In vitro translation and processing of rat kidney gamma-glutamyl transpeptidase

Rat kidney gamma-glutamyl transpeptidase is composed of two nonidentical glycosylated subunits. The enzyme is localized on the lumenal surface of the brush-border membranes of proximal tubule epithelial cells; it is attached to the membranes via an NH2-terminal segment of the larger of the two subunits. Tissue-labeling experiments followed by immunoprecipitation with antibodies directed against the enzyme and its two subunits demonstrate that a glycosylated single chain precursor (Mr = 78,000), containing the elements of both the subunits, is initially synthesized. Pulse-chase studies in the presence of pactamycin, and inhibitor of protein synthesis initiation, indicate that the larger of the two subunits is located at the NH2 terminus of the Mr = 78,000 precursor. The initial events in the biosynthesis and processing of gamma-glutamyl transpeptidase were investigated by in vitro translation of rat kidney mRNA. Such translation results in the synthesis of a Mr = 63,000 unglycosylated polypeptide which has been shown immunologically to contain the domains for both subunits. The Mr = 63,000 species is processed to a Mr = 78,000 core-glycosylated polypeptide when translation of mRNA is carried out in the presence of dog pancreas microsomes. This processing does not appear to be associated with cleavage of an NH2-terminal leader sequence. The Mr = 78,000 polypeptide is integrated into the microsomal membranes with an orientation that is analogous to that found on the brush-border membranes. Glycosylation and membrane integration of transpeptidase are cotranslational events. Upon longer incubation, the Mr = 78,000 species sequestered within the microsomal vesicles is cleaved to species corresponding in size to the two subunits of the kidney enzyme.     

Biosynthesis of the insulin receptor in rat adipose cells. Intracellular processing of the Mr-190 000 pro-receptor.

We investigated the biosynthesis of the insulin receptor in primary cultures of isolated rat adipose cells. Cells were pulse-chase-labelled with [3H]mannose, and at intervals samples were homogenized. Three subcellular membrane fractions were prepared by differential centrifugation: high-density microsomal (endoplasmic-reticulum-enriched), low-density microsomal (Golgi-enriched), and plasma membranes. After detergent solubilization, the insulin receptors were immunoprecipitated with anti-receptor antibodies and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and autoradiography. After a 30 min pulse-label [3H]mannose first appeared in a band of Mr 190 000. More than 80% of the Mr-190 000 component was recovered in the microsomal fractions. Its intensity reached a maximum at 1 h in the high-density microsomal fraction and at 2 h in the low-density microsomal fraction, and thereafter declined rapidly (t 1/2 approx. 3 h) in both fractions. In the plasma-membrane fraction, the radioactivity in the major receptor subunits, of Mr 135 000 (alpha) and 95 000 (beta), rose steadily during the chase and reached a maximum at 6 h. The Mr-190 000 precursor could also be detected in the high-density microsomal fraction by affinity cross-linking to 125I-insulin. In the presence of monensin, a cationic ionophore that interferes with intracellular transport within the Golgi complex, the processing of the Mr-190 000 precursor into the alpha and beta subunits was completely inhibited. Our results suggest that the Mr-190 000 pro-receptor originates in the endoplasmic reticulum and is subsequently transferred to the Golgi complex. Maturation of the pro-receptor does not seem to be necessary for the expression of the insulin-binding site. Processing of the precursor into the mature receptor subunits appears to occur during the transfer of the pro-receptor from the Golgi complex to the plasma membrane.     

Biogenesis, transit, and functional properties of the insulin proreceptor and modified insulin receptors in 3T3-L1 adipocytes. Use of monensin to probe proreceptor cleavage and generate altered receptor subunits

