Dog pancreatic microsomal-membrane polypeptides analysed by two-dimensional gel electrophoresis.

The two-dimensional polyacrylamide-gel electrophoresis technique of O'Farrell [(1975) J. Biol. Chem 250, 4007-4021] was applied to resolve and analyse the polypeptide composition of dog pancreatic rough microsomal membranes, which were shown to be active in co-translational processing of preprolactin synthesized from pituitary mRNA in a translation system in vitro. About 100 polypeptides are resolved. Treatment of rough microsomal membranes with EDTA and high KCl concentration yielded membranes stripped of their ribosomes with retention of activity for translocation and processing. Stripped microsomal membranes showed a selective concentration of approximately 25 polypeptides in the membranes when analysed by two-dimensional polyacrylamide-gel electrophoresis. The two-dimensional electrophoretic profile was catalogued into polypeptides that are glycoproteins, those that contain free thiol groups disposed at the cytosolic surface of microsomal vesicles and those that are of secretory origin but have been entrapped in the microsomal preparation. Several secretory components, including amylase, procarboxypeptidases, lipase and anionic trypsinogen, were tentatively identified among the microsomal polypeptides. The rough and stripped microsomal membranes from dog pancreas show a characteristic set of seven major acidic polypeptides, which are also identifiable in microsomal-membrane preparations isolated from dog liver and rat liver. One of these polypeptides was identified as protein disulphide-isomerase (EC 5.3.4.1).     

RIBOSOME-MEMBRANE INTERACTION : Nondestructive Disassembly of Rat Liver Rough Microsomes into Ribosomal and Membranous Components

In a medium of high ionic strength, rat liver rough microsomes can be nondestructively disassembled into ribosomes and stripped membranes if nascent polypeptides are discharged from the bound ribosomes by reaction with puromycin. At 750 mM KCl, 5 mM MgCl₂, 50 mM Tris·HCl, pH 7 5, up to 85% of all bound ribosomes are released from the membranes after incubation at room temperature with 1 mM puromycin. The ribosomes are released as subunits which are active in peptide synthesis if programmed with polyuridylic acid. The ribosome-denuded, or stripped, rough microsomes (RM) can be recovered as intact, essentially unaltered membranous vesicles Judging from the incorporation of [³H]puromycin into hot acid-insoluble material and from the release of [³H]leucine-labeled nascent polypeptide chains from bound ribosomes, puromycin coupling occurs almost as well at low (25–100 mM) as at high (500–1000 mM) KCl concentrations. Since puromycin-dependent ribosome release only occurs at high ionic strength, it appears that ribosomes are bound to membranes via two types of interactions: a direct one between the membrane and the large ribosomal subunit (labile at high KCl concentration) and an indirect one in which the nascent chain anchors the ribosome to the membrane (puromycin labile). The nascent chains of ribosomes specifically released by puromycin remain tightly associated with the stripped membranes. Some membrane-bound ribosomes (up to 40%) can be nondestructively released in high ionic strength media without puromycin; these appear to consist of a mixture of inactive ribosomes and ribosomes containing relatively short nascent chains. A fraction (~15%) of the bound ribosomes can only be released from membranes by exposure of RM to ionic conditions which cause extensive unfolding of ribosomal subunits, the nature and significance of these ribosomes is not clear.     

Transfer to proteins across membranes. II. Reconstitution of functional rough microsomes from heterologous components

