Biosynthesis of rat-liver pI-5.0 esterases in cell-free systems and in cultured hepatocytes

Rat liver esterases focusing at pH 5.0 (referred to below as pI-5.0 esterases) are structurally related glycoproteins which differ slightly in their mobility in sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE). They reside in the lumen of the endoplasmic reticulum. We have studied their biosynthesis in cell-free systems programmed by total liver RNA, using sheep and rabbit antibodies to isolate the translation products related to these enzymes. Our results show that they are assembled as a precursor polypeptide chain (62 kDa) larger than the mature proteins. The pI-5.0 esterase mRNA could be extracted from bound but not free polysomes. Reticulocyte lysates supplemented with dog pancreas microsomes produced four esterase-related components in segregated form (61, 60, 58 and 56 kDa). The largest three correspond in electrophoretic mobility to the mature enzymes. They are glycoproteins that bind to concanavalin A, and can be reduced to the size of the shortest component by endo-beta-N-acetylglucosaminidase H (endo-H). Immunoprecipitation after biosynthetic labeling of the proteins in cultured hepatocytes also gave three glycosylated components that had the same mobility in SDS-PAGE as the mature enzymes. When tunicamycin was present in the culture medium, a single immunoprecipitable form was observed. Its apparent Mr was similar to that of the unglycosylated pI 5.0 esterase form synthesized in vitro in the presence of dog pancreas microsomes. Thus the biosynthesis of these esterases has characteristics in common with that of numerous secretory proteins, except for the rather large difference in size (approximately equal to 6 kDa) resulting from the proteolytic processing of their in-vitro-synthesized precursor.     

Biosynthesis of rat liver pI-6.1 esterase, a carboxylesterase of the cisternal space of the endoplasmic reticulum.

Biosynthesis of the rat liver microsomal esterase with pI 6.1 was investigated in cell-free systems and in cultured hepatocytes, by using a rabbit antiserum. Protein synthesis directed by total rat liver RNA in wheatgerm extract or reticulocyte lysate generated a single immunoprecipitable product, also found with the RNA extracted from bound, but not from free, polysomes. When dog pancreas microsomal fractions were included, reticulocyte lysates gave two processed products, a prominent one slightly larger, and another slightly smaller, than the precursor, both resistant to exogenous proteinases and, hence, segregated within vesicles. The processing was co-translational; it consisted of the removal of a peptide fragment and, for the large component, the addition of a single oligosaccharide chain. Indeed, this component bound to concanavalin A-Sepharose and gave the small one (approximately 2000 Mr loss) by cleavage with endo-beta-N-acetylglucosaminidase H (endo-H). A single labelled peptide was precipitated from hepatocytes incubated with [35S]methionine. Its apparent Mr was decreased by approximately 2000 after treatment with endo-H; it was then identical with that of an unglycosylated form produced in hepatocytes poisoned with tunicamycin. Even in that case, immunoreactive peptides were not detected in the culture medium. Whether synthesized in reticulocyte lysate or in hepatocytes, the glycosylated forms migrated in SDS/polyacrylamide-gel electrophoresis as the purified enzyme labelled with [3H]di-isopropyl fluorophosphate. Thus, although pI-6.1 esterase is not secreted, its biosynthesis is, as yet, indistinguishable from that of secretory proteins. Its oligosaccharide moiety is apparently not the structural element that retains it in the endoplasmic reticulum.     

Intracellular site of synthesis of microsomal heme oxygenase in pig spleen

In the pig spleen the specific activity of heme oxygenase was two to three times higher in smooth microsomes than in rough microsomes, whereas the total heme oxygenase activities recovered in the two microsomal fractions were similar. Free and bound polysomes were isolated from pig spleen and nascent peptides on these polysomes were analyzed by employing [3H]puromycin and a heme oxygenase-specific rabbit antibody (IgG). It was shown that free polysomes are the major site of heme oxygenase synthesis. In addition, cell-free synthesis of heme oxygenase was performed in a reticulocyte lysate system with free and bound polysomes isolated from pig spleen, and the results obtained again indicated that heme oxygenase is synthesized predominantly on free polysomes. The heme oxygenase newly synthesized on free polysomes may be incorporated first into the rough portion of endoplasmic reticulum either before or after its release from polysomes, although the specific activity of this enzyme at the steady state is considerably higher in the smooth region.     

