The rough endoplasmic reticulum is the site of reserve-protein synthesis in developing Phaseolus vulgaris cotyledons

Cytyledons of the common bean, Phaseolus vulgaris L., were incubated with radioactive amino acids at different stages of seed development. The proteins were fractionated by ion-exchange chromatography, sucrose gradients, and sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis. From 16 to 28 d after flowering about 40% of the incorporated radioactivity was associated with the polypeptides of vicilin and 10% with those of phytohemagglutinin.Polysomes were isolated from developing cotyledons 20–25 d after flowering and free polysomes were separated from membrane-bound polysomes. Aurintricarboxylic acid, an inhibitor of initiation in cell-free translation systems, did not inhibit the incorporation of amino acids into in-vitro synthesized proteins, indicating that synthesis was limited to the completion of already initiated polypeptides. Autofluorography of SDS-polyacrylamide gels showed that the two classes of polysomes made two different sets of polypeptides and that there was little overlap between these two sets.Four polypeptides similar in size to the 4 polypeptides of vicilin were made by membrane-bound polysomes and not by free polysomes. Antibodies specific for vicilin bound to those 4 polypeptides. Free polysomes made only polypeptides which did not bind to antibodies specific for vicilin. Antibodies against phytohemagglutinin did not bind to any of the invitro synthesized polypeptides.The membranes to which the polysomes were bound were characterized on sucrose gradients and by electron microscopy. Polysomes recovered from membranes which banded on top of 35 and 50% sucrose synthesized the vicilin polypeptides most rapidly. These membrane fractions were rich in vesicles of rough endoplasmic reticulum (ER). The ER marker-enzyme NADH-cytochrome-c reductase banded with an average density of 1.18 g/cm³ (40% w/w sucrose) on continuous gradients. These experiments demonstrate that the ER is the site of vicilin synthesis in developing bean cotyledons. Quantitative determinations of several ER parameters (RNA and lipid-phosphate content, NADH-cytochrome-c-reductase activity) show that expansion of the cotyledons is accompanied by a 4-6-fold increase in ER.     

The termination of the synthesis of proteins in vitro with extracts from the undeveloped cyst of Artemia salina

Soluble fractions (S-100) from both undeveloped cysts and developing embryos of Artemia salina promoted elongation of polypeptides initiated in vivo on polysomes of developing embryos or nauplius larvae. The ability of the extract from the undeveloped cyst to terminate correctly the synthesis of polypeptides has been determined indirectly from the distribution of polysomes before and after in vitro translation and, more directly, from the nature of the protein product released from rabbit reticulocyte polysomes. The extract from the undeveloped cyst and also, as expected, that from the developing embryo catalyzed a reduction in the amount of the polysomes of larger size and an increase in the amount of 80 S ribosomes. The soluble extract from the undeveloped cyst can terminate the synthesis of rabbit globin on reticulocyte polysomes. The major polypeptide product released from the polysomes had an electrophoretic mobility identical with that of the subunit of isolated rabbit globin. This indicated that the cyst contained the components necessary to complete and terminate the synthesis of polypeptides correctly and that the released protein product was not predominantly as a result of premature chain termination. The size distribution of Artemia salina proteins released from polysomes from developing embryos was similar when the synthesis was directed by the S-100 at each stage of development.     

THE SECRETORY PATHWAYS OF RAT SERUM GLYCOPROTEINS AND ALBUMIN : Localization of Newly Formed Proteins within the Endoplasmic Reticulum

