Association of vesicular stomatitis virus proteins with HeLa cell membranes and released virus.

The association of vesicular stomatitis virus proteins with intracellular and plasma membranes was examined by pulse and pulse-chase labeling of virus-infected HeLa cells with [35S]methionine and separation of cell homogenates into three major membrane fractions in discontinuous sucrose gradients. The glycoprotein G was primarily associated with rough endoplasmic reticulum-like membranes after short radioactive pulses (2 to 4 min) but accumulated in the plasma membrane-enriched fraction and the smooth internal membrane fraction with longer pulse or chase periods. The nucleocapsid protein N and the matrix protein M accumulated in the rough endoplasmic reticulum and plasma membrane-like fractions but not in the smooth internal membrane fraction. Only a fraction (35 to 40%) of the viral protein synthesized during a short pulse in the mid-cycle of infection was apparently utilized in released virus. The newly synthesized virus proteins first appeared in released virus in the order: M, N and L, and G.     

Intracellular distribution of Sindbis virus membrane proteins in BHK-21 cells infected with wild-type virus and maturation-defective mutants.

The association of Sindbis virus proteins with cellular membranes during virus maturation was examined by utilizing a technique for fractionating the membranes of BHK-21 cells into three subcellular classes, which were enriched for rough endoplasmic reticulum, smooth endoplasmic reticulum, and plasma membrane. Pulse-chase experiments with wild-type (strain SVHR) virus-infected cells showed that virus envelope proteins were incorporated initially into membranes of the rough endoplasmic reticulum and subsequently migrated to the smooth and plasma membrane fractions. Large amounts of capsid protein were associated with the plasma membrane fraction even at the earliest times postpulse, and relatively little was found associated with the other membranes, suggesting a rapid and preferential association of nucleocapsids with the plasma membrane. We also examined the intracellular processing of the proteins of two temperature-sensitive Sindbis virus mutants in pulse-chase experiments at the nonpermissive temperature. Labeled virus proteins of mutant ts-20 (complementation group E) first appeared in the rough endoplasmic reticulum and were then transported to the smooth and plasma membrane fractions, as in wild-type (strain SVHR) virus-infected cells. In cells infected with ts-23 (complementation group D), the pulse-labeled virus proteins appeared initially in the rough membrane fraction and were transported to the smooth membrane fraction, but only limited amounts reached the plasma membrane. Thus, in ts-23-infected cells, the transport of the virus-encoded proteins from the smooth membranes seemed to be defective. In both ts-20- and ts-23-infected cells the envelope precursor polypeptide PE2 was not processed to E2, and no label was incorporated into free virus at the nonpermissive temperature.     

Cytoplasmic Compartmentalization of the Protein and Ribonucleic Acid Species of Vesicular Stomatitis Virus

The cytoplasmic sites of synthesis in L cells of the protein and ribonucleic acid species of vesicular stomatitis virus were studied by polyacrylamide gel electrophoresis after fractionation of membrane and other cytoplasmic components by the Caliguiri-Tamm technique. The viral spike protein (glycoprotein G) was found primarily associated with a smooth membrane fraction which is rich in plasma membrane; the G protein was also present in fractions containing rough endoplasmic reticulum. The nonglycosylated envelope protein S (also called M) was found in the smooth membrane fractions but was more abundant in endoplasmic reticulum-enriched fractions. Longer labeling resulted in detection of nucleoprotein N, as well as other minor nucleocapsid proteins L and NS₁, in the cellular membrane fractions. The N protein appeared to be made in membrane-free cytoplasm along with progeny ribonucleic acid and later became associated with membrane containing G and S viral proteins.     