The biogenesis, intracellular transport, and functional properties of the insulin proreceptor and modified insulin receptors were studied in hormone-responsive 3T3-L1 adipocytes. After control cells were labeled with [35S]Met for 7 min, the principal polypeptide that was precipitated by anti-insulin receptor antibodies had a molecular weight (Mr) of 180 000. This initial precursor was rapidly converted (t1/2 = 35 min) to a 200-kilodalton (kDa) polypeptide, designated the insulin proreceptor, by the apparent posttranslational addition of N-linked, high mannose core oligosaccharide units. Mature alpha (Mr 130 000) and beta (Mr 90 000) subunits were derived from sequences within the proreceptor by proteolytic cleavage and late processing steps, and these subunits appeared on the cell surface 2-3 h after synthesis of the 180-kDa precursor. The cation ionophore monensin was used in combination with metabolic labeling, affinity cross-linking, and external proteolysis to probe aspects of proreceptor function, transit, and the development of insulin sensitivity at the target cell surface. At 5 micrograms/mL, monensin potently inhibited the proteolytic cleavage step, and the 200-kDa polypeptide accumulated. Lower concentrations of the ionophore selectively blocked late processing steps in 3T3-L1 adipocytes so that apparently smaller alpha' (Mr 120 000) and beta' (Mr 85 000) subunits were produced. Proreceptor and alpha' and beta' subunits were translocated to the cell surface, indicating that the signal for intracellular transit occurs in the 200-kDa polypeptide and is independent of the posttranslational proteolysis and late processing steps. The alpha' subunit bound insulin both at the surface of intact cells and after solubilization with Triton X-100; the beta' subunit was phosphorylated in an insulin-stimulated manner. The detergent-solubilized 200-kDa proreceptor also exhibited both functional properties. However, the proreceptor that was transported to and exposed on the cell surface was incapable of binding insulin in intact adipocytes. Thus, late processing is not essential for the expression of functions associated with mature alpha and beta subunits. In contrast, it appears that the proteolytic generation of subunits is required for the correct orientation of the hormone binding site in the plasma membrane bilayer and the development of insulin responsiveness in 3T3-L1 adipocytes.     

Ricin and Ricinus communis agglutinin subunits are all derived from a single-size polypeptide precursor

Antibodies have been raised in rabbits against the individually purified A and B subunits of the toxic castor bean lectin, ricin, and against the A' and B' subunits of Ricinus communis agglutinin type I. Each of the antisera recognised a single polypeptide species of Mr 60 500 when maturing castor bean endosperm mRNA was translated in vitro in a rabbit-reticulocyte-derived system. When dog pancreatic microsomal vesicles were included in the translational system, each subunit antiserum precipitated a group of 66 000-68 000-Mr core-glycosylated polypeptides which had been translocated into the lumen of the vesicles. The 60 500-Mr polypeptide appeared to be a common precursor to all four individual lectin subunits since (a) its glycosylated (66 000-68 000-Mr) forms were readily detected in the endoplasmic reticulum fraction isolated from maturing castor bean endosperm and (b) pulse-chase studies showed that the glycosylated precursors disappeared from the endoplasmic reticulum fraction with the concomittant appearance of authentic lectin subunits in a soluble protein fraction which included protein body matrix components. Antiserum prepared against whole R. communis agglutinin, type I, also precipitated the 65 000-Mr precursor in vitro and in vivo, but in addition precipitated a non-glycosylated 34 000-Mr polypeptide. This smaller protein is not a lectin subunit precursor, contradicting an earlier suggestion. It is most probably a precursor to the 2-S albumin storage proteins found in castor bean endosperm protein bodies.     

Mosquito vitellogenin subunits originate from a common precursor

Using a cell-free translation system, we demonstrated that the two subunits of mosquito vitellogenin (VG), 200 kDa and 65 kDa, originate from a common precursor. The precursor polypeptide of 220 kDa is a translation product specific to mRNA from vitellogenic mosquitoes. In immunoprecipitation analysis, the 220-kDa polypeptide was recognized by monoclonal antibodies directed either to the large or the small VG subunit. Peptide mapping showed homology between the 220-kDa polypeptide and both subunits, thus providing further proof that the 220-kDa product of translation is the precursor for both VG subunits. In the presence of microsomal membranes, the molecular size of the VG precursor increased to 235 kDa suggesting this as a first step in co-translational modifications of VG.     

Vitellogenesis in the hematophagous Dipetalogaster maxima (Hemiptera: Reduviidae), a vector of Chagas' disease

Oocyte extracts of anautogenous Dipetalogaster maxima were chromatographed on an ion-exchange column in order to purify vitellin (Vt), the main insect yolk protein precursor. Purified Vt (Mr ~443 kDa) was composed of four subunits with approximate molecular weights of 174, 170, 50, and 44 kDa. Polyclonal anti-Vt antibody, which cross-reacted equally with fat body extracts and hemolymph vitellogenin (Vg), was used to measure the kinetics of Vg expression in the fat body and the levels in hemolymph. In addition, morphological and immunohistochemical changes that took place in the ovary during vitellogenesis were analyzed. The study was performed between 2 and 8 days post-ecdysis and between 2 and 25 days post-blood feeding. During the post-ecdysis period, D. maxima showed decreased synthesis of Vg and concomitantly, low levels of Vg in hemolymph (4.5 x 10(-3) microg/microl at day 4). After a blood meal, Vg synthesis in the fat body and its levels in hemolymph increased significantly, reaching an average of 19.5 microg/microl at day 20. The biochemical changes observed in the fat body and hemolymph were consistent with the histological and immunohistochemical finds. These studies showed noticeable remodeling of tissue after blood feeding.     

