A polysome-membrane binding system from rat liver. I. Basic characterization of the binding system.

A simple reaction system was developed to examine the binding of polysomes to membranes of the endoplasmic reticulum and to investigate the fate of ribosomes and nascent chains during protein synthesis in vitro. The system conssited of Sephadex G-25 treated post-mitochondrial fraction prepared from rat liver (Sephadex-PM) as a source of membranes, and radioactive free polysomes prepared from another rat liver. The following results were obtained. 1. Nascent chains on free polysomes labeled in vivo were transferred to membranes in vitro. The process required protein synthesis. 2. This reaction occurred in two steps: a) Binding of the free polysomes to membranes in the absence of protein synthesis. b) Release of ribosomes, leaving nascent chains on the membranes, requiring protein syntehsis. 3. A portion of the ribosomes found on membranes in vivi (membrane-bound ribosomes) was also released from the membranes during incubation in vitro, leaving their nascent chains on the membranes. The significance of the transfer of nascent chains from free polysomes to membranes in vitro is discussed in the light of known polysome-membrane interaction in vivo.     

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.     

The chloroplast 32 kDa protein is synthesized on thylakoid-bound ribosomes in Chlamydomonas reinhardtii

A cloned cpDNA fragment containing a portion of the gene for the 32–36 kDa thylakoid protein of Chlamydomonas (polypeptide D-1) was isolated. Hybridization probing of RNA from soluble and membrane fractions of Chlamydomonas showed that the mRNA for D-1 is bound to thylakoid membranes. Run-off translation of thylakoid-bound polysomes (rough thylakoids) with [³⁵S]-methionine yields polypeptide D-1 as the major product. Peptide mapping with S. aureus V-8 protease of D-1 synthesized (1) in vivo, (2) in vitro by rough thylakoids and (3) in the reticulocyte lysate directed by non-polyadenylated RNA showed that D-1 is synthesized as a precursor in the reticulocyte lysate but as the mature polypeptide by rough thylakoids.     

Recombinant rabbit uteroglobin expressed at high levels in E. coli forms stable dimers and binds progesterone

In order to express uteroglobin in Escherichia coli we have constructed a DNA coding for complete mature rabbit uteroglobin by fusing genomic sequences from the second exon of the gene to an incomplete cDNA. This DNA was inserted into various positions of the polylinker cloning region of pDS expression vectors and the uteroglobin gene was expressed in E. coli by IPTG induction. Four different uteroglobin-derived proteins were produced containing 1, 3, 5 and 7 more N-terminal amino acids than the naturally occurring mature protein. The yield of soluble protein strongly increased with increasing length of the N-terminal additions. Protein and RNA analysis showed that this variation is most likely due to progressively higher translation efficiencies of the larger recombinants. UG7, the most efficiently synthesized recombinant protein, carrying seven additional N-terminal amino acids, was purified and further characterized. Like natural uteroglobin, UG7 forms a dimer and binds progesterone with an affinity indistinguishable to the natural protein. This bacterially produced protein can be used for detailed structure-function investigations of uteroglobin.     

Juvenile Hormone Induction of the Insect Yolk Protein Precursor

In Leucophaea maderae the female specific protein, or vitellogenin,is being synthesized and secreted exclusively by the adult femalefat bodies. This specific protein, which is induced by thejuvenilehormones makes up approximately 90% of the total yolk proteins.It is a lipophosphoprotein of low phosphorus (0.14) content.The female specific protein is synthesized on polysomes boundto membranes of the ergastoplasmic reticulum (ER). Microsomevesicles obtained from active tissues are heavily studded withribosomes and are considerably more dense than those from "inactive"tissues. The nascent polypeptide chains of the vitellogeninare secreted into the cisternae of the ER and can be releasedby Na deoxycholate digestion of the membranes. Similarly, thenon-specific serum proteins are also secreted into the cisternaeof the ER. All evidence points to the fact that microsomes maycarry mixed populations of polysomes, those associated withspecific and those associated with non-sex-specific proteinsynthesis. The significance of the polysomal association withmembranes for the synthesis of exportable sex-specific proteinis discussed.     