Characterization of the integral membrane polypeptides of rat liver peroxisomes isolated from untreated and clofibrate-treated rats.

We have characterized the integral membrane polypeptides of liver peroxisomes from untreated rats and rats treated with clofibrate, a peroxisome proliferator. Membranes, prepared by treatment of purified peroxisomes with sodium carbonate, were used to raise an antiserum in rabbits. Immunoblot analysis demonstrated the reaction of this antiserum with six peroxisomal integral membrane polypeptides (molecular masses, 140, 69, 50, 36, 22, and 15 kDa). Treatment of rats with the hypolipidemic drug clofibrate caused a 4- to 10-fold induction in the 69-kDa integral membrane polypeptide, while the other integral membrane polypeptides remained unchanged or varied to a lesser extent. The anti-peroxisomal membrane serum reacted with two integral membrane polypeptides of the endoplasmic reticulum which co-migrated with the 50- and 36-kDa integral membrane polypeptides of the peroxisome. Biochemical and immunoblot analyses indicated that these integral membrane polypeptides were co-localized to peroxisomes and endoplasmic reticulum. Immunoprecipitation of in vitro translation products of RNA isolated from free and membrane-bound polysomes indicated that the 22-, 36-, and 69-kDa integral membrane polypeptides were synthesized on free polysomes, while the 50-kDa integral membrane polypeptide was predominantly synthesized on membrane-bound polysomes. The predominant synthesis of the 50-kDa integral membrane polypeptide on membrane-bound polysomes raises interesting possibilities concerning its biosynthesis.     

Erythrocyte membrane protein band 3: its biosynthesis and incorporation into membranes

Band 3, a transmembrane protein that provides the anion channel of the erythrocyte plasma membrane, crosses the membrane more than once and has a large amino terminal segment exposes on the cytoplasmic side of the membrane. The biosynthesis of band 3 and the process of its incorporation into membranes were studied in vivo in erythroid spleen cells of anemic mice and in vitro in protein synthesizing cell-free systems programmed with polysomes and messenger RNA (mRNA). In intact cells newly synthesized band 3 is rapidly incorporated into intracellular membranes where it is glycosylated and it is subsequently transferred to the plasma membrane where it becomes sensitive to digestion by exogenous chymotrypsin. The appearance of band 3 in the cell surface is not contingent upon its glycosylation because it proceeds efficiently in cells treated with tunicamycin. The site of synthesis of band 3 in bound polysomes was established directly by in vitro translation experiments with purified polysomes or with mRNA extracted from them. The band-3 polypeptide synthesized in an mRNA- dependent system had the same electrophoretic mobility as that synthesized in cells treated with tunicamycin. When microsomal membranes were present during translation, the in vitro synthesized band-3 polypeptide was cotranslationally glycosylated and inserted into the membranes. This was inferred from the facts that when synthesis was carried out in the presence of membranes the product had a lower electrophoretic mobility and showed partial resistance to protease digestion. Our observations indicate that the primary translation product of band-3 mRNA is not proteolytically processed either co- or posttranslationally. It is, therefore, proposed that the incorporation of band 3 into the endoplasmic reticulum (ER) membrane is initiated by a permanent insertion signal. To account for the cytoplasmic exposure of the amino terminus of the polypeptide we suggest that this signal is located within the interior of the polypeptide. a mechanism that explains the final transmembrane disposition of band 3 in the plasma membrane as resulting from the mode of its incorporation into the ER is presented.     

Mechanism of biosynthesis of soluble and membrane-bound forms of dopamine beta-hydroxylase in PC12 pheochromocytoma cells