These studies compare the secretory pathways of newly formed rat serum glycoproteins and albumin by studying their submicrosomal localization at early times after the beginning of their synthesis and also by determining the submicrosomal site of incorporation of N-acetylglucosamine, mannose, galactose, and leucine into protein. N-acetylglucosamine, mannose, and galactose were only incorporated in vitro into proteins from membrane-attached polysomes and not into proteins from free polysomes. Mannose incorporation occurred in the rough endoplasmic reticulum, was stimulated by puromycin but not by cycloheximide, and 90% of the mannose-labeled protein was bound to the membranes. Galactose incorporation, by contrast, occurred in the smooth microsome fraction and 89% of the radioactive protein was in the cisternae. Albumin was mostly recovered (98%) in the cisternae, with negligible amounts in the membranes. To determine whether the radio-active sugars were being incorporated into serum proteins or into membrane protein, the solubilized in vivo-labeled proteins were treated with specific antisera to rat serum proteins or to albumin. Immunoelectrophoresis of the ¹⁴C-labeled leucine membrane and cisternal proteins showed that the membranes contained radioactive serum glycoprotein but no albumin, while the cisternal fraction contained all of the radioactive albumin and some glycoproteins. The results indicate that newly formed serum glycoproteins remain attached to the membranes of the rough endoplasmic reticulum after they are released from the membrane-attached polysomes, while albumin passes directly into the cisternae.     

Role of endoplasmic reticulum in biosynthesis of oat globulin precursors

Oat (Avena sativa L.) groats were labeled with radioactive leucine and salt-soluble proteins were extracted and analyzed. Polyacrylamide gel electrophoresis followed by fluorography indicated two radioactive polypeptides with molecular weight 58 to 62 kilodaltons which were similar in size to unreduced globulin α-β dimers. The role of endoplasmic reticulum in the synthesis of these globulin polypeptides was investigated by in vivo and in vitro protein synthesis studies. Labeled tissue was fractionated by centrifugation and rough endoplasmic reticulum was isolated. Two polypeptides which had molecular weights of 58 to 62 kilodaltons and were immunoprecipitable with antiglobulin immunoglobulin G were found to be transiently associated with the endoplasmic reticulum. Rough endoplasmic reticulum, as well as membrane-bound polysomes, directed the in vitro synthesis of two polypeptides with molecular weight 58 to 62 kilodaltons corresponding in size to unreduced α-β dimers and could be immunoprecipitated with antiglobulin immunoglobulin G. The translation products of free polysomes did not show this. In pulse-labeling, globulin polypeptides with molecular weight 58 to 62 kilodaltons, as well as the α + β subunits, were labeled in protein bodies.

The data suggest that oat globulin polypeptides are synthesized as higher molecular weight precursors on ER-associated polysomes. These precursors are probably transported into protein bodies and cleaved into smaller α and β subunits.

     

Controlled proteolysis of nascent polypeptides in rat liver cell fractions. II. Location of the polypeptides in rough microsomes

Rough microsomes were incubated in an in vitro amino acid-incorporating system for labeling the nascent polypeptide chains on the membrane-bound ribosomes. Sucrose density gradient analysis showed that ribosomes did not detach from the membranes during incorporation in vitro. Trypsin and chymotrypsin treatment of microsomes at 0° led to the detachment of ribosomes from the membranes; furthermore, trypsin produced the dissociation of released, messenger RNA-free ribosomes into subunits. Electron microscopic observations indicated that the membranes remained as closed vesicles. In contrast to the situation with free polysomes, nascent chains contained in rough microsomes were extensively protected from proteolytic attach. By separating the microsomal membranes from the released subunits after proteolysis, it was found that nascent chains are split into two size classes of fragments when the ribosomes are detached. These were shown by column chromatography on Sephadex G-50 to be: (a) small (39 amino acid residues) ribosome-associated fragments and (b) a mixture of larger membrane-associated fragments excluded from the column. The small fragments correspond to the carboxy-terminal segments which are protected by the large subunits of free polysomes. The larger fragments associated with the microsomal membranes depend for their protection on membrane integrity. These fragments are completely digested if the microsomes are subjected to proteolysis in the presence of detergents. These results indicate that when the nascent polypeptides growing in the large subunits of membrane-bound ribosomes emerge from the ribosomes they enter directly into a close association with the microsomal membrane.     

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.     