Role of the nuclear envelope in synthesis, processing, and transport of membrane glycoproteins

The outer nuclear membrane is morphologically similar to rough endoplasmic reticulum. The presence of ribosomes bound to its cytoplasmic surface suggests that it could be a site of synthesis of membrane glycoproteins. We have examined the biogenesis of the vesicular stomatitis virus G protein in the nuclear envelope as a model for the biogenesis of membrane glycoproteins. G protein was present in nuclear membranes of infected Friend erythroleukemia cells immediately following synthesis and was transported out of nuclear membranes to cytoplasmic membranes with a time course similar to transport from rough endoplasmic reticulum (t 1/2 = 5-7 min). Temperature-sensitive mutations in viral membrane proteins which block transport of G protein from endoplasmic reticulum also blocked transport of G protein from the nuclear envelope. Friend erythroleukemia cells and NIH 3T3 cells differed in the fraction of newly synthesized G protein found in nuclear membranes, apparently reflecting the relative amount of nuclear membrane compared to endoplasmic reticulum available for glycoprotein synthesis. Nuclear membranes from erythroleukemia cells appeared to have the enzymatic activities necessary for cleavage of the signal sequence and core glycosylation of newly synthesized G protein. Signal peptidase activity was detected by the ability of detergent-solubilized membranes of isolated nuclei to correctly remove the signal sequence of human preplacental lactogen. RNA isolated from the nuclear envelope was highly enriched for G protein mRNA, suggesting that G protein was synthesized on the outer nuclear membrane rather than redistributing to nuclear membranes from endoplasmic reticulum before or during cell fractionation. These results suggest a mechanism for incorporation of membrane glycoproteins into the nuclear envelope and suggest that in some cell types the nuclear envelope is a major source of newly synthesized membrane glycoproteins.     

Effect of ribosome stripping procedures on antigenicity and conformation of endoplasmic reticulum membrane.

The effects of 3 different procedures for stripping ribosomes from membranes on theantigeniticity and conformation of isolated rough and smooth endoplasmic reticulum from rat liver were examined by microcomplement fixation and circular dichroism. Some of the blocked antigenic binding sites in rough endoplasmic reticulum became available after stripping of ribosomes. None of the 3 methods used is capable of stripping ribosomes completely from rough endoplasmic reticulum without the concomitant removal of protein from the membrane. Such loss of membrane protein by the stripping treatments is probably involved in the observed changes in rough endoplasmic reticulum, since a marked reduction in complement fixing capacity and in ellipticity of circular dichroism is observed also in smooth endoplasmic reticulum after similar treatments.     

Protein phosphorylation of the smooth and rough endoplasmic reticulum in normal and neoplastic liver of the rat.

Smooth and rough endoplasmic reticulum from rat liver and hepatomas exhibited endogenous protein kinase activity independent of adenosine 3':5'-monophosphate. The phosphorylation of smooth membranes by this process was consistently higher than that of rough membranes. When histone was added along with the smooth endoplasmic reticulum, cyclic AMP stimulated protein phosphorylation. Analysis of membrane-phosphorylated proteins by gel electrophoresis showed 5 major phosphorylated bands with estimated molecular weights of 155 000, 62 000, 50 000, 46 000 and 43 000, whereas major bands having estimated molecular weights of 62 000, 50 000 and 43 000 were found in membranes of the smooth endoplasmic reticulum of the Morris hepatoma 5123 C. Since previous studies in this and other laboratories have demonstrated the similarity of the protein components of membranes of the endoplasmic reticulum of normal liver and hepatoma, our findings indicate an inability of the protein kinase of hepatoma intracellular membranes to phosphorylate protein species that are found in membranes of both liver and the neoplasm.     

A comparative study of the molecular structures of the plasma membranes and the smooth and the rough endoplasmic-reticulum membranes from rat liver

Plasma-membrane as well as smooth-, rough- and degranulated-endoplasmic-reticulum-membrane fractions were isolated from the microsomal pellet of rat liver. The purity of these fractions, as determined by marker-enzyme activities, electron microscopy, cholesterol content and RNA content, was found to be adequate for a comparative structural study. Major differences in lipid and protein composition were found to exist between the plasma membrane and the endoplasmic reticulum, but not between the smooth and the rough fractions of the endoplasmic reticulum. Differences in the location of membrane protein thiol groups and the mobility of the membrane phospholipids were observed between the plasma membranes and the endoplasmic reticulum, and these could be explained by differences in protein and lipid composition. However, by employing fluorescence and spin-labelling techniques structural changes were also observed between the smooth and the rough endoplasmic-reticulum fractions. These results suggest that the structural heterogeneity existing between the two latter membrane fractions occurs near or on their membrane surfaces and is not due to the greater number of ribosomes bound to the rough endoplasmic-reticulum fraction.     