Dopamine beta-hydroxylase was present as 2 subunit forms (apparent Mr = 77,000 and 73,000) in the PC12 pheochromocytoma cell line as detected by immunoprecipitation from [35S]methionine-labeled cultures, and analyzed by sodium dodecyl sulfate gel electrophoresis and fluorography. The Mr = 77,000 form was present in a crude membrane fraction, while the Mr = 73,000 form was soluble. Both forms appeared to be present in approximately equal amounts, and both were glycosylated. Treatment of PC12 cells with tunicamycin, a potent inhibitor of core glycosylation in the endoplasmic reticulum, completely inhibited the appearance of the Mr = 77,000 and Mr = 73,000 forms, and 2 new immunoreactive polypeptides were obtained (apparent Mr = 67,000 and 63,000). Pulse-chase experiments suggested that the Mr = 77,000 form is initially synthesized (by 5 min) and a portion is converted in 15-90 min to the Mr = 73,000 form. Thereafter, the ratio between forms remains relatively constant, at least for several hours. Translation of mRNA from bovine and rat adrenals, and immunoprecipitation, indicated that dopamine beta-hydroxylase is initially synthesized as a single polypeptide (apparent Mr = 67,000). The subcellular site of biosynthesis of dopamine beta-hydroxylase was determined by isolation of mRNA from free and membrane-bound polysomes from bovine adrenal medulla. Translation in a cell free system and immunoprecipitation localized the synthesis of dopamine beta-hydroxylase on membrane-bound polysomes. These experiments suggest that both soluble and membrane-bound forms of dopamine beta-hydroxylase are synthesized and core glycosylated in the endoplasmic reticulum, and that there probably is a precursor-product relationship between the Mr = 77,000 and the Mr = 73,000 subunit forms of dopamine beta-hydroxylase.     

Precursors of ricin and Ricinus communis agglutinin. Glycosylation and processing during synthesis and intracellular transport

During synthesis in vivo the castor bean lectin precursors initially appear in the endoplasmic reticulum as a group of core glycosylated polypeptides of relative molecular mass 64 000-68 000. Pretreatment of intact castor bean endosperm tissue with tunicamycin partially inhibits the cotranslational core glycosylation step and results in the accumulation of a single sized unglycosylated precursor polypeptide of relative molecular mass 59 000. The glycosylated precursors in the endoplasmic reticulum were enzymically converted to the 59 000-Mr form by incubation with endoglucosaminidase H. Intracellular transport of the glycosylated lectin precursors from the endoplasmic reticulum to a denser vesicle fraction was accompanied by modifications to the oligosaccharide moieties which conferred resistance to the action of endoglucosaminidase H. The post-translational addition of fucose to the carbohydrate chain was identified as one of the oligosaccharide modification steps. Fucose addition was catalysed by a glycosyltransferase associated with a smooth-surfaced membrane fraction which was distinct from the endoplasmic reticulum and which was tentatively identified as the Golgi apparatus. Glycosylation was not essential for intracellular transport of the lectin precursors: unglycosylated precursor synthesized in the presence of tunicamycin gave rise to unglycosylated lectin subunits in the protein bodies.     

Role of beta2-microglobulin in the intracellular processing of HLA antigens

The biosynthesis of HLA-A, -B, and -C antigens was examined in the two lymphoblastoid cell lines DAUDI and RAJI. In RAJI cells the HLA-A, -B, and -C antigen heavy chains become core-glycosylated in the endoplasmic reticulum as evidenced by their sensitivity to endo-H digestion and tunicamycin treatment. Beta2-Microglobulin is present in excess in the endoplasmic reticulum of the RAJI cells and associates with the heavy chain at the time of synthesis of the heavy chain. Pulse-chase experiments demonstrated that the RAJI HLA-A, -B, and -C antigen heavy chains become terminally glycosylated since their changed characteristics included resistance to endo-H digestion, sensitivity to neuraminidase treatment, and incorporation fucose. DAUDI HLA-A, -B, and -C antigen heavy chains are synthesized normally and become core-glycosylated but not terminally glycosylated. Other glycosylated cell surface proteins, like the HLA-DR antigens, display normal glycosylation in DAUDI cells. Therefore it is unlikely that the absence of terminally glycosylated HLA-A, -B, and -C antigen heavy chains is the result of a general defect in the biosynthetic machinery of DAUDI cells. However, DAUDI cells lack the ability to synthesize beta2-microglobulin, the common subunit of all HLA-A, -B, and -C antigens. Therefore, it seems reasonable to conclude that beta2-microglobulin is of importance for intracellular transport of newly synthesized HLA-A, -B, and -C antigens.     

Differences in the incorporation of [3H]-glucosamine into nascent polypeptide chains on free polysomes and two fractions of membrane-bound polysomes in mouse myeloma cells

The incorporation of [³H]-glucosamine into polypeptides of three fractions of polysomes in MPC-11 cells was studied. After short term incubation greatest incorporation was observed in a fraction of membrane-bound polysomes, which after nitrogen cavitation of cells, remained bound to the endoplasmic reticulum (ER) associated with the nucleus (fraction 2). Polypeptide chains on membrane-bound polysomes in the microsomal fraction (fraction 1) and free polysomes contained much less radioactivity. Since nascent polypeptide chains contained within membrane-bound polysomes of fraction 2 are glycosylated at an earlier stage than those in fraction 1 it is likely that this represents a difference in type of proteins synthesized in the respective fractions of ER.     