Compartmentation of the rough endoplasmic reticulum

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

In vivo and in vitro processing of seed reserve protein in the endoplasmic reticulum: evidence for two glycosylation steps

Cotyledons of the common bean (Phaseolus vulgaris L.) synthesize large amounts of the reserve protein phaseolin. The polypeptides are synthesized on membrane-bound polysomes, pass through the endoplasmic reticulum (ER) and accumulate in protein bodies. For a study of the biosynthesis and processing of phaseolin, developing cotyledons were labeled with radioactive amino acids, glucosamine and mannose, and isolated fractions (polysomal RNA, polysomes, and rough ER) were used for in vitro protein synthesis. Newly synthesized phaseolin present in the ER of developing cotyledons can be fractioned into four glycopolypeptides by SDS PAGE. In vitro synthesis with polysomal RNA results in the formation of two polypeptides by polysome run-off shows that glycosylation is a co-translational event. The two unglycosylated polypeptides formed by polysome run-off are slightly smaller than the two polypeptides formed by in vitro translation of isolated RNA, indicating that a signal peptide may be present on these polypeptides. Run-off synthesis with rough ER produces a pattern of four polypeptides similar to the one obtained by in vivo labeling. The two abundant glycopolypeptides formed by polysome run-off. This result indicates the existence of a second glycosylation event for the abundant polypeptides. Inhibition of glycosylation by Triton X-100 during chain-completion with rough ER was used to show that these two glycosylation steps normally occur sequentially. Both glycosylation steps are inhibited by tunicamycin. Analysis of carhohydrate to protein ratios of the different polypeptides and of trypsin digests of polypeptides labeled with [(3)H]glucosamine confirmed the conclusion that some glycosylated polypeptides contain two oligosaccharide chains, while others contain only one. An analysis of tryptic peptide maps shows that each of the unglycosylated polypeptides is the precursor for one glycosylated polypeptide with one oligosaccharide chain and one with two oligosaccharide chains.     

In Vitro Synthesis of Wheat (Triticum aestivum L.) Storage Proteins

Free and membrane-associated polysomes were isolated in approximately equal amounts from endosperm of wheat kernels harvested 20 days after anthesis. The presence of heparin in the homogenizing buffer minimized polysome degradation. Ribonucleic acid from the isolated polysomes, when translated in vitro in a wheat germ system, yielded products ranging in size from about 12,000 to about 80,000 daltons, including at least two polypeptides that co-migrated with seed extract proteins in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The nature of the translation products of free and membrane-associated RNA are distinctly different, with membrane-associated RNA yielding a higher proportion of polypeptides in the size range of 30,000 to 37,000 daltons. Analysis of membrane-associated 3′-terminal polyadenylyl-containing RNA in vitro translation products, by solubility in 70% ethanol and by immunoprecipitation, indicates that the 33,000- to 37,000-dalton polypeptides contain gliadins, and the analysis provides evidence that these proteins are synthesized in association with membranous cell organelles. Gliadin polypeptides synthesized in vitro are larger than authentic gliadins and probably are precursors which, in vivo, undergo modification to yield the smaller final products.     

Surface biochemical changes accompanying primary infection with Rous sarcoma virus. I. Electrokinetic properties of cells and cell surface glycoprotein:glycosyl transferase activities

A special class of polysomes synthesizing tubulin was determined using embryos of the sea urchin, Hemicentrotus pulcherrimus. Three criteria were established for identification of polysomes carrying nascent tubulin, i.e., nascent tubulin on polysomes should have (i) colchicine binding activity, (ii) precipitability with vinblastine and (iii) coincidence in mobility by electrophoresis with tubulin. Two classes of polysomes had polypeptides which accorded with the three criteria. One was tetramers and the other was composed of 15–20 ribosomes. From data reported previously on the molecular weight and amino acid composition of completed microtubule proteins, it was suggested that the class of polysomes composed of 15–20 ribosomes constituted the polysome-synthesizing tubulin of sea urchin embryos. The nature of the nascent polypeptides carried by the tetramer polysomes having colchicine binding activity and precipitability with vinblastine could not be clarified.     

Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution

By in vitro translation of mRNA’s isolated from free and membrane-bound polysomes, direct evidence was obtained for the synthesis of two lysosomal hydrolases, β-glucuronidase of the rat preputial gland and cathespin D of mouse spleen, on polysomes bound to rough endoplasmic reticulum (ER) membranes. When the mRNA’s for these two proteins were translated in the presence of microsomal membranes, the in vitro synthesized polypeptides were cotranslationally glycosylated and transferred into the microsomal lumen. Polypeptides synthesized in the absence of microsomal membranes were approximately 2,000 daltons larger than the respective unglycosylated microsomal polypeptides found after short times of labeling in cultured rat liver cells treated with tunicamycin. This strongly suggests that nascent chains of the lysosomal enzymes bear transient amino terminal signals which determine synthesis on bound polysomes and are removed during the cotranslational insertion of the polypeptides into the ER membranes. In the line of cultured rat liver cells used for this work, newly synthesized lysosomal hydrolases showed a dual destination; approximately 60 percent of the microsomal polypeptides detected after short times of labeling were subsequently processed proteolytically to lower molecular weight forms characteristic of the mature enzymes. The remainder was secreted from the cells without further proteolytic processing. As previously observed by other investigations in cultured fibroblasts (A. Gonzalez-Noriega, J.H. Grubbs, V. Talkad, and W.S. Sly, 1980, J Cell Biol. 85: 839-852; A. Hasilik and E.F. Neufeld, 1980, J. Biol. Chem., 255:4937-4945.) the lysosomotropic amine chloroquine prevented the proteolytic maturation of newly synthesized hydrolases and enhanced their section. In addition, unglycosylated hydrolases synthesized in cells treated with tunicamycin were exclusively exported from the cells without undergoing proteolytic processing. These results support the notions that modified sugar residues serve as sorting out signals which address the hydrolases to their lysosomal destination and that final proteolytic cleavage of hydrolase precursors take place within lysosome itself. Structural differences in the carbohydrate chains of intracellular and secreted precursors of cathespin D were detected from their differential sensitivity to digestion with endoglycosidases H and D. These observations suggest that the hydrolases exported into the medium follow the normal secretory route and that some of their oligosaccharides are subject to modifications known to affect many secretory glycoproteins during their passage through the Golgi apparatus.     

Cell-free Synthesis of Pea Seed Proteins

Both polysomes and polysomal RNA, isolated from cotyledons of ripening pea (Pisum sativum) seeds and supplemented respectively with wheat germ S-100 and S-30 fractions, were used to program the cell-free synthesis of polypeptides. The relationship of these polypeptide products to seed storage proteins has been investigated. When fractionated on sucrose density gradients the translation products did not coincide with native storage proteins, nor were they exactly coincident with the subunits of storage proteins on dissociating gels. Treatment with antiserum prepared against storage proteins precipitated only a very small proportion of these products. Nonetheless, tryptic peptide mapping showed that a significant proportion (up to 65%) of the in vitro products from cell-free systems were related to the storage proteins. Alternative interpretations of these results are that either the translatable mRNAs for storage proteins make up a small proportion of the total template isolated from pea cotyledon polysomes, or that storage protein polypeptides are made in significant amounts in vitro but lack major antigenic determinants which in vivo may be acquired during chain completion or post-translational modification.     

Studies on the Biosynthesis of Intermediate Filament Proteins in the Rat CNS

Abstract: The biosynthesis of brain intermediate filament proteins [neurofilament proteins and glial fibrillary acidic protein (GFA)] was studied with cell-free systems containing either rat spinal cord polysomes (free polysomes or rough microsomes) and rabbit reticulocyte factors or wheat germ homogenate containing spinal cord messenger RNA. The products of translation were isoated by immunoaffinity chromatography and then analyzed by two-dimensional gel electrophoresis (2DGE) followed by fluorography. The free polysome population was found to synthesize two neurofilament proteins (MW 145K, p15.4, and MW 70K, pl 5.3) and three isomers of GFA (α, β, and γ) that differ in isoelectric point. Wheat germ homogenate containing messenger RNA extracted from free cord polysomes synthesized two proteins that comigrated with neurofilament protein standards at 145K 5.4 and 70K 5.3; these proteins were partially purified by neurofilament affinity chromatography. The wheat germ system also synthesized the α, β, and γ isomers of GFA as characterized by immunoaffinity chromatographic purification and comigration with standards in 2DGE analysis. Our data are consistent with the conclusion that synthesis of neurofilament proteins requires multiple messenger RNAs. Also, synthesis of intermediate filament proteins occurs in the free polysome population; detectable amounts of these proteins were not synthcsized by the rough microsomes.     