Identification of the primary translation product of atrial natriuretic peptide receptor mRNA in a cell-free system using anti-receptor antiserum

The primary translation product of mRNA encoding atrial natriuretic peptide (ANP) receptor has been shown to have an Mr of 58,000. Poly(A)+ RNA was isolated from the bovine kidney and lung and translated in a rabbit reticulocyte lysate system containing [35S]methionine. Immunoprecipitation of the labeled translation products, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, identified a 58-kDa protein as the primary translation product which is the unglycosylated precursor to be processed to the glycosylated mature 70-kDa form found in the plasma membranes. The result lends strong support to our previous proposal that mature ANP receptor is composed of two disulfide-linked 70-kDa subunits, eliminating the possibility that the two 70-kDa subunits arise from a larger 140-kDa precursor by proteolytic cleavage.     

A potential mucus precursor in Tetrahymena wild type and mutant cells.

By using an antibody to a specific mucus polypeptide (34 kDa) to study whole cell extracts of both a secretory mutant (SB281) and wild type (wt) Tetrahymena, we demonstrate that a 57-kDa polypeptide is a probable precursor to the 34-kDa secretory polypeptide. We postulate that the precursor accumulates in the mutant cells because it cannot be cleaved. This mutant contains no recognizable mature secretory granules (mucocysts). By immunoelectron microscopy, the 34-kDa polypeptide was localized in wt cells specifically to the mature mucocysts and to their released products. Localization in mutant cells occurred in two different types of cytoplasmic vesicles: small electron dense vesicles (0.3-0.5 microns in diameter) and large electron lucent vacuoles (1.2-3.5 microns in diameter). Immunoblot analyses of homogenates of mutant and wt cells with the anti-34-kDa serum revealed a dominant band in the mutant at Mr 57 kDa whereas the wt showed a dominant band only at Mr 34 kDa. Furthermore, the 57-kDa polypeptide is immunoprecipitated with anti-34-kDa serum from the mutant cell. Further evidence for a precursor relation of the 57-kDa polypeptide in mutant cells to the 34-kDa mucus polypeptide of wt cells was obtained by the use of drugs (monensin, chloroquine, NH4Cl) that block secretory product processing in wt cells. Extracts of drug-treated wt cells showed the presence of a 57-kDa cross reacting band even after 18 h of incubation in growth medium whereas untreated control cells contained the 34-kDa mature protein almost exclusively. These results indicate that processing of the precursor to the 34-kDa polypeptide occurs in an acidic compartment(s) possibly in either the trans Golgi network, or condensing vacuoles or both.     

The role of the fat body, midgut and ovary in vitellogenin production and vitellogenesis in the female tick, Dermacentor variabilis.

Polyclonal antibodies directed against D. variabilis vitellin were utilized for immunocytochemistry at the ultrastructural level. We localized vitellogenin (Vg) in rough endoplasmic reticulum cisternae, secretory granules and secreted products of fat body trophocytes and midgut vitellogenic cells from feeding and ovipositing females. Vg was localized in the oocyte Golgi bodies and in the yolk bodies of both feeding and ovipositing females. Uptake of exogenous Vg was indicated by the presence of immunospecific gold probe in coated pits and coated vesicles at the apical plasma membrane of oocytes from females in rapid engorgement and oviposition. In unmated females little detectable evidence of Vg uptake by developing oocytes suggests that mating and host detachment signal the beginning of vitellogenesis. We conclude that fat body trophocytes, midgut vitellogenic cells and oocytes are involved in the synthesis and/or processing of Vg and that feeding is the signal associated with the initiation of Vg synthesis and/or processing.     

A Nicotiana tabacum mRNA encoding a 69-kDa glycoprotein occurring abundantly in pollen tubes is transcribed but not translated during pollen development in the anthers

Regulation of expression of a 69-kDa glycoprotein which occurs abundantly in tobacco (Nicotiana tabacum L.) pollen tubes but is absent in ungerminated pollen has been studied in vitro by means of a coupled translation/glycosylation system with RNA isolated from various stages of pollen development. Pollen mRNA could be translated in a rabbit reticulocyte lysate and the products glycosylated with canine pancreatic microsomal membranes. The electrophoretic pattern of translation products obtained with pollen-tube RNA showed a prominent polypeptide with an apparent molecular mass of 58 kDa. In the presence of the canine pancreatic microsomal membranes this polypeptide was glycosylated, producing the 69-kDa glycoprotein. The presence of mRNA encoding the 58-kDa precursor polypeptide was also demonstrated in ungerminated pollen and in young mid-binucleate pollen isolated from anthers. Initiation of synthesis of the 69-kDa glycoprotein at the onset of pollen germination thus occurs through unmasking of the mRNA transcribed during pollen differentiation and stored during pollen maturation and dormancy in an inactive state.Abbreviation pI isoelectric point     