Biosynthesis of microsomal nicotinamide–adenine dinucleotide phosphate–cytochrome c reductase by membrane-bound and free polysomes from rat liver

1. NADPH-ferricytochrome c oxidoreductase (EC 1.6.2.3) was purified from the endoplasmic reticulum of rat liver cells. The methods, which involved digestion of membrane with Steapsin, a crude pancreatic extract containing diastase and trypsin, gel filtration and preparative electrophoresis on polyacrylamide, provided an enzyme with a high specific activity in good yield. 2. The incorporation of (14)C-labelled amino acids into the purified reductase by the incubation of various subcellular fractions was studied. The microsome fraction, bound polysomes, free polysomes and detergent-treated polysomes effected the synthesis of the enzyme. 3. The reductase that had been synthesized by the polysomes was tightly bound to preparations of smooth-surfaced endoplasmic reticulum that were added to the incubation medium. 4. Reductase activity could be detected on both free and detergent-treated polysomes. Evidence is presented to show that this activity was due, at least in part, to the presence on the ribosomes of nascent enzyme. The association of enzyme with detergent-treated polysomes did not appear to be due to contamination of the ribosomes with either membrane or cell sap but it is possible for such ribosomes to adsorb some enzyme. 5. The amount of reductase activity associated with the detergent-treated polysomes was increased when the rats from which the polysomes were derived had been previously injected with phenobarbitone. 6. The results are discussed with respect to their relevance for the question of the existence of two functionally different groups of polysomes in the liver and for current ideas on the biogenesis of membranes.     

Active site-directed photoaffinity labeling and partial characterization of oligosaccharyltransferase

Oligosaccharyltransferase, the enzyme that catalyzes the transfer of the oligosaccharide chain of dolichol-P-P-GlcNAc2Man9Glc3 to asparagine residues in -Asn-X-Thr/Ser- sites within polypeptides, has been radiolabeled using a photoactivatable azido tripeptide acceptor, N alpha-[3H]Ac-Asn-Lys(N epsilon-p-azidobenzoyl)-Thr-NH2. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular mass of the oligosaccharyltransferase polypeptide from hen oviduct microsomes is 60 kDa. Radiolabeling of the 60-kDa polypeptide was completely dependent upon photolysis of hen oviduct endoplasmic reticulum preparations in the presence of the azido peptide and Mn2+, which is required for enzymatic activity. Labeling of the enzyme was not inhibited in the presence of a 10-fold excess of the nonacceptor peptides, unacetylated Asn-Lys(N epsilon-p-azidobenzoyl)-Thr-NH2 or unacetylated Asn-Leu-Thr-NH2, whereas it was completely abolished by the presence of a 10-fold excess of the competing acceptor peptide, N alpha-Bz-Asn-Leu-Thr-NH2. Thermal inactivation of oligosaccharyltransferase was achieved by heating endoplasmic reticulum preparations to 60 degrees C. This loss of enzyme activity at 60 degrees C paralleled a comparable decrease in radiolabeling of the 60-kDa polypeptide, whereas temperatures of 50 degrees C and lower had no effect on either process. Oligosaccharyltransferase itself may be an N-linked glycoprotein, because the 60-kDa radiolabeled polypeptide binds to concanavalin A-agarose and is susceptible to digestion by beta-endohexosaminidase H.     

The effect of a single intraperitoneal dose of the hepatocarcinogen 4-dimethylaminoazobenzene on the rough-surfaced endoplasmic reticulum of the liver of the rat

1. Changes in the structure and function of the rough-surfaced endoplasmic reticulum of the rat liver as deduced by electron microscopy, polysome analysis and the amino acid-incorporating activity of microsome fractions have been followed at various time-intervals after a single intraperitoneal dose of the hepatocarcinogen 4-dimethylaminoazobenzene. 2. The earliest effect observed was detachment of polysomes and disorganization and vesiculation of the cisternae of the rough-surfaced endoplasmic reticulum. This occurred after 6hr. 3. Subsequent to this was a phase of polysome disaggregation accompanied by impaired amino acid-incorporating activity by microsomes together with a much enhanced stimulating effect of polyuridylic acid on amino acid uptake. This reached a maximum at 24hr. 4. By 40hr. polysomes had re-formed and the amino acid-incorporating activity, together with the polyuridylic acid effect, were similar to those in controls. 5. It was not until 112hr. that something like the normal structure of the rough-surfaced endoplasmic reticulum was re-established. 6. It is not yet possible to relate these changes specifically with the process of azo-dye carcinogenesis.     