In vitro synthesis of superoxide dismutases of rat liver

The syntheses of copper, zinc-superoxide dismutase (Cu,Zn-SOD) and manganese-superoxide dismutase (Mn-SOD) in vitro were studied. Both Cu,Zn-SOD and Mn-SOD were preferentially synthesized by free polysomes. Mn-SOD was synthesized as a large precursor (26,000 daltons), which was processed to the mature size (22,500 daltons) by in vitro incubation with a rat liver mitochondrial fraction. On the other hand, Cu,Zn-SOD was synthesized as the mature size product. It was shown that Cu,Zn-SOD and Mn-SOD synthesized in vitro represented 0.018% and 0.016% of the total translation products of free polysomes, respectively.     

Precursor of beta-lactamase is enzymatically inactive. Accumulation of the preprotein in Saccharomyces cerevisiae

Synthesis and properties of the bacterial precursor of beta-lactamase (E.C.3.5.2.6) were studied in Saccharomyces cerevisiae transformants. A protease-deficient yeast mutant was transformed with the plasmid pADH040-2 conferring high expression of the bla gene. Besides precisely processed beta-lactamase, transformed yeast cells contained mainly bla precursor up to the amount of 2% of total cellular protein. The precursor was shown to be synthesized on free polysomes in vivo but could be processed with rough microsomal membranes in a cell-free translation system. By applying an isolation procedure using high-salt conditions, the labile precursor could be separated in a native form from the mature beta-lactamase. Thereby it could be shown that the pre-beta-lactamase had virtually no enzymatic activity in contrast to the mature enzyme, which was indistinguishable from bacterial beta-lactamase. Furthermore, the precursor was highly susceptible to proteolytic degradation by trypsin under conditions which did not affect the mature enzyme. Accordingly, the protein conformation of the precursor must be substantially different from that of the authentic beta-lactamase, demonstrating that specific processing and transport of beta-lactamase is associated with directing the protein to a distinct conformation.     

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.     

Studies on biosynthesis, assembly and expression of human major transplantation antigens

Biosynthesis and regulation of expression of transplantation as detected by a monoclonal antibody to HLA-A,B,C antigens (human leucocytic antigen) and a polyclonal antiserum to beta 2-microglobulin have been investigated using radioactive amino acids and sugars to label human lymphoid cells. We found unbalanced synthesis of HLA heavy chains and beta 2-microglobulin, the latter being in excess and secreted to the extracellular medium. In DAUDI cells, which are defective in beta 2-microglobulin, no HLA-A,B,C could be detected intracellularly even in the presence of added beta 2-microglobulin. Treatment of BRI-8 cells with tunicamycin, an antibiotic which inhibits glycosylation of polypeptides, almost had no effect on the levels of beta 2-microglobulin, while it markedly decreased that of HLA heavy chains, both on the cell surface and intracellularly. Glycosylation of the HLA heavy chains appeared to be an essential requirement for the normal expression of HLA-A,B,C antigens. The translation in vitro in a messenger-dependent reticulocyte system with total polysomes obtained from BRI-8 cells showed that beta 2-microglobulin was synthesized as a precursor. This larger polypeptide was converted into mature beta 2-microglobulin when protein synthesis was performed with microsomes instead of polysomes.     

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.     