Protein fatty acyltransferase is located in the rough endoplasmic reticulum

The fatty acid acylation of polypeptides was studied in vivo and in vitro by incorporation of radiolabeled palmitic acid into Semliki Forest viral polypeptides. Utilizing a cell-free system for acylation protein fatty acyltransferase was characterized as an integral membrane protein. No acylation activity was detected in the cytosol. During subcellular fractionation of a variety of mammalian or avian cells the enzyme was localized to the rough endoplasmic reticulum. Therefore this posttranslational hydrophobic modification starts earlier in the biosynthesis of acylated polypeptides than previously believed.     

ONTOGENETIC CHANGES OF PROTEINS OF ENDOPLASMIC RETICULUM

The proteins of the smooth and rough endoplasmic reticulum from fetal, immature, and adult male rats were compared after incorporation of two radioactively labeled precursors, ¹⁴C-labeled amino acids and δ-aminolevulinic acid-³H by means of gel electrophoresis. The labeling patterns indicated that protein components present in two major electrophoretic bands underwent significant synthesis in fetal tissue while three actively incorporating protein bands were noted in adult tissue. Although the uptake of the amino acids-¹⁴C decreased for the smooth and rough elements of the endoplasmic reticulum as a whole during liver development, the qualitative patterns were not significantly different in adult and fetal livers. The over-all incorporation (disintegrations per minute per milligram protein) of the heme precursor into the smooth and rough elements also did not change with development. However, a change was noted in the distributional electrophoretic patterns with development. The estimation of molecular weight (by disc electrophoresis) and the incorporation of the heme precursor suggested the similarity of the two major protein bands to cytochrome P-450 and cytochrome b₅, components of the endoplasmic reticulum, thought to be involved in the mixed-function oxidase system. The evidence indicated that in fetal liver, at a time when the oxidase capability was low, the amino acid incorporation into these two protein groups was the same as in the adult. The incorporation of the heme moiety, however, was different, decreasing in the cytochrome b₅ region and increasing in the cytochrome P-450 region during development. These results correlate with the increase in oxidase activity associated with liver development.     

Passage of viral membrane proteins through the Golgi complex

BHK-21 cells, infected with Semliki Forest virus, were treated with cycloheximide to stop further synthesis but not intracellular transport of the viral membrane proteins. These proteins were then localized in thin, frozen sections using specific antibodies labelled indirectly with ferritin or gold. Quantitation of the labelling on micrographs showed the movement of spike proteins from the rough endoplasmic reticulum and through the Golgi stacks. The spike proteins spent about 15 minutes in each of these intracellular organelles and their final destination was the plasma membrane. Parallel biochemical studies showed that most of the simple oligosaccharides on the viral spike proteins were modified to the complex form at the same time as these membrane proteins were passing through the Golgi stacks. Cell fractionation studies revealed the same pattern; the proteins passed from the rough endoplasmic reticulum to the plasma membrane via a vesicle fraction isolated according to its content of galactosyl transferase. Independent evidence that this fraction was derived at least in part from the Golgi complex in BHK cells was obtained by showing that it reacted specifically with an antibody raised to rat liver Golgi membranes.     

Subcellular location of the major protein antigens of paramyxoviruses revealed by immunoperoxidase cytochemistry

The major structural proteins of Newcastle disease virus and Sendai virus were localized in infected BHK-21 and MDBK cells by ultrastructural immunoperoxidase cytochemistry using antibodies against the individual viral protein antigens. The intracellular glycoproteins were strictly membrane bound, being localized in the rough endoplasmic reticulum (RER), perinuclear spaces, smooth membrane vesicles, and presumed Golgi apparatus. The nucleocapsid proteins were detected exclusively in membrane free cytosol and accumulated there, forming inclusions. The membrane (M) protein was found both in cytosol and on RER. The viral proteins on RER exhibited a distinct site specificity; the glycoproteins were facing the lumen of RER whereas M protein was present at the outer cytoplasmic surface. All the viral proteins were detectable at the plasma membrane where virus assembly takes place. However, their modes of distribution differed remarkably. The glycoproteins were spread widely over the entire cell surface including the areas of virus budding and those of normal morphology, whereas M protein was localized in restricted areas of the membrane, frequently forming a patch of virus specific membrane. The presence of nucleocapsids was confined to the virus particles budding from the plasma membrane. These results complement and extend the earlier morphological and biochemical data on the assembly or morphogenesis of paramyxoviruses.     