The data presented in this paper demonstrate that native small ribosomal subunits from reticulocytes (containing initiation factors) and large ribosomal subunits derived from free polysomes of reticulocytes by the puromycin-KCl procedures can function with stripped microsomes derived from dog pancreas rough microsomes in a protein-synthesizing system in vitro in response to added IgG light chain mRNA so as to segregate the translation product in a proteolysis- resistant space. No such segregation took place for the translation product of globin mRNA. In addition to their ability to segregate the translation product of a specific heterologous mRNA, native dog pancreas rough microsomes as well as derived stripped microsomes were able to proteolytically process the larger, primary translation product in an apparently correct manner, as evidenced by the identical mol wt of the segregated translation product and the authentic secreted light chain. Segregation as well as proteolytic processing by native and stripped microsomes occurred only during ongoing translation but not after completion of translation. Attempts to solubilize the proteolytic processing activity, presumably localized in the microsomal membrane by detergent treatment, and to achieve proteolytic processing of the completed light chain precursor protein failed. Taken together, these results establish unequivocally that the information for segregation of a translation product is encoded in the mRNA itself, not in the protein- synthesizing apparatus; this provides strong evidence in support of the signal hypothesis.     

Ribosomal-membrane interaction: in vitro binding of ribosomes to microsomal membranes

Rat liver rough microsomal membranes were stripped of bound ribosomes by treatment with puromycin and high concentrations of monovalent ions. Ribosomal subunits labeled in the RNA were detached from rough microsomes by the same procedure, recombined into monomers, and then incubated with stripped membranes in a medium of low ionic strength (25 mm-KCl, 50 mm-Tris-HCl, 5 mm-MgCl₂). These ribosomes readily attached to the stripped membranes, as determined by isopycnic flotation of the reconstituted microsomes. The binding reaction was complete after incubation for five minutes at 37 °C, but also proceeded at 0 °C, at a lower rate. Scatchard plots showed a binding constant of ~8 × 10⁷m^?1 and ~5 × 10^?8 mol binding sites per gram of membrane protein. Native rough microsomes showed a much lower binding capacity at 0 °C than stripped rough microsomes, but showed considerable uptake of ribosomes at 37 °C. Smooth microsomes, treated for stripping and incubated at 0 °C, accepted less than half as many ribosomes as stripped rough microsomes. Erythrocyte ghosts were incapable of binding ribosomes. Microsomal binding sites were heat sensitive, were destroyed by a brief incubation with a mixture of trypsin and chymotrypsin in the cold, and were unaffected by incubation with phospholipase C.Ribosome binding was decreased by increasing the concentration of monovalent ions and was strongly inhibited by 10^?4m-aurintricarboxylic acid. Experiments with purified ribosomal subunits revealed that at concentrations of monovalent ions close to physiological concentrations (100 to 150 mm-KCl), microsomal binding sites had a greater affinity for 60 S than for 40 S subunits.Stripped rough microsomes were also capable of accepting polysomes obtained from rough microsomes by detergent treatment. Although this binding presumably involves the correct membrane binding sites, polypeptides discharged from re-bound polymers were not transferred to the vesicular cavities, as in native microsomes. The released polypeptides remained firmly associated with the outer microsomal face, as shown by their accessibility to proteases.     

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.     

Release of poly a(+) messenger RNA from rat liver rough microsomes upon disassembly of bound polysomes

Several procedures were used to disassemble rat liver rough microsomes (RM) into ribosomal subunits, mRNA, and ribosome-stripped membrane vesicles in order to examine the nature of the association between the mRNA of bound polysomes and the microsomal membranes. The fate of the mRNA molecules after ribosome release was determined by measuring the amount of pulse-labeled microsomal RNA in each fraction which was retained by oligo-dT cellulose or by measuring the poly A content by hybridization to radioactive poly U. It was found that ribosomal subunits and mRNA were simultaneously released from the microsomal membranes when the ribosomes were detached by: (a) treatment with puromycin in a high salt medium containing Mg++, (b) resuspension in a high salt medium lacking Mg++, and (c) chelation of Mg++ by EDTA or pyrophosphate. Poly A-containing mRNA fragments were extensively released from RM subjected to a mild treatment with pancreatic RNase in a medium of low ionic strength. This indicates that the 3' end of the mRNA is exposed on the outer microsomal surface and is not directly bound to the membranes. Poly A segments of bound mRNA were also accessible to [(3)H] poly U for in situ hybridization in glutaraldehyde-fixed RM. Rats were treated with drugs which inhibit translation after formation of the first peptide bonds or interfere with the initiation of protein synthesis. After these treatments inactive monomeric ribosomes, as well as ribosomes bearing mRNA, remained associated with their binding sites in microsomes prepared in media of low ionic strength. However, because there were no linkages provided by nascent chains, ribosomes, and mRNA, molecules were released from the microsomal membranes without the need of puromycin, by treatment with a high salt buffer containing Mg++. Thus, both in vivo and in vitro observations are consistent with a model in which mRNA does not contribute significantly to the maintenance of the interaction between bound polysomes and endoplasmic reticulum membranes in rat liver hepatocytes.     