ISOLATION OF TWO DISTINCT CLASSES OF POLYSOMES FROM A NUCLEAR FRACTION OF RAT LIVER

Isolated rat liver nuclei were washed with Triton-X-100 in the presence of liver cell sap. This treatment liberated a fraction of polysomes which were isolated by differential centrifugation and were designated "outer membrane polysomes." The outer membrane polysomes synthesized protein in vivo. Shortly after injection of orotic acid-¹⁴C, the RNA of outer membrane polysomes had a higher specific activity than that of cytoplasmic polysomes. It was postulated that outer membrane polysomes may be an intermediate in the transfer of newly synthesized RNA from the nucleus to the cytoplasm. In other experiments, Triton-washed rat liver nuclei were lysed in the presence of deoxycholate and deoxyribonuclease. A ribonucleoprotein fraction was isolated from the lysate by differential centrifugation. This fraction contained "intranuclear ribosomes," which sedimented like partially degraded polysomes in sucrose gradients. This degradation could be partially prevented if intranuclear ribosomes were purified by sedimentation through heavy sucrose. The resulting pellets were termed "intranuclear polysomes" because they contained some undergraded polysomes. Intranuclear polysomes were highly radioactive after a brief pulse with orotic acid-¹⁴C, but did not appear to synthesize protein rapidly in vivo. Intranuclear polysomes may represent the initial stage of assembly of polyribosomes in the nucleus.     

Synthesis of metallothionein in a polysomal cell-free system.

A new covalent chromatography system utilizing Activated Thiol Sepharose 4B was employed to quantitate the content of thionein chains synthesized in a polysomal cell-free system. Liver polysomes from zinc injected rats directed the translation of more thionein-like polypeptide chains than polysomes from control rats. The increase was similar to the stimulation in MT synthesis $\text{in vivo}$ following a zinc injection. This evidence supports the concept that metallothionein synthesis is regulated by changes in the pool of translatable thionein mRNA.     

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.     

Transport of newly synthesized proteins into mitochondria — a review

Summary This review examines the mechanism of translocation of cytoplasmically synthesized proteins into mitochondria. Approximately 10% of the mitochondrial proteins are synthesized within the organelles while most mitochondrial proteins are coded for by nuclear genes and synthesized on cytoplasmic ribosomes. Those mitochondrial proteins synthesized on cytoplasmic ribosomes have to be transferred at some point into one of the mitochondrial compartments, a process which would require their insertion through one or both mitochondrial membranes. Data accumulated during the past five years indicate that the cytoplasmically synthesized mitochondrial proteins are synthesized on free polysomes then released into the cytoplasm. Most of the proteins examined so far are synthesized in the cytoplasm as larger precursors whose conformations may differ from the conformations of their respective mature forms. These precursor proteins become translocated into mitochondrial post-translationally and processed to their mature forms either during or immediately following translocation into the organelles. The translocation step appears to require mitochondrial ATP. Some processing activities have been localized in the matrix fractions of mitochondria from liver and yeast and they appear to be associated with soluble endopeptidases which act selectively on precursors of mitochondrial proteins. Although it is not clear how the precursor proteins interact with or recognize mitochondrial membranes, studies in yeast indicate that the interactions occur at specific regions on the outer mitochondrial membranes.     

Acyl-Coa oxidase and hydratase-dehydrogenase, two enzymes of the peroxisomal beta-oxidation system, are synthesized on free polysomes of clofibrate-treated rat liver

We investigated the site of synthesis of two abundant proteins in clofibrate-induced rat hepatic peroxisomes. RNA was extracted from free and membrane-bound polysomes, heated to improve translational efficiency, and translated in the mRNA-dependent, reticulocyte-lysate- cell-free, protein-synthesizing system. The peroxisomal acyl-CoA oxidase and enoyl-CoA hydratase-beta-hydroxyacyl-CoA dehydrogenase 35S- translation products were isolated immunochemically, analyzed by SDS PAGE and fluorography, and quantitated by densitometric scanning. The RNAs coding for these two peroxisomal proteins were found predominantly on free polysomes, and the translation products co-migrated with the mature proteins. As in normal rat liver, preproalbumin and catalase were synthesized mainly by membrane-bound and by free polysomes, respectively. mRNAs for a number of minor 35S-translation products also retained by the anti-peroxisomal immunoadsorbent were similarly found on free polysomes. These results, together with previous data, allow the generalization that the content proteins of rat liver peroxisomes are synthesized on free polysomes, and the data imply a posttranslational packaging mechanism for these major content proteins.     