Subcellular fractionation and subcellular localization of aminopeptidase N in the rabbit enterocytes

Summary A fast and easy procedure is proposed for preparing concomitantly from the same sample of intestinal mucosa of A⁺ rabbits, four fractions high enriched in the brush-border and basolateral plasma membrane domains, rough endoplasmic reticulum, and smooth endoplasmic reticulum plus Golgi apparatus membranes, respectively. This is the first time the technique of flow fluorometry has been applied to characterize the brush-border and basolateral membrane fractions using polyclonal or monoclonal antibodies against antigens common to or specific for these two plasma membrane domains. This technique definitely proves the presence of aminopeptidase in at least 60% of the basolateral membrane vesicles, where its level is about 4.5% of that in the brush-border membrane vesicles. The endoglycosidase H-sensitive intermediate of glycosylation of aminopeptidase N in the steady state is accumulated in both the rough and smooth endoplasmic reticulum membranes. Although the rough membrane is more extensive it contains only about 40% of this transient form.     

Subcellular fractionation of actively growing protoplasts of Saccharomyces cerevisiae

Cell homogenates obtained from partially regenerated Saccharomyces cerevisiae protoplasts were fractionated by a procedure using a combination of continuous and discontinuous sucrose gradients, under experimental conditions that minimize possible artifacts due to centrifugation and resuspension. At least five different membranous organelle fractions (plasma membrane, mitochondria, rough endoplasmic reticulum, smooth endoplasmic reticulum-like structures and small-sized particulated structures) were isolated. Subcellular fractions were characterized by assaying established marker enzymes. Radioactive labelled ([U-³H]uracil) ribosomes were also used as a further characterization criterion of the rough endoplasmic reticulum. Comparative SDS-polyacrylamide gel electrophoresis of the protein constituents of the isolated membrane-bound organelles suggest that the polypeptide pattern could also be used as an additional marker for some of these structures. Finally, subcellular distribution of chitin synthase was determined using this fractionation procedure, and two partially zymogenic enzyme pools (one inside the cell associated to particles which sediments at high speed, and the second one associated to the plasma membrane) were found.     

Properties of the scrapie agent-endomembrane complex from hamster brain.

Subcellular fractionation of scrapie-infected hamster brain indicated the association of the scrapie agent with a component of the endomembrane system. Characterization by equilibrium density gradient centrifugation, electron microscopy, and marker enzymes suggested a primary association with rough and smooth endoplasmic reticulum and a possible incorporation into the plasma membrane. DNA polymerase activity demonstrated a direct correlation with regions of scrapie activity from the gradient fractions. A scrapie-related product was detected after (3H)TMP incorporation and analysis on 2.2% polyacrylamide gels. Analysis of nucleic acid species extracted from subcellular fractions resulted in a greater quantity from healthy brain; however, no qualitative distinctions were detected.     

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.     

Subcellular structure of bovine thyroid gland. A study on bovine thyroid membranes by buoyant-density-gradient centrifugation in a B-XIV zonal rotor.

A combined mitochondrial and light mitochondrial fraction and a microsomal fraction were isolated from bovine thyroid gland and fractionated further in a B-XIV zonal rotor. A density gradient ranging from 20 to 50% (w/w) sucrose was used. The rotor was operated for 3 h at 45 000 rev./min. All manipulations were performed at 4 degrees C and at pH 7.4. 2. Membranous material was recovered in two zones: zone I, containing microsomal material derived from both smooth endoplasmic reticulum and plasma membranes and probably also from other smooth membranes; zone II, containing material from rough endoplasmic reticulum. 3. Increasing the pH of the medium up to 8.6, or the addition of Mg2+ to the medium resulted in the formation of a single zone at intermediate densities (aggregation of membranes?). An analogous effect was obtained after treatment with Pb (NO3) 2. 4. In the presence of heparin (50 i.u./ml) the bulk of the membranes was found in zone I. This was due to the release of ribosomes from the rough endoplasmic reticulum.     