Saponin permeabilization of rough microsomes from rat liver reveals a novel prothrombin pool

Saponin permeabilization of rough microsomes in the presence of high salt revealed a novel pool of prothrombin associated by ionic interactions to the microsomal membrane. The lumenal content was obtained by treating rough microsomes with 0.32% saponin in a low salt (0.05 M KCl) buffer. By a subsequent treatment with 0.32% saponin in a slightly alkaline high salt buffer a fraction of peripherally associated membrane prothrombin was released from rough microsomes. Finally, the membrane-bound fraction was solubilized with 2.5% Triton X-100. The lumenal content fraction, the peripherally membrane-associated and the membrane-bound fraction from normal rats contained 55%, 29% and 16% of the total rough microsomal prothrombin, respectively. The corresponding fractions from warfarin-treated rats contained 86%, 5% and 9% of the total prothrombin. Following ¹⁴C-γ-carboxylation of intact microsomes for 30 min, the novel membrane-associated and the membrane-bound pool contained 42% and 33%, respectively, of labeled prothrombin. A similar distribution was found with warfarin-treated rats.     

Mobility of ribosomes bound to microsomal membranes. A freeze-etch and thin-section electron microscope study of the structure and fluidity of the rough endoplasmic reticulum

The lateral mobility of ribosomes bound to rough endoplasmic reticulum (RER) membranes was demonstrated under experimental conditions. High- salt-washed rough microsomes were treated with pancreatic ribonuclease (RNase) to cleave the mRNA of bound polyribosomes and allow the movement of individual bound ribosomesmfreeze-etch and thin-section electron microscopy demonstrated that, when rough microsomes were treated with RNase at 4 degrees C and then maintained at this temperature until fixation, the bound ribosomes retained their homogeneous distribution on the microsomal surface. However, when RNase- treated rough microsomes were brought to 24 degrees C, a temperature above the thermotropic phase transition of the microsomal phospholipids, bound ribosomes were no longer distributed homogeneously but, instead, formed large, tightly packed aggregates on the microsomal surface. Bound polyribosomes could also be aggregated by treating rough microsomes with antibodies raised against large ribosomal subunit proteins. In these experiments, extensive cross-linking of ribosomes from adjacent microsomes also occurred, and large ribosome-free membrane areas were produced. Sedimentation analysis in sucrose density gradients demonstrated that the RNase treatment did not release bound ribosomes from the membranes; however, the aggregated ribosomes remain capable of peptide bond synthesis and were released by puromycin. It is proposed that the formation of ribosomal aggregates on the microsomal surface results from the lateral displacement of ribosomes along with their attached binding sites, nascent polypeptide chains, and other associated membrane proteins; The inhibition of ribosome mobility after maintaining rough microsomes at 4 degrees C after RNase, or antibody, treatment suggests that the ribosome binding sites are integral membrane proteins and that their mobility is controlled by the fluidity of the RER membrane. Examination of the hydrophobic interior of microsomal membranes by the freeze-fracture technique revealed the presence of homogeneously distributed 105-A intramembrane particles in control rough microsomes. However, aggregation of ribosomes by RNase, or their removal by treatment with puromycin, led to a redistribution of the particles into large aggregates on the cytoplasmic fracture face, leaving large particle-free regions.     