The COOH terminus of several liver carboxylesterases targets these enzymes to the lumen of the endoplasmic reticulum

To investigate the potential role of the COOH-terminal peptides in retaining a family of soluble carboxylesterases in the lumen of the endoplasmic reticulum, the pI 6.1 esterase cDNA has been cloned into the pKCR3 vector for transient expression in COS cells. The plasmid-encoded product appeared to be identical to the authentic enzyme: it was active on alpha-naphthyl acetate and behaved as a homotrimer of noncovalently bound 60-kDa subunits which contain a single, endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharide chain. This enzyme was retained in the transgenic COS cells. In contrast, a mutated form ending in HVER-COOH was secreted, indicating that the natural terminus HVEL-COOH contains topogenic information, with the ultimate Leu residue as an essential part. Variants of pI 6.1 esterase ending in HIEL-COOH, or HTEL-COOH were retained in cells to the same extent as the wild-type protein. Therefore, the sequences HIEL and HTEL present at the COOH termini of several liver esterases (rabbit forms 1 and 2, human esterase, mouse egasyn, and rat pI 6.4 esterase) most likely have a function in their localization in the endoplasmic reticulum. Finally, an HDEL-COOH variant of pI 6.1 esterase was also normally retained, demonstrating that this signal can be correctly decoded by the sorting machinery of mammalian cells. Cell retention signals of the type HXEL-COOH appear to be common in higher eukaryotes and tolerate considerable variation at the antepenultimate X residue.     

Glycosyltransferase activities in Golgi complex and endoplasmic reticulum fractions isolated from African trypanosomes

Highly enriched Golgi complex and endoplasmic reticulum fractions were isolated from total microsomes obtained from Trypanosoma brucei, Trypanosoma congolense, and Trypanosoma vivax, and tested for glycosyltransferase activity. Purity of the fractions was assessed by electron microscopy as well as by biochemical analysis. The relative distribution of all the glycosyltransferases was remarkably similar for the three species of African trypanosomes studied. The Golgi complex fraction contained most of the galactosyltransferase activity followed by the smooth and rough endoplasmic reticulum fractions. The dolichol- dependent mannosyltransferase activities were highest for the rough endoplasmic reticulum, lower for the smooth endoplasmic reticulum, and lowest for the Golgi complex. Although the dolichol-independent form of N-acetylglucosaminyltransferase was essentially similar in all the fractions, the dolichol-dependent form of this enzyme was much higher in the endoplasmic reticulum fractions than in the Golgi complex fraction. Inhibition of this latter activity in the smooth endoplasmic reticulum fraction by tunicamycin A1 suggests that core glycosylation of the variable surface glycoprotein may occur in this organelle and not in the rough endoplasmic reticulum as previously assumed.     

Biosynthesis and localization of rat liver microsomal carboxyesterase E1

One of the microsomal carboxyesterases, carboxyesterase E1, was purified from rat liver to homogeneity. Carboxyesterase E1 is a glycoprotein of high mannose type, and is composed of three identical subunits of 59 kDa each. It is very similar to "esterase pI 6.0" described by Menthein et al. (Arch. Biochem. Biophys. 200, 547-559 (1980)) in molecular weight, amino acid composition, and enzymic activities. Carboxyesterase E1 was found to be evenly distributed between rough and smooth microsomes. The content of the enzyme in microsomes was about 1.5% of total microsomal protein. It was exclusively located on the luminal side of microsomes, and was not detected immunologically in Golgi fractions or serum. In vitro translation of rat liver RNA by reticulocyte lysate showed that carboxyesterase E1 was synthesized preferentially on the bound ribosomes, as a precursor peptide larger than the peptide of the mature enzyme. Carboxyesterase E1 was solubilized from microsomes by treatment with low concentrations of detergents. However, it was not released from microsomes by treatment with a synthetic peptide which made the microsomal membrane permeable to soluble protein molecules. Carboxyesterase E1 is not a soluble luminal protein, and seems to be bound to the luminal surface of the membrane.     

