B which is enriched in the endoplasmic reticulum and the inner mitochondrial markers. Digitonin inhibited the ATP-dependent Ca uptake by the N1 fraction in a concentration-dependent manner (I50 = 0.3 mg/mL). Digitonin (0.5 mg/mL) inhibited the ATP-dependent azide-insensitive Ca uptake by all the fractions. The results support the hypothesis that (a) N1 and N2 are subfractions of plasma membrane, and (b) ATP-dependent azide-insensitive Ca uptake in rat myometrium is a property of plasma membranes.     

Domains of rough endoplasmic reticulum (a review)

The rough endoplasmic reticulum isolated from several eukaryotic cell lines can be separated into subfractions. These subfractions possess different properties indicating that they represent separate domains of the endoplasmic reticulum system.     

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.     

Subcellular metabolism of exogenous GM1 ganglioside in normal human fibroblasts

The metabolization of exogenous GM1 in normal human fibroblasts at a subcellular level is investigated in the present paper. For this a GM1 ganglioside, radiolabelled on the sphingosine moiety, was given to the cells and all the formed metabolites analyzed, in a time-course study, in enriched fractions of lysosomes, plasma membrane and microsomes. After feeding the cells, the radioactivity incorporation was relevant in the enriched lysosomal and plasma membrane subfractions whereas it was modest in the enriched microsomal fraction. The kinetic curves obtained for each enriched fraction, following a 3-day chase period, suggested a translocation of exogenous GM1 from the plasma membrane to the lysosomal apparatus and, of GM1 itself together with its metabolites, to the Golgi or endoplasmic reticulum and finally again to the plasma membrane.     

Calcium-ion-transporting activity in two microsomal subfractions from rat pancreatic acini. Modulation by carbamylcholine

Two microsomal subfractions from isolated rat pancreatic acini were produced by centrifugation through a discontinuous sucrose density gradient and characterized by biochemical markers. The denser fraction ( SF2 ) was a highly purified preparation of rough endoplasmic reticulum; the less-dense fraction ( SF1 ) was heterogeneous and contained Golgi, endoplasmic reticulum and plasma membranes. 45Ca2+ accumulation in the presence of ATP and its rapid release after treatment with the bivalent-cation ionophore A23187 were demonstrated in both fractions. The pH optimum for active 45Ca2+ uptake was approx. 6.8 for the rough endoplasmic reticulum ( SF2 ) and approx. 7.5 for SF1 . Initial rate measurements were used to determine the affinity of the rough-endoplasmic-reticulum uptake system for free Ca2+. An apparent Km of 0.16 +/- 0.06 microM and Vmax. of 21.5 +/- 5.6 nmol of Ca2+/min per mg of protein were obtained. 45Ca2+ uptake by SF1 was less sensitive to Ca2+, half-maximal uptake occurring at 1-2 microM-free Ca2+. When fractions were prepared from isolated acini stimulated with 3 microM-carbamylcholine, 45Ca2+ uptake was increased in the rough endoplasmic reticulum. The increased uptake was due to a higher Vmax. with no significant change in Km. No effect was observed on 45Ca2+ uptake by SF1 . In conclusion, two distinct non-mitochondrial, ATP-dependent calcium-uptake systems have been demonstrated in rat pancreatic acini. One of these is located in the rough endoplasmic reticulum, but the precise location of the other has not been determined. We have shown that the Ca2+-transporting activity in the rough endoplasmic reticulum may have an important role in maintaining the cytosolic free Ca2+ concentration in resting acinar cells and is involved in Ca2+ movements which occur during stimulation of enzyme secretion.     

Heterogeneous distribution of glycosyltransferases in the endoplasmic reticulum of castor bean endosperm

Endoplasmic reticulum membranes stripped of attached ribosomes were isolated from homogenates of germinating castor bean (Ricinus communis L.) endosperm by sucrose density gradient centrifugation. The isolated endoplasmic reticulum fraction was further separated into two major membrane subfractions by centrifugation on a flotation gradient. Both subfractions appeared to be derived from the endoplasmic reticulum inasmuch as they share several enzymic markers including cholinephosphotransferase, NADH-cytochrome c reductase, and glycoprotein fucosyl-transferase and phase separation of membrane polypeptides using Triton X-114 revealed a striking similarity in both their hydrophilic and hydrophobic protein components. The endoplasmic reticulum membrane subfractions contain glycoproteins which were readily labeled by incubating intact endosperm tissue with radioactive sugars prior to fractionation.

Castor bean endosperm endoplasmic reticulum apparently exhibits a degree of enzymic heterogeneity, however, since the enzymes responsible for the synthesis of dolicholpyrophosphate N-acetylglucosamine and dolicholmonophosphate mannose together with their incorporation into the oligosaccharide-lipid precursor of protein N-glycosylation were largely recovered in a single endoplasmic reticulum subfraction.

     

Effect of diabetes on the rat hepatic glucose-6-phosphatase system in endoplasmic reticulum subfractions

Diabetes-induced alterations in the activities of the components of the glucose-6-phosphatase system (i.e., the enzyme, the glucose-6-P translocase (T(1)), and the phosphate translocase (T(2)) were examined in smooth and rough subfractions of hepatic endoplasmic reticulum from streptozotocin-injected rats. A significant effect of diabetes on the maximal velocity of glucose-6-P hydrolysis by the enzyme was present in both endoplasmic reticulum subfractions (3.1-fold increase in rough endoplasmic reticulum; 3.8-fold increase in smooth endoplasmic reticulum). Based on latency values, diabetes did not result in a proportional increase in capacity of T(1) or T(2). In contrast to the control condition, the relationship between transport capacity and hydrolytic capacity was not significantly different in the two subfractions from diabetic animals. Elucidation of the effects of diabetes on the components of the glucose-6-phosphatase system associated with smooth and rough endoplasmic reticulum membranes enhances our understanding of the hepatic contribution to diabetic hyperglycemia.     