Localization of ATP-dependent calcium transport activity in mouse pancreatic microsomes

Electron-dense deposits representing calcium oxalate crystals which result from ATP-dependent calcium uptake have been localized within vesicles of of a heavy microsomal fraction prepared from mouse pancreatic acini. In the absence of either ATP or oxalate, no electron-dense deposits could be observed. By subfractionation of microsomes on discontinuous sucrose gradients, it could be shown that the highest energy-dependent calcium transport activity was associated with the rough endoplasmic reticulum. In rough microsomes, the 45Ca2+-uptake measured was 7 times greater than that of smooth microsomes in the presence of ATP and oxalate and about 3 times greater in he presence of ATP alone. When ribosomes were released from the rough endoplasmic reticulum vesicles by treatment with KCl in the presence of puromycin, the stripped microsomes showed a 40% increase in the specific 45Ca2+-uptake activity measured in he presence of ATP and oxalate and an increase of 80 to 90% in the presence of ATP alone. From these results it can be concluded that the calcium transport activity of microsomes prepared from mouse pancreatic acini is located predominantly in the rough endoplasmic reticulum membrane.     

Proteins of rough microsomal membranes related to ribosome binding. I. Identification of ribophorins I and II, membrane proteins characteristics of rough microsomes

Rat liver rough microsomes (RM) contain two integral membrane proteins which are not found in smooth microsomes (SM) and appear to be related to the presence of ribosome-binding sites. These proteins, of molecular weight 65,000 and 63,000, were designated ribophorins I and II, respectively. They were not released from the microsomal membranes by alkali or acid treatment, or when the ribosomes were detached by incubation with puromycin in a high salt medium. The anionic detergent sodium deoxycholate caused solubilization of the ribophorins, but neutral detergents led to their recovery with the sedimentable ribosomes. Ribosomal aggregates containing both ribophorins, but few other membrane proteins, were obtained from RM treated with the nonionic detergent Kyro EOB (2.5 X10(-2) M) in a low ionic strength medium. Sedimentation patterns produced by these aggregates resembled those of large polysomes but were not affected by RNase treatment. The aggregates, however, were dispersed by mild trypsinization (10 microgram trypsin for 30 min at 0 degrees C), incubation with deoxycholate, or in a medium of high salt concentration. These treatments led to a concomitant degradation or release of the ribophorins. It was estimated, from the staining intensity of protein bands in acrylamide gels, that in the Kyro EOB aggregates there were one to two molecules of each ribophorin per ribosome. Sedimentable complexes without ribosomes containing both ribophorins could also be obtained by dissolving RM previously stripped of ribosomes by puromycin- KCl using cholate, a milder detergent than DOC. Electron microscope examination of the residue obtained from RM treated with Kyro EOB showed that the rapidly sedimenting polysome-like aggregates containing the ribophorins consisted of groups of tightly packed ribosomes which were associated with remnants of the microsomal membranes.     

High affinity specific antiestrogen binding sites are concentrated in rough microsomal membranes of rat liver

Saturation and competitive binding analyses demonstrated the presence of a high affinity (K_D = 0.92 nM), specific antiestrogen binding site (AEBS) in rat liver microsomes and at least 75% of total liver AEBS was recovered in this fraction. When microsomes were further separated into smooth and rough fractions, AEBS was concentrated in the latter. Subsequent dissociation of ribosomes from the rough membranes revealed that AEBS was associated with the membrane and not the ribosomal fraction. Antiestrogen binding activity could not be extracted from membranes with 1 M KCl or 0.5 M acetic acid but could be solubilized with sodium cholate. These data indicate that AEBS is an integral membrane component of the rough microsomal fraction of rat liver.     