Biosynthesis and turnover of carnitine acetyltransferase in rat liver

Male Wistar rats were fed on a diet with and without di(2-ethylhexyl)phthalate (DEHP) for 2 weeks. Carnitine acetyltransferase in the liver was increased about 100-fold by administration of DEHP. The results of in vivo experiments showed that the incorporation of L-[4,5-3H]leucine into the enzyme was 12-fold higher and the half-life of the labeled enzyme was elongated by a factor 4.6. The results of in vitro translation experiments with total hepatic RNA in a rabbit reticulocytelysate system and the results concerning the synthesis of the enzyme in isolated hepatocytes indicate that the translatable mRNA for the enzyme was increased upon administration of DEHP and that the enzyme is synthesized as a precursor (Mw = 69,000) larger than the mature enzyme (Mw = 67,500). RNA in the free polysomes directed the synthesis of the enzyme precursor five times more actively than RNA in membrane-bound polysomes.     

Glycosylation of nascent polypeptide chains in Aspergillus niger

Polysomes were isolated from Aspergillus niger and were characterized on sucrose gradients in several ways. First, they were found to be susceptible to degradation by treatment with RNase or EDTA. Second, they were labeled after treating mycelia with short pulses of [³H]uridine or [³H]leucine prior to polysome isolation. Third, they were capable of stimulating incorporation of [³H]leucine into trichloroacetic acid-precipitable material in a chick reticulocyte cell-free protein-synthesizing system. When isolated [³H]leucine pulse-labeled polysomes were treated with either EDTA-RNase or puromycin, 80–90% of the radioactivity was released, indicating that only the nascent polypeptide chains were labeled. After exposing mycelia for 1 min to [¹⁴C]mannose, the polysomes were exclusively labeled, indicating that initial glycosylation takes place on nascent polypeptide chains. Preincubation of mycelia with 2-deoxyglucose followed by pulse-labeling with [³H]leucine and [¹⁴C]mannose showed that 2-deoxy-d-glucose inhibits both protein synthesis and glycosylation. However, similar preincubation with tunicamycin caused an 80% drop in [¹⁴C]mannose label in the polysomes, but only a 10–20% drop of [³H]leucine label, suggesting that glycosylation of nascent chains in A. niger involves an oligosaccharide-lipid intermediate, since it has been shown that tunicamycin inhibits the synthesis of such an intermediate. When isolated polysomes were placed into an in vitro glycosylating mixture containing Mn²⁺, GDP-[¹⁴C]mannose, and smooth membranes from A. niger nascent chains were labeled. This reaction was shown to be dependent on addition of polysomes to the mixture and was not inhibited by 2-deoxy-d-glucose or tunicamycin. Both in vivo and in vitro glycosylated nascent chains were found to have about the same size range, and so it is suggested that in vitro no new oligosaccharide chains were synthesized, but preexisting chains were extended.     

In vitro synthesis of adrenodoxin and adrenodoxin reductase: Existence of a putative large precursor form of adrenodoxin

Cytoplasmic free and bound polysomes were isolated from bovine adrenal cortex, and used to program $\text{in}\text{vitro}$ protein synthesis in rat liver cell sap and wheat germ lysate systems. Synthesis of adrenodoxin(Ad) and adrenodoxin reductase(AdR) in the cell-free systems was determined by immunoprecipitation using monospecific antibodies, and the sizes of the $\text{in}\text{vitro}$ products were analyzed by SDS-polyacrylamide gel electrophoresis. Ad was synthesized by both free and bound polysomes as a putative large precursor having molecular weight of approximately 20,000 daltons, which was processed to mature size Ad (MW 12,000 daltons) by $\text{in}\text{vitro}$ incubation with adrenal cortex mitochondria. On the other hand, AdR was synthesized only by free polysomes apparently as the mature size product.