Characterization of molecules involved in protein translocation using a specific antibody

The vectorial translocation of nascent proteins through the membrane of the rough endoplasmic reticulum has been shown to require a specific membrane-bound protein whose cytoplasmic domain can be proteolytically cleaved and isolated as an active peptide of mol wt 60,000 (Meyer and Dobberstein, 1980, J. Cell Biol. 87:503-508). Rabbit antibodies raised against this peptide were used to further characterize the membrane- bound molecule. Immunoprecipitation of solubilized, radiolabeled rough microsomal proteins yielded a single polypeptide of mol wt 72,000, representing the membrane-bound protein from which the 60,000-mol wt peptide was proteolytically derived. The antibody could also be used to remove exclusively the 60,000-mol wt peptide, and thus the translocation activity, from elastase digests tested in a reconstituted system. Moreover, immunoprecipitation of elastase extracts alkylated with [14C] N-ethylmaleimide selected a single species of mol wt 60,000. Immunoprecipitation of in vivo radiolabeled proteins from the appropriate cell type yielded the 72,000-mol wt membrane protein irrespective of the duration of labeling, or if followed by a chase. Subsequent treatment with protease generated the 60,000-mol wt fragment. In addition, the antibody could be used to visualize reticular structures in intact cells which correspond to endoplasmic reticulum at the ultrastructural level. It is thus clear that one membrane component required in the vectorial translocation of nascent secretory (and membrane) proteins is a peptide of mol wt 72,000.     

The role of components of the endoplasmic reticulum in the biosynthesis of cytochrome P-450.

1. Antibodies have been prepared to rat hepatic cytochrome P-450 and their specificity demonstrated. These antibodies have been used to investigate the biosynthesis of cytochrome P-450 in vitro and in situ in various components of the endoplasmic reticulum. 2. A preparation of heavy rough endoplasmic reticulum translocates proteins newly biosynthesized in vitro vectorially into the luminal space and these are released by low concentrations of deoxycholate. A significant proportion of the radioactivity found in this released fraction is incorporated into cytochrome P-450. 3. Following incorporation of [14C]leucine by perfused rat liver, radioactively labelled cytochrome P-450 can be found in the intrascisternal content of heavy rough, light rough and smooth endopalsmic reticulum and also in a solublized Golgi preparation. 4. We suggest that at least part of the newly biosynthesized cytochrome P-450 is translocated into the intracisternal space of the rough endoplasmic and then passes through the other components of the endoplasmic reticulum before insertion at its ultimate membrane locus.     

Depression of cytochrome P-450 and alterations of protein metabolism in mice treated with the interferon inducer polyriboinosinic acid X polyribocytidylic acid

Treatment of mice with the interferon inducer polyriboinosinic acid X polyribocytidylic acid [poly(IC)] results in the depression of several hepatic proteins. In this study we examined synthesis and degradation of the proteins of liver cell organelles in mice treated with poly(IC). Effects on synthesis were determined by using [14C]- and L-[3H]leucine incorporation into control and poly(IC)-treated mice, respectively. At selected times after poly(IC) treatment the 3H/14C ratio was established for preparations of nuclei, mitochondria, lysosomes, smooth endoplasmic reticulum, rough endoplasmic reticulum, and 105,000g supernatant (cytosol). Time-dependent alterations in de novo protein synthesis were greatest in lysosomal and rough endoplasmic reticular fractions; both were depressed 9 h after treatment. The effects of poly(IC) on protein degradation were determined with [14C]bicarbonate. Poly(IC) treatment decreased the time required for disappearance of 50% of 14C-labeled protein (t1/2) of smooth and rough endoplasmic reticula. Examination of endoplasmic reticulum marker enzymes showed depression of cytochromes P-450 and b5 from 9 h onward after poly(IC) administration. Tyrosine aminotransferase activity was elevated 6 h after treatment with poly(IC), and then depressed after 9 h. The other organelle marker enzymes were not affected significantly. We conclude that poly(IC) decreases the content of proteins of the hepatic endoplasmic reticulum, including certain cytochrome P-450 isozymes, by decreasing rates of protein synthesis and increasing rates of protein degradation.