Study of the regulation of plastid development in Euglena gracilis. II. Functional localisation and synthesis of ribosomal chloroplast particles (author's transl)]

Chloroplast ribosomes in greening cells of Euglena gracilis are found either in the stroma or bound to thylakoid membranes. The membrane-bound chloroplast ribosomes are of two main types: those which can be released by 0.5 M KCl or by puromycin and 0.5 M KCl, and those which are released by detergent (deoxycholate or Triton X-100) and KCl. The ribosomes which are released by puromycin are presumably bound to chloroplast membrane by nascent peptide chains. Ribosomes released by puromycin are found only during the course of plastidial differentiation at the time of active thylacoid membrane synthesis. Following greening, those ribosomes remain bound to the membranes but can be removed by KCl alone. An analysis of RNA labelling showed that 30-S but not 53-S subunits of membrane-bound ribosomes are of uniform specific activity. This suggests that 30-S subunit exchange in a common pool while 53 S subunits remain membrane bound and do not exchange in a common pool. Membrane-bound chloroplast ribosomes which are released either by puromycin or by detergent are originally derived from loosely bound particles, released by 0.5 M KCl.     

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.     

The isolation of microsomal subfractions of mouse plasmacytoma cells: The effect of salt concentration during nitrogen cavitation

Following disruption of MPC-11 cells by nitrogen cavitation the microsomes have been fractionated by centrifugation on discontinuous sucrose gradients. When the homogenization buffer contained 25 mm KCl three fractions were observed: smooth microsomes, light rough microsomes, and heavy rough microsomes. When it contained 100 mm KCl, however, only smooth and light microsomal fractions were found. Under the latter conditions the heavy rough microsomal vesicles were apparently not released as separate organelles but instead sedimented together with the endoplasmic reticulum which remains attached to the nuclei.     

Biogenesis of endoplasmic reticulum membrane in rat liver cells. III. Biosynthesis of NADPH-cytochrome c reductase and cytochrome b5 by loosely-bound ribosomes.

Membrane-bound ribosomes were separated into two distinct classes (loosely-bound and tightly-bound ribosomes) by treatment with 0.6 M KCl, 1 mM puromycin, 0.05% DOC, or 10 mM EDTA. It was also confirmed that any one of these reagents except for EDTA dissociated the same class of ribosomes from the membrane. A population of lighter microsomal vesicles was formed from rough microsomes upon the dissociation of loosely-bound ribosomes by treatment with these chemicals. Rough microsomes were subfractionated into lighter and heavier fractions, L-rMs and H-rMs, by centrifugation using a discontinuous gradient of sucrose consisting of 1.3 M, 1.5 M, and 2.1 M solutions. It was found that L-rMs was rich in loosely-bound ribosomes, whereas H-rMs contained a high proportion of tightly-bound ribosomes. It is likely that loosely-bound and tightly-bound ribosomes are heterogeneously distributed among rough microsomal vesicles. Loosely-bound ribosomes and tightly-bound ribosomes synthesize different kinds of proteins. Two microsomal membrane proteins, NADPH-cytochrome c reductase and cytochrome b5, were exclusively synthesized by loosely-bound ribosomes, whereas serum albumin, which is a major component of the secretory proteins of hepatocytes, was synthesized only by tightly-bound ribosomes. Since the nascent peptides of NADPH-cytochrome c reductase and cytochrome b5 are released from bound ribosomes to the cytoplasmic surface of endoplasmic reticulum, while those of secretory proteins are discharged into the lumen across the membrane, the strength of the association between ribosomes and microsomal membrane seems to be correlated with the direction of release of nascent peptides.     