Alteration of membrane barrier in stripped rough microsomes from rat liver on incubation with GTP: its relevance to the stimulation by this nucleotide of the dolichol pathway for protein glycosylation

The membrane barrier of stripped rough microsomes from rat liver is markedly altered on incubation with GTP at 37 degrees C: after 30 min the structure-linked latency of mannose-6-phosphatase was considerably reduced, and esterase and nucleoside diphosphatase were partly released into the suspension medium. This phenomenon was already maximal with 30 microM GTP and was specific for this nucleotide. Similar conditions enhance the dolichol-mediated glycosylation of protein in microsomes incubated with uridine diphosphate N-acetylglucosamine and guanosine diphosphate mannose (Godelaine, D., H. Beaufay, M. Wibo, and A. Amar- Costesec, 1979, Eur. J. Biochem., 96:17-26; Godelaine, D., H. Beaufay, and M. Wibo, 1979, Eur. J. Biochem., 96:27-34). The GTP-induced permeability and glycosylation activities evolved in parallel in rough microsomes subjected to various treatments to detach the ribosomes and were maximal after removal of congruent to 60% of the RNA. In addition, GTP had no effect of this type in smooth microsome subfractions. Triton X-100, in spite of complex inhibitory effects on glycosylation reactions, mimicked the action of GTP by increasing the amount of microsomal dolichylphosphate that reacts with uridine diphosphate N- acetylglucosamine and by enhancing synthesis of dolichylpyrophosphoryl- chitobiose at concentrations greater than 2 mg/ml. Thus, GTP may activate dolichol-mediated glycosylation reactions in stripped microsomes by lowering the permeability barrier that prevents access of sugar nucleotides to the inner aspect of the membrane. The genuine role of GTP in the functioning of the endoplasmic reticulum membrane in situ remains unknown. Because GTP seems to act only on rough microsomes, we hypothesize that this role is somehow related to biosynthesis of protein by the rough endoplasmic reticulum.     

Primary structure and possible origin of the non-glycosylated basic proline-rich protein of human submandibular/sublingual saliva.

As a first step in determining the molecular mechanism of membrane fusion stimulated by GTP in rough endoplasmic reticulum (RER), we have looked for GTP-binding proteins. Rough microsomes from rat liver were treated for the release of ribosomes, and the membrane proteins were separated by SDS/polyacrylamide-gel electrophoresis. The polypeptides were then blotted on to nitrocellulose sheets and incubated with [alpha-32P]GTP [Bhullar & Haslam (1987) Biochem. J. 245, 617-620]. A doublet of polypeptides (23 and 24 kDa) was detected in the presence of 2 microM-MgCl2. Binding of [alpha-32P]GTP was blocked by 1-5 mM-EDTA, 10-10,000 nM-GTP or 10 microM-GDP. Either guanosine 5'-[gamma-thio]triphosphate or guanosine 5'-[beta gamma-imido]triphosphate at 100 nM completely inhibited binding, but ATP, CTP or UTP at 10 mciroM did not. Pretreatment of microsomes by mild trypsin treatment (0.5-10 micrograms of trypsin/ml, concentrations known not to affect microsomal permeability) led to inhibition of [alpha-32P]GTP binding, suggesting a cytosolic membrane orientation for the GTP-binding proteins. Two-dimensional gel-electrophoretic analysis revealed the 23 and 24 kDa [alpha-32P]GTP-binding proteins to have similar acid isoelectric points. [alpha-32P]GTP binding occurred to similar proteins of rough microsomes from rat liver, rat prostate and dog pancreas, as well as to a 23 kDa protein of rough microsomes from frog liver, but occurred to distinctly different proteins in a rat liver plasma-membrane-enriched fraction. Thus [alpha-32P]GTP binding has been demonstrated to two low-molecular-mass (approx. 21 kDa) proteins in the rough endoplasmic reticulum of several varied cell types.     