The human receptor for T-cell growth factor. Evidence for variable post-translational processing, phosphorylation, sulfation, and the ability of precursor forms of the receptor to bind T-cell growth factor

The T-cell growth factor (TCGF) receptor on phytohemagglutinin-activated normal peripheral blood T-cells is characterized as a glycoprotein with an apparent Mr = 55,000 that contains N-linked and O-linked carbohydrate with only approximately 33,000 daltons of peptide structure (p33) as evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There are two N-linked glycosylated intermediate precursor forms (apparent Mr = 35,000 (p35) and 37,000 (p37]. This receptor differs from the TCGF receptor on HUT-102B2 cells (apparent Mr = 50,000) because of differences in post-translational processing. Experiments with the carboxylic ionophore monensin demonstrate blockade of the transition of the p35 and p37 receptor precursor forms to the mature receptor, presumably secondary to inhibition of Golgi-associated receptor processing. We identify the primary translation product of TCGF receptor mRNA as intermediate in size between the p33 and the p35/p37 forms. We further demonstrate that the p33, p35, and p37 precursor forms, but not the primary translation product, are all capable of binding TCGF. Thus, the removal of the signal peptide and/or conformational changes of the primary translation product are necessary for ligand binding; however, the extensive post-translational modifications are not. Lastly, we demonstrate that at least some TCGF receptors are phosphorylated and sulfated, and that TCGF receptors on phytohemagglutinin-activated normal T-cells are more heavily sulfated than those on HUT-102B2 cells.     

A novel protein produced by the vitellogenic fat body and accumulated in mosquito oocytes

Summary The fat body of vitellogenic mosquitoes was found to synthesize and secrete another protein in addition to vitellogenin, that accumulated in developing oocytes. In the tissues, this protein has M_r = 53000 on SDS-PAGE under reducing or non-reducing conditions. This protein is glycosylated as shown by [³H]mannose incorporation and experiments with tunicamycin. Polyclonal antibodies were produced using the ovarian 53-kDa peptide. Immunoblot analysis demonstrated the immunological identity of 53 kDa peptides from the fat body and the ovary. Furthermore, the 53-kDa protein (53KP) is synthesized and secreted exclusively by the vitellogenic fat body. Radioimmunoassay showed that 53KP is produced by the female fat body as early as 4 h and reaches its peak near 24 h after the initiation of vitellogenesis. Synthesis then drops to low levels by 36 h and declines to background levels by 48 h. In vitro experiments conducted on fat bodies of previtellogenic females demonstrated that the synthesis and secretion of 53KP can be stimulated by a physiological dose of 20-hydroxyecdysone (10^–6 M). Immunocytochemical studies of the ovary demonstrate that 53KP is present in channels between follicle cells, in the perioocytic space and in yolk granules of the developing oocytes. This suggests that 53KP is accumulated in the oocytes by a pathway similar to that of vitellogenin.     

Storage proteins of the fall webworm,Hyphantria cunea drury

Two storage proteins, storage protein-1 (SP1) and storage protein-2 (SP2), were found in hemolymph and fat body during the development of Hyphantria cunea, the fall webworm. Both storage proteins show similiar quantitative changes during development in males and females; however, SP1 is more abundant. The hemolymph of last instar larvae contains high concentrations of the storage proteins. However, following pupation, the storage proteins accumulate in fat bodies. SP1 peaks in the hemolymph of males and females late in last instar larvae (8-day-old 7th instar larvae). SP1 has a native molecular weight of 460,000 and consists of six identical subunits (Mr = 76,700), while SP2 has a molecular weight of 450,000 and is composed of two different subunits (Mr = 74,100 and 72,400). Both SP1 and SP2 are hexamers and are phosphorylated glycolipoproteins. The pl values of SP1 and SP2 were determined to be 5.70 and 5.50, respectively. Antibodies raised against SP1 react positively with vitellogenin and ovary extract, as well as with proteins in the hemolymph from last instar larvae and proteins in pupal fat bodies. Storage protein synthesis starts in fat bodies of a 4-day-old 7th instar larvae and in female peaks at 6–8 days of the 7th instar.     

The Processing of a 57-kDa Precursor Peptide to Subunits of Rice Glutelin

The processing of a 57-kDa peptide into 37- and 22-kDa subunitsof glutelin, a major storage protein of rice, was confirmedby the immunological compatibility between the precursor andglutelin subunits. The 57-kDa peptide reacted with the antiseraraised against purified 37-kDa and 22-kDa subunits of glutelin.The processing was further confirmed by alteration of an invivo protein synthesis by monensin, a sodium ionophore whichinhibits the intracellular transport of secretory and membraneproteins. Infusion of monensin into developing rice grains resultedin suppressed formation of mature glutelin subunits with concomitantaccumulation of the 57-kDa peptide. The present results indicatethat both subunits of rice glutelin were produced by post-translationalcleavage of the 57-kDa peptide. (Received July 9, 1986; Accepted October 1, 1986)     

Evidence for the involvement of an acidic compartment in the processing of myeloperoxidase in human promyelocytic leukemia HL-60 cells

The observation that myeloperoxidase precursor and larger intermediate (Mr 91,000 and 81,000, respectively) were extracted in the presence of detergent from isolated granule fractions of human promyelocytic leukemia HL-60 cells under mildly acidic conditions was investigated. In contrast, under conditions of neutral pH, only the Mr 74,000 intermediate and mature species were extracted. Extraction of the Mr 91,000 and 81,000 forms was also enhanced in the presence of EDTA. Kinetic studies of the processing of the different myeloperoxidase species confirmed the intermediate nature of the Mr 81,000 and 74,000 forms. Support for a role of an acidic intracellular compartment was obtained through evidence that the acid-extractable precursor and intermediates accumulated in HL-60 cells which had been treated with 1 microM monensin. Under these conditions, the production of mature heavy (Mr 63,000) and light (Mr 13,500) subunits of myeloperoxidase was consistently inhibited by greater than 40% over a 16-h period. The effects of monensin on processing of myeloperoxidase were completely reversed if monensin was removed during this 16-h period. These data support the idea that an acidic compartment may be involved in the transport of myeloperoxidase precursors to azurophil granules and/or their processing to a smaller intermediate form (Mr 74,000) of the enzyme.     

Cyclic AMP-dependent phosphorylation and regulation of the cardiac dihydropyridine-sensitive Ca channel.

A polyclonal antibody, CR2, prepared using the C-terminal peptide of the alpha 1 subunit of the rabbit cardiac DHP-sensitive Ca channel, specifically immunoprecipitated the [3H]PN200-110-labeled Ca channel solubilized from cardiac microsomes. The antibody recognized 250 and 200-kDa cardiac microsomal proteins as determined by immunoblotting, and cAMP-dependent protein kinase phosphorylated the 250-kDa, but not the 200-kDa protein in vitro. CHO cells, transfected with the cardiac alpha 1 subunit cDNA carried by an expression vector, synthesized a 250-kDa protein which was recognized by CR2. Adding db-cAMP or forskolin to the transformed CHO cells induced phosphorylation of the 250-kDa protein and stimulated the DHP-sensitive Ba current under patch-clamp conditions. These results suggested that the cardiac DHP-sensitive Ca channel was regulated by cAMP-dependent phosphorylation of the alpha 1 subunit.     

Glycosylation and membrane insertion of newly synthesized rat dopamine beta-hydroxylase in a cell-free system without signal cleavage.

Dopamine beta-hydroxylase (DBH, EC 1.14.17.1) is present in both membrane-bound and soluble forms in neurosecretory vesicles. This study was designed to investigate the differences between membrane-bound and soluble DBH and how they may arise from translation of a single mRNA. Antisera to a peptide corresponding to the carboxyl terminus of rat DBH was found to specifically immunoprecipitate the 77- and 73-kDa subunits of newly synthesized DBH in rat brain. Thus, both soluble and membrane-bound forms contain the same carboxyl terminus. To investigate differences at the amino terminus, full-length rat DBH mRNA, translated in a cell-free system, produced a 66-kDa peptide. An additional higher molecular mass product was synthesized upon co-translational addition of microsomal membranes. This product was glycosylated since it bound to concanavalin A-Sepharose and reverted to the 66-kDa polypeptide after treatment with endoglycosidase H. This glycosylated product was resistant to protease digestion and fractionated with microsomal membranes on sucrose gradients, indicating that it is incorporated into the microsomal membranes. Amino-terminal sequencing of the glycosylated translation product indicated that the amino-terminal "signal" sequence was not cleaved. The results indicate that in the cell-free system newly synthesized DBH undergoes glycosylation and incorporation into microsomal membranes without cleavage of the NH2-terminal signal sequence.