Mechanism of compartmentation of secretory proteins: transport of exocrine pancreatic proteins across the microsomal membrane

The mechanism by which secretory proteins are segregated within the cisternal space of microsomal vesicles was studied using dog pancreas mRNA which directs the synthesis of 14 well-characterized nonglycosylated pancreatic exocrine proteins. In the absence of microsomal membranes, each of the proteins was synthesized as larger polypeptide chains (presecretory proteins). 1,000-2,000 daltons larger than their authentic counterparts as judged by polyacrylamide gel electrophoresis in SDS. Conditions optimal for the study of reconstituted rough microsomes in the reticulocyte lysate system were examined in detail using mRNA and microsomal membranes isolated from dog pancreas. Functional reconstitution of rough microsomes was considerably more efficient in the presence of micrococcal nuclease- treated membranes than in the presence of EDTA-treated membranes. Analysis for segregation of nascent secretory proteins by microsomal vesicles, using post-translational incubation in the presence of trypsin and chymotrypsin, 50 μg/ml each, was shown to be inadequate, because of the disruption of vesicles by protease activity. Addition of 1-3 mM tetracaine or 1 mM dibucaine stabilized microsomal membranes incubated in the presence of trypsin and chymotrypsin at either 0 degrees or 22 degrees C. Each of the pancreatic presecretory proteins studied was correctly processed to authentic secretory proteins by nuclease-treated microsomal membranes, as judged by both one-dimensional and two-dimensional gel electophoresis. Post-translational addition of membranes did not result in either segregation or processing of nascent polypeptide chains. Post- translational proteolysis, carried out in the presence of 3 mM tetracaine, indicated that each of the 14 characterized dog pancreas secretory proteins was quantitatively segregated by nuclease-treated microsomal vesicles. Segregation of nascent secretory proteins was irreversible, since radioactive amylase, as well as the other labeled secretory proteins, remained quantitatively sequestered in microsomal vesicles during a 90-min incubation at 22 degrees C after the cessation of protein synthesis. Studies employing synchronized protein synthesis and delayed addition of membranes indicated that all pancreatic presecretory proteins contain amino terminal peptide extensions. These peptide extensions are shown to mediate the cotranslational binding of presecretory proteins to microsomal membranes and the transport of nascent secretory proteins to the vesicular space. The maximum chain lengths which, during synthesis, allow segregation of nascent polypeptide chains varied between 61 (pretrypsinogen 2 + 3) and 88 (preprocarboxypeptidase A1) amino acid residues among dog pancreas presecretory proteins. Reconstitution studies using homologous and heterologous mixtures of mRNA (dog, guinea pig, and rat pancreas; rat liver) and micrococcal nuclease-treated microsomal membranes (dog, guinea pig, and rat liver; dog pancreas), in the presence of placental ribonuclease inhibitor, suggest that the translocation mechanism described is common to the rough endoplasmic reticulum of all mammalian tissues.     

Coalescence of microsomal vesicles from rat liver: a phenomenon occurring in parallel with enhancement of the glycosylation activity during incubation of stripped rough microsomes with GTP

Rough microsomes from rat liver have been subjected to various treatments and incubated afterwards with UDP-N-acetyl-[14C]glucosamine and GDP-mannose in the presence of GTP (0.5 mM), or of other nucleotides. In agreement with earlier results from this laboratory, the preparations previously treated to strip off the ribosomes and incubated in the presence of GTP assembled dolichol-linked oligosaccharides and transferred these oligosaccharides to endogenous protein acceptors much more actively than untreated preparations, or stripped preparations incubated in the absence of GTP. Thin-section and freeze-fracture electron microscopy have revealed that pyrophosphate- treated preparations incubated with GTP are aggregated and contain numerous vesicles as large as 1-4 micrometer, or more. Such large vesicles were not present before incubation and thus were considered to have been formed through coalescence of regular-sized ones. Like glycosylation, the coalescence phenomenon depends upon the removal of ribosomes, because it occurred whether ribosomes had been stripped, at least partly, with pyrophosphate, KCl, or puromycin, but not when rough microsomes had been washed with 0.25 M sucrose or with KCl and MgCl2. Like glycosylation, it also depends on the addition of GTP and was not induced by ATP, UTP, CTP, and nonhydrolysable analogues of GTP. Rough microsomes coalesced, however, when pyrophosphate-treated preparations were incubated with GTP in the absence of nucleotide sugars, or in the presence f tunicamycin, indicating that the coalescence phenomenon does not result from the glycosylation of some membrane constituents.     