Segregation of specific classes of messenger RNA into free and membrane-bound polysomes

Analysis of mRNA populations from rat liver rough microsomes and free polysomes by homologous and heterologous cDNA . mRNA hybridization shows that the two mRNA populations are distinct, demonstrating that specific mRNA classes are efficiently segregated for translation in association with endoplasmic reticulum membranes. We estimate that approximately 90% of the mRNA in membrane-bound polysomes contains a diverse set of messengers with a minimum of 500--2000 different species although approximately 5--8 messengers may constitute 25--30% of the mRNA mass. The complexity of the mRNA population of free polysomes appears to be comparable to that estimated for total liver poly(A) + mRNA by other investigators, and is likely to be substantially greater than that of the bulk of bound mRNA. In addition, mRNA in free polysomes lacks the high abundance class characteristic of mRNA-bound polysomes. The substantial complexity of the bound mRNA population suggests that the segregation of polysomes in rough microsomes is not limited to a small class specialized in manufacturing secretory proteins, but extends to polysomes engaged in the synthesis of proteins for intracellular distribution. The segregation of specific messengers into the free and membrane-bound classes was abolished when polysome disassembly was induced by administration of ethionine. Thus, messenger RNA molecules themselves lacked the capacity for segregation, although they contain information for segregation which is expressed during translation. These findings are consistent with the presence of signal sequences in nascent polypeptides which determine the attachment of ribosomes to endoplasmic reticulum membranes.     

Identification of a G protein in rough endoplasmic reticulum of canine pancreas

Employing [32P]ADP-ribosylation by pertussis toxin we have identified a G protein that is located in the rough endoplasmic reticulum of canine pancreas and therefore termed it GRER. Identification of GRER is based on the following data. A 41-kDa polypeptide was the only polypeptide that was [32P]ADP-ribosylated by pertussis toxin in pancreas rough microsomes. Guanosine 5'-(gamma-thio)triphosphate (GTP gamma S) and 1 mM ATP, 6 mM MgCl2, 10 mM NaF (AMF) inhibited ADP-ribosylation of this polypeptide. The [32P]ADP-ribosylated 41-kDa polypeptide was immunoprecipitated by antisera which specifically recognized the C-terminal residues of the alpha subunits of Gi and transducin, indicating that the 41-kDa polypeptide is immunologically related to the alpha subunits of heterotrimeric G proteins. Treatment with GTP gamma S resulted in a reduction in the sedimentation rate of the [32P]ADP-ribosylated, detergent-solubilized GRER. It also induced the release of the [32P]ADP-ribosylated 41-kDa polypeptide from rough microsomes in the absence of detergent, unlike ADP-ribosylated alpha subunits of plasma membrane-associated G proteins. These data are consistent with an oligomeric nature of GRER. The codistribution of GRER with an endoplasmic reticulum marker protein during subcellular fractionation and the lack of plasma membrane contamination of the rough microsomal fraction, combined with the isodensity of GRER with rough microsomes as well as the isodensity of GRER with "stripped" microsomes after extraction of rough microsomes with EDTA and 0.5 M KCl, localized GRER to the rough endoplasmic reticulum. Preliminary experiments suggest that GRER appears not to be involved in translocation of proteins across the rough endoplasmic reticulum membrane.     

Intracellular maturation and secretion of acid phosphatase of Saccharomyces cerevisiae

To elucidate intracellular maturation and secretion of acid phosphatase of Saccharomyces cerevisiae we prepared a monoclonal antibody that recognizes specifically the protein moiety of this cell surface glycoprotein. With this antibody membranes and soluble fractions of wild-type cells, grown in low-phosphate medium in the presence and absence of tunicamycin, were examined by the immunoblot technique. Similarly, secretory mutants, blocked at distinct steps in the secretory pathway at the restrictive temperature as well as a strain harboring several copies of the structural gene PHO5 for repressible acid phosphatase, were analyzed. The data suggest the following sequence of events in acid phosphatase maturation and secretion: three unglycosylated precursors with molecular masses of 60 kDa, 58 kDa and 56 kDa are synthesized into membranes of the endoplasmic reticulum, where these are core glycosylated in a membrane-bound form. They appear on sodium dodecyl sulfate gels as bands with molecular masses of 76 kDa and 80 kDa. Owing to a rate-limiting maturation step, occurring after core glycosylation, they can accumulate in a membrane-bound form. At the Golgi apparatus outer carbohydrate chains are attached to the core and the enzyme appears in a soluble form, indicating a release of acid phosphatase from the membrane between the endoplasmic reticulum and the Golgi. Pulse-chase experiments suggest that the time for acid phosphatase synthesis and its transport to the Golgi is about 5 min.