A procedure for the quantitative recovery of homogeneous populations of undegraded free and bound polysomes from rat liver.

A procedure is described for the preparation of free and bound polysomes from whole homogenates of rat liver tissue. Liver is homogenized in a conventional medium containing glutathione; then after a 12-min centrifugation at 131000g, the free polysomes in the supernatant are saved, while the membrane-bound polysomes in the pellet are suspended in a mixture of ribonuclease inhibitors (cell sap, 250 mM KCl, and glutathione), homogenized in the presence of detergent (Triton X-100), centirfuged for 5 min at 1470g, decanted, and treated with deoxycholate; the polysomes in the two supernatants are harvested by centrifugation through sucrose gradients containing 250 mM KCl and cell sap. Free and bound polysomes prepared in this manner are undegraded, equally active in cell-free protein synthesis, and virtually free of ribonuclease, membranous material, glycogen, deoxycholate, completed protein, and cross-contamination. The recovery of polysomes is approximately 95% and the distribution between the free and membrane-bound state is 25 and 75%, respectively. The molecular weight profiles after sodium dodecyl sulfate-acrylamide gel electrophoresis of the polypeptides completed and released by free and bound polysomes in vitro are different, indicating that there are quantitative differences in the synthesis of various size polypeptides between the two polysome classes. The differential centrifugation procedure is rapid and reproducible, requires much less ultracentrifugation than the isopycnic technique, and provides a nearly quantitative means of separating free and bound polysomes.     

Association of messenger ribonucleic acid with mammalian microsomal membranes: characterization by analysis of cell-free translation products.

Total rough microsomes, isolated from the dog pancreas, were stripped of membranes-bound polysomes by treatment with either EDTA or puromycin and 0.5 M KCl. The stripped microsomal membranes were isolated relatively free from contamination, by using buoyant density centrifugation, and mRNA was isolated from both the membrane fraction and the released material. Depending on the method used to strip the rough microsomes, we found a variable but small percentage (3--15%) of the cellular poly(A)-containing mRNA attached to the microsomal membranes. Reextraction of isolated microsomal membranes with puromycin and 0.5 M KCl reduced the content of membrane-associated mRNA by approximately 50%, resulting in less than 2% of the total membrane-bound polysomal mRNA remaining associated with the microsomal membranes. The membrane-associated mRNA was characterized by translation in the wheat germ cell-free protein synthesizing system, and the products were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The translation products of the membrane-associated mRNA were identical with those from the total pancreas mRNA and also with those obtained by using mRNA isolated from material released directly from the rough microsomes.     

Secretory protein synthesis in Chironomus salivary gland cells is not coupled with protein translocation across endoplasmic reticulum membranes. Electron microscopic evidence

Fragments of rough endoplasmic reticulum containing polysomes bound to the membranes only at the 5 end were visualized in electron microscopic spreads from Chironomus thummi salivary gland cells. The length of the nascent protein molecules in the polysomes increased from the 5 to the 3 (free) polysome end. The data obtained disagree with the generally accepted model according to which synthesis of secretory proteins is concomitant with the protein transport across the endoplasmic membrane.     

Analysis of pea chloroplast inner and outer envelope membrane proteins by two-dimensional gel electrophoresis and their comparison with stromal proteins

Analysis of inner and outer pea (Pisum sativum var. Laxtons Progress No. 9) chloroplast envelope membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that, although the two membranes have distinct polypeptide compositions, there are several comigrating polypeptides in the two membrane fractions. To determine whether these comigrating polypeptides were identical by criteria other than molecular weight, the membrane proteins were analyzed by two-dimensional gel electrophoresis. The results demonstrated that an 86-kilodalton band found in both membranes represents at least two different polypeptides, one an outer membrane protein and the other an inner membrane protein. Several other polypeptide bands found in both membranes appear to be of stromal origin. Two of these polypeptides were shown to be the large and small subunits of ribulose 1,5-bisphosphate carboxylase. The large subunit was identified by two-dimensional electrophoresis of envelope membranes to which stromal proteins were added. Additionally, the large and small subunits of ribulose 1,5-bisphosphate carboxylase were immunologically identified using an electrophoretic transfer procedure coupled with an enzyme-linked immunosorbent assay. Various treatments, including sonication, resulted in no significant loss of the stromal polypeptides from the outer envelope membranes. Based on these results, it is suggested that the stromal proteins are not simply bound to the outer surface of the vesicles.     