Sequestration of pea reserve proteins by rough microsomes

Free polysomes, polysomes released from membranes, and rough microsomal vesicles isolated from developing cotyledons of Pisum sativum L. cv. Burpeeana were used to direct cell-free protein synthesis in a wheat germ system. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the polypeptide products had molecular weights ranging from 12,000 to 74,000. Some of the polypeptides migrated during electrophoresis with the same mobility as polypeptides present in legumin and vicilin preparations. By the use of rabbit antibodies raised against pea reserve proteins it was established that polysomes released from membranes and rough microsomes directed the synthesis of polypeptides that were related to reserve proteins whereas free polysomes did not.     

Purification of protein fatty acyltransferase and determination of its distribution and topology

Studies reported from this laboratory have demonstrated that O-glycosidic glycoproteins of salivary, pulmonary, and gastrointestinal origin are acylated by fatty acyltransferase residing in Golgi and microsome-enriched fraction (Slomiany, A., Liau, Y.H., Takagi, A., Laszewicz, W., and Slomiany, B.L. (1984) J. Biol. Chem. 259, 13304-13308). Here we report on the successful purification of this enzyme from rough microsomal membranes of rat gastric mucosa and its identification in a number of diverse tissues and organs, such as heart, liver, pancreas, lung, kidney, salivary glands, and lymphoblasts. The enzymatic activity has been released from the stripped and salt-extracted microsomes with 0.5% Triton X-100 and recovered from 100,000 x g supernatant by affinity chromatography on Cibacron blue F3GA column. The retained fatty acyltransferase protein was selectively displaced from the column with 50 microM palmitoyl-CoA. On nonreducing polyacrylamide gel electrophoresis, the enzymatic activity was associated with a 234-kDa complex, and on sodium dodecyl sulfate polyacrylamide gel electrophoresis, the complex afforded 65- and 67-kDa protein bands. Incubation of microsomes with trypsin prior to enzyme extraction resulted in a 50% inactivation of the fatty acyltransferase and generation of 53- and 55-kDa protein bands, which also had affinity to Cibacron blue F3GA and were displaced from the column together with the active (intact) enzyme. We suggest that the fatty acyltransferase is an integral rough microsomal protein partially exposed to cytosol, which catalyzes the fatty acyl-CoA-protein reaction on the cytosolic site of the rough endoplasmic reticulum and that this enzyme is responsible for processing of the group of protein which are entering rough endoplasmic reticulum-Golgi secretory pathway.     

Proteolytic processing of presecretory proteins is required for development of biological activities in pancreatic exocrine proteins

The biological activities of pancreatic presecretory and secretory proteins synthesized in vitro were compared in studies of (a) the binding of nascent amylase to its substrate, glycogen, (b) the binding of nascent trypsinogen 1, trypsinogen 2+3, and chymotrypsinogen 1 to Sepharose-bound soybean trypsin inhibitor, and (c) the activation of nascent trypsinogen by porcine enterokinase. Nascent secretory proteins synthesized in vitro using a mRNA-dependent gel-filtered reticulocyte lysate translation system supplemented with canine pancreas rough microsomes or canine pancreas mRNA and micrococcal nuclease-treated microsomal membranes showed biological activities similar to authentic secretory proteins if oxidized glutathione was added during their synthesis. Proteins synthesized in the presence of membranes and the absence of glutathione showed significantly less biological activity due to incorrect development of conformation. Presecretory proteins synthesized in vitro with canine pancreas mRNA in the absence of microsomal membranes had little or no activity after translation in either the absence or presence of glutathione. These and previous findings (Scheele, G. A., and Jacoby, R. (1982) J. Biol. Chem. 257, 12277-12282) indicate that proteolytic removal of the NH2-terminal transport peptide is necessary to allow correct conformational development, including the formation of native disulfide bonds, which not only stabilizes the molecule but allows expression of authentic biological and probiological activity.