Membrane flow in plants: Fractionation of growing pollen tubes of tobacco by preparative free-flow electrophoresis and kinetics of labeling of endoplasmic reticulum and golgi apparatus with [3H]leucine

Summary Tobacco (Nicotiana tabacum L.) pollen, germinated 4 hours in suspension culture, was labeled with radioactive leucine and fractionated into constituent membranes by the technique of preparative free-flow electrophoresis. Tubes were ruptured by sonication directly into the electrophoresis buffer. Unfortunately, the Golgi apparatus of the rapidly elongating pollen tubes did not survive the sonication step. However, it was possible to obtain useful fractions of endoplasmic reticulum and mitochondria. To obtain Golgi apparatus, glutaraldehyde was added to the homogenization buffer during sonication. Plasma membrane, which accounted for only about 3% of the total membrane of the homogenates as determined by staining with phosphotungstate at low pH, was obtained in insufficient quantity and fraction purity to permit analysis. Results show rapid incorporation of [³H]leucine into endoplasmic reticulum followed by rapid chase out. The half-time for loss of radioactivity from the pollen tube endoplasmic reticulum was about 10 minutes. Concomitant with the loss of radioactivity from endoplasmic reticulum, the Golgi apparatus fraction was labeled reaching a maximum 20 minutes post chase. The findings suggest flow of membranes from endoplasmic reticulum to the Golgi apparatus during pollen tube growth.     

Glycosylation of pea cotyledon membranes

Pea cotyledons were injected with d-[(14)C]mannose or d-[(14)C]-glucosamine and incubated for 1 to 1.5 hours. Cotyledons were homogenized and subcellular fractions were isolated by differential centrifugation followed by linear sucrose density gradient centrifugation.Radioactivity that was precipitated by trichloroacetic acid was associated most extensively with rough endoplasmic reticulum, Golgi membranes, a membrane with a density of 1.14 grams per cubic centimeter (possibly plasma membrane) and an unidentified subcellular component with a density of 1.22 grams per cubic centimeter. Lower levels of incorporation were observed in protein bodies and mitochondria.Isolated membrane fractions were lipid-extracted to determine which components of the membrane contained the label. Rough endoplasmic reticulum contained the most extensively labeled lipids which had similar properties to the lipid intermediates thought to be involved in glycoprotein assembly. The lipid free residues of the various membrane fractions contained radioactivity that was released by protease treatment. Acid hydrolysis of the residues indicated that most of the radioactivity was associated with mannose or glucosamine. It appears that various subcellular components of the pea cotyledon possess glycoproteins that contain mannose and glucosamine.     

Secretion kinetics of hydroxyproline-containing macromolecules in carrot root discs

Short-term pulse-chase studies using radioactive L-proline on carrot tissue support the classical endomembrane route for secretory proteins. Labelled hydroxyproline residues were first detectable in fractions containing the endoplasmic reticulum (ER) after a 5 min pulse. Upon chase-out this fraction looses and, initially, the Golgi apparatus (GA) fraction gains radioactivity. Unlike ER and GA fractions which show chase-out characteristics a plasma membrane (PM) containing-fraction reveals retention of some of the radioactivity.     

Intracellular transport and processing of lysosomal cathepsin B

Intracellular transport and processing of lysosomal cathepsin B was investigated in the subcellular fractions of rat liver by pulse-labeling experiments with [35S]methionine in vivo. A newly synthesized procathepsin B with a molecular weight of 39 kDa firstly appeared in the rough microsomal fraction at 10 min postinjection of label. This procathepsin B moved from the microsomal fractions to the Golgi subfractions at 30 min postinjection, and then a processed mature enzyme appeared in the lysosomal fraction at 60 min. These results suggest that the propeptide-processing of procathepsin B takes place in lysosomes in the course of intracellular transport from endoplasmic reticulum through Golgi complex to lysosomes.     

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)     

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.     

Albumin secreted by rat liver bypasses Golgi apparatus cisternae

Albumin was isolated immunologically from various subcellular fractions from livers of adult male rats receiving an intraperitoneal injection of [³H]leucine to investigate the kinetics and pathway of subcellular transfer of newly synthesized albumin during secretion. At appropriate time intervals, livers were excised and fractionated into endoplasmic reticulum and Golgi apparatus. Golgi apparatus were further subfractionated into cisternae and secretory vesicles. In endoplasmic reticulum fractions, labeled albumin appeared within 7.5 min of injection of isotope, followed by a rapid decline in specific activity. Albumin in Golgi apparatus was labeled and concentrated in secretory vesicles over 25 min. The radioactivity in albumin per mg total protein was highest in secretory vesicles and insignificant in the cisternal fraction. Labeled albumin was present in serum by 30 min and radioactivity in serum albumin reached a plateau within 60–90 min after injection of isotope. Results provide evidence for the migration of albumin from its site of synthesis on endoplasmic reticulum membrane-bound polyribosomes to its site of secretion into the circulation via the Golgi apparatus. The pathway of albumin transport to secretory vesicles is suggested to involve peripheral elemenst of the Golgi apparatus. Secretory vesicle formation and maturation required 20 to 30 min for completion, via a mechanism whereby the inner spaces of the central saccules may be bypassed.