Ribosome-membrane association in fat body tissues from reproductively active females of Leucophaea maderae

Polysomal and microsomal profiles from fat body tissues of Leucophaea analysed by a combination of equilibrium sedimentation and sedimentation velocity centrifugations on sucrose density gradients revealed that microsomes from egg-maturing females are considerably more dense than those from allatectomized (reproductively inactive) females or from males. This greater density is conferred on the microsomes of reproductively active females by the binding of many more ribosomes (polysomes) to the membranes. Treatment of the microsomes with 500 mM K⁺ or 1 mM puromycin resulted in a removal of only a few ribosomes and polysomes from the membranes, as is documented with the electron microscope. Incubation of microsomes with a combination of 500 mM K⁺ and 1 mM puromycin resulted in a complete degranulation of the membranes. Such microsomes attained a density similar to those of the inactive tissues. From the available evidence it is concluded that the female specific protein (vitellogenin), which is induced by the juvenile hormone, is synthesized on ergastoplasmic membranes and released into the cisternae as is known for the synthesis of exportable proteins in several vertebrate systems.     

Effect of Nitrogen Starvation on Polypeptide Composition, Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase, and Thylakoid Carotenoprotein Content of Synechocystis sp. Strain PCC6308

Synechocystis sp. strain PCC6308 cells were starved for nitrogen for 5 days. The polypeptide compositions of whole cell extracts and washed membranes of nitrogen-replete and nitrogen-starved cells were compared by one- and two-dimensional electrophoresis. Immunoblotting of one-dimensional gels indicated that pelletable ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was depleted in cells starved for nitrogen, while levels of soluble Rubisco were comparable in nitrogen-starved and nitrogen-replete cells. This is consistent with the hypothesis that pelletable Rubisco may serve as a nitrogen reserve in Synechocystis 6308. Other polypeptides were differentially enriched in the membrane or soluble fractions of nitrogen-replete cells or nitrogen-starved cells, suggesting nitrogen starvation may alter partitioning of polypeptides into soluble and membrane fractions. Degradation of abundant polypeptides during nitrogen starvation appeared to cause an effective magnification of less abundant polypeptides in the molecular mass range of 20 to 40 kilodaltons, as shown by two-dimensional electrophoresis. A 42-kilodalton thylakoid carotenoid protein identified by immunoblotting was conserved in membranes from nitrogen-starved cells. This may be functional for cells depleted of pigment and thus exposed to higher light levels because of decreased self-shading.     

The products of mitochondria-bound cytoplasmic polysomes in yeast

Experiments were undertaken to examine the fate and composition of polypeptides synthesized on cytoplasmic polysomes associated with the outer mitochondrial membrane of Saccharomyces cerevisiae. Mitochondria with their associated cytoplasmic polysomes were isolated from growing yeast spheroplasts and placed in a polypeptide chain completion system together with [35S]methionine. Of the total products synthesized in the readout system, 80 to 85% remain associated with the mitochondria after sucrose gradient centrifugation. Most of the labeled products are resistant to papain digestion unless the membranes are disrupted by treatment with detergent or shaking with glass beads. When free cytoplasmic polysomes were translated in the presence of [35S]methionine and incubated with mitochondria, only about 20% of the labeled polypeptides remain associated with the mitochondria; furthermore, most of these products are equally sensitive to papain digestion in the presence or absence of detergent. These results support the view that the cytoplasmic polysomes associated with the outer mitochondrial membrane of yeast facilitate the segregation of newly synthesized proteins into the organelle. The proportion of the alpha, beta, and gamma subunits of the F1-ATPase was determined among the products synthesized by mitochondria-bound and free cytoplasmic polysomes. By double antibody precipitation and immunoreplicate electrophoresis, we find that the proportion of the subunits of F1-ATPase is much greater among the products of the mitochondria-bound polysomes than those synthesized on free polysomes.