Effect of tunicamycin on epidermal glycoprotein and glycosaminoglycan synthesis in vitro

1. When pig ear skin slices were cultured for 18h in the presence of 1μg of tunicamycin/ml the incorporation of d-[³H]glucosamine into the epidermis, solubilized with 8m-urea/5% (w/v) sodium dodecyl sulphate, was inhibited by 45–55%. This degree of inhibition was not increased by using up to 5μg of tunicamycin/ml or by treating the skin slices with tunicamycin for up to 8 days. The incorporation of (U-¹⁴C)-labelled l-amino acids under these conditions was not affected by tunicamycin. Polyacrylamide-gel electrophoresis indicated that the labelling of the major glycosaminoglycan peak with d-[³H]glucosamine was unaffected, whereas that of the faster migrating glycoprotein components was considerably decreased in the presence of tunicamycin. 2. Subcellular fractionation indicated that tunicamycin specifically inhibited the incorporation of d-[³H]glucosamine but not of (U-¹⁴C)-labelled l-amino acids into particulate (mainly plasma-membrane) glycoproteins by about 70%. The labelling of soluble glycoproteins was hardly affected. Polyacrylamide-gel electrophoresis of the plasma-membrane fraction showed decreased d-[³H]glucosamine incorporation into all glycoprotein components, indicating that the plasma-membrane glycoproteins contained mainly N-asparagine-linked oligosaccharides. 3. Cellulose acetate electrophoresis of both cellular and extracellular glycosaminoglycans showed that tunicamycin had no significant effect on the synthesis of the major component, hyaluronic acid. However, the incorporation of both d-[³H]glucosamine and ³⁵SO₄²⁻ into sulphated glycosaminoglycans was inhibited by about 50%. This inhibition was partially overcome, at least in the cellular fraction, by 2mm-p-nitrophenyl β-d-xyloside indicating that tunicamycin-treated epidermis retained the ability to synthesize sulphated glycosaminoglycan chains. Tunicamycin may affect the synthesis and/or degradation of proteoglycan core proteins or the xylosyltransferase. 4. Electron-microscopic examination of epidermis treated with tunicamycin for up to 4 days revealed no significant changes in cell-surface morphology or in epidermal-cell adhesion. Either N-asparagine-linked carbohydrates play little role in epidermal-cell adhesion or more probably there is little turnover of these components in epidermal adhesive structures such as desmosomes and hemidesmosomes during organ culture.     

Effect of tunicamycin on the glycosylation of rhodopsin

The incorporation of [³H]glucosamine, [³H]mannose, and [³⁵S]methionine into rhodopsin was investigated in retinas which had been incubated in the presence and absence of the antibiotic, tunicamycin. In its presence, the incorporation of glucosamine was inhibited 70% and mannose, 96% compared to controls. In the presence of tunicamycin the attachment of glucosamine to core-region sites was virtually eliminated. The formation of unglycosylated rhodopsin was also indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and concanavalin A-Sepharose chromatography. These findings are consistent with the participation of the lipid-linked pathway in the glycosylation of this well-characterized intrinsic glycoprotein of the membranes of the disk of the rod outer segment. As indicated by the incorporation of [³⁵S]methionine, the synthesis of rhodopsin apoprotein was inhibited by a much lesser amount. This suggests that the glycosylation of rhodopsin is not required for its insertion into the disk membrane.     

Seed reserve-protein glycosylation in an in vitro preparation from developing cotyledons of Phaseolus vulgaris

A particulate preparation from developing cotyledons of Phaseolus vulgaris L. was incubated with uridine-5-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc; [6-³H]glucosamine), and by polyacrylamide gel electrophoretic analysis it was shown that the labeled (N-acetyl)glucosamine (GlcNAc) was incorporated into the principal reserve protein of the cotyledons, vicilin, and also into phytohemagglutinin. Some of the labeled product also reacted with antiserum to vicilin from mature seeds. In contrast it was not possible to detect the incorporation of labeled mannose from guanosine-5-diphospho-D-mannose (GDP-mannose; [U-¹⁴C]mannose) into either of these proteins by gel-electrophoretic analysis of the mannose-labeled products, but we did observe a low incorporation of mannose into material which reacted with antiserum to vicillin. The predominant glycosylation reaction in vitro was therefore probably a transfer of GlcNAc alone, rather than in combination with mannose as preformed oligosaccharide.Abbreviations GlcNAc N-acetyl-D-glucosamine - GDP guanosine 5-diphospho - IEF isoelectric focusing - PHA phytohemagglutinin - SDS sodium dodecylsulfate - UDP uridine-5-diphospho     

Effect of Inhibition of Glycosylation on the Appearance of a 60 kD Membrane Glycopolypeptide in Endomembrane Fractions of Soybean Root

Endomembrane (endoplasmic reticulum, Golgi apparatus, plasma membrane) proteins of soybean (Glycine max) root cells are highly glycosylated. We investigated whether N-linked oligosaccharide moieties are essential for the correct intracellular transport of plant endomembrane glycoproteins. Excised roots were incubated with tunicamycin, to block cotranslational glycosylation of proteins, and dual labeled with [³H]glucosamine and [³⁵S] (methionine, cysteine). In the presence of tunicamycin, the incorporation of glucosamine into membrane proteins was inhibited by 60 to 90% while amino acid incorporation was only slightly affected. Autoradiograms of two-dimensionally separated polypeptides from each endomembrane fraction revealed the presence of at least one new polypeptide in tunicamycin-treated tissue. The new polypeptide was of the same isoelectric point but lower molecular weight than a preexisting polypeptide. The new polypeptide was unreactive to concanavalin A, as opposed to the preexisting polypeptide, suggesting the absence of the glycan portion. Trifluoromethanesulfonic acid and N-glycanase were used to cleave the carbohydrate from the preexisting concanavalin A binding polypeptide. In each case a deglycosylated polypeptide of the same isoelectric point and molecular weight as the new polypeptide from tunicamycin-treated tissue resulted. Since the absence of carbohydrate from the new endomembrane polypeptide did not prevent its appearance on autoradiograms of Golgi and plasma membrane, intracellular transport and intercalation of newly synthesized glycoproteins into plant cell membranes may not require the presence of polysaccharide moieties.     

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.     

Inhibitory effects of tunicamycin on procollagen biosynthesis and secretion

Chick embryo cells were briefly exposed to the antibiotic, tunicamycin. Pre-exposed cells, compared to control cultures, showed a severe, progressive inhibition of the incorporation of glucosamine and mannose into total cellular macromolecules. Inhibition of the incorporation of glycine, leucine and proline was also progressive but not as marked as for the carbohydrates. Cellular secretion of all macromolecules was severely impaired. while comparison of the procollagens showed no difference in their subunit size or in their degree of glycosylation; the intracellular content of procollagen polypeptides was similar for both types of cells. In vitro studies showed that tunicamycin selectively inhibited glucosamine, but not mannose, incorporation into macromolecules. The composite results indicate that tunicamycin effectively inhibits protein synthesis, protein glycosylation and protein secretion in chick embryo cells.     

Inhibitory effects of tunacamycin on procollagen biosynthesis and secretion

Chick embryo cells were briefly exposed to the antibiotic, tunicamycin. Pre-exposed cells, compared to control cultures, showed a severe, progressive inhibition of the incorporation of glucosamine and mannose into total cellular macromolecules. Inhibition of the incorporation of glycine, leucine and proline was also progressive but not as marked as for the carbohydrates. Cellular secretion of all macromolecules was severely impaired, while comparison of the procollagens showed no difference in their subunit size or in their degree of glycosylation; the intracellular content of procollagen polypeptides was similar for both types of cells. In vitro studies showed that tunicamycin selectively inhibited glucosamine, but not mannose, incorporation into macromolecules. The composite results indicate that tunicamycin effectively inhibits protein synthesis, protein glycosylation and protein secretion in chick embryo cells.     

Role of the endoplasmic reticulum in the synthesis of reserve proteins and the kinetics of their transport to protein bodies in developing pea cotyledons

Developing pea (Pisum sativum L.) cotyledons were labeled with radioactive amino acids, glucosamine, and mannose in pulse an pulse- chase experiments to study the synthesis, glycosylation, and transport of the reserve proteins vicilin and legumin to the protein bodies. Tissue extracts were fractionated on sucrose gradients to isolate either the endoplasmic reticulum (ER) or the protein bodies. Immunoaffinity gels were used to determine radioactivity in the reserve proteins (legumin and vicilin). After pulse-labeling for 45 min with amino acids, about half the total incorporated radioactivity coincided closely with the position of the ER marker enzyme NADH-cytochrome c reductase at a density of 1.13 g . cm-3 on the sucrose gradient. Both radioactivity and enzyme activity shifted to a density of 1.18 g . cm-3 in the presence of 3 mM MgCl2 indicating that the radioactive proteins were associated with the rough ER. Approximately half of the incorporated radioactivity associated with the rough ER was in newly synthesized reserve protein and this accounted for 80% of the reserve protein synthesized in 45 min. Trypsin digestion experiments indicated that these proteins were sequestered within the ER. In pulse-chase experiments, the reserve proteins in the ER became radioactive without appreciable lag and radioactivity chased out of the ER with a half-life of 90 min. Radioactive reserve proteins became associated with a protein body-rich fraction 20-30 min after their synthesis and sequestration by the ER. Pulse-chase experiments with radioactive glucosamine and mannose in the presence and absence of tunicamycin indicated that glycosylation of vicilin occurs in the ER. However, glycosylation is not a prerequisite for transport of vicilin from ER to protein bodies. Examination of the reserve protein polypeptides by SDS PAGE followed by fluorography showed that isolated ER contained legumin precursors (Mr 60,000-65,000) but not the polypeptides present in mature legumin (Mr 40,000 and 19,000) as well as the higher molecular weight polypeptides of vicilin (Mr 75,000, 70,000, 50,000, and 49,000). The smaller polypeptides of vicilin present in vicilin extracted from protein bodies (Mr 12,000-34,000) were absent from the ER. The results show that newly synthesized reserve proteins are preferentially and transiently sequestered within the ER before they move to the protein bodies, and that the ER is the site of storage protein glycosylation.     

Cloning and sequencing of a cDNA encoding the major storage proteins of Theobroma cacao

The major storage proteins, polypeptides of 31 and 47 kilodaltons (kDa), from the seeds of cocoa (Theobroma cacao L.), have been identified and partially purified by preparative gel electrophoresis. The polypeptides were both N-terminally blocked, but some N-terminal amino-acid sequence was obtained from a cyanogen bromide peptide common to both polypeptides, permitting the construction of an oligonucleotide probe. This probe was used to isolate the corresponding copy-DNA (cDNA) clone from a library made from poly(A)⁺ RNA from immature cocoa beans. The cDNA sequence has a single major open reading frame, that translates to give a 566-amino-acid polypeptide of M_r 65 612. The existence of a common precursor to the 31- and 47-kDa polypeptides of this size was confirmed by immunoprecipitation from total poly(A)⁺RNA translation products. The precursor has an N-terminal hydrophobic sequence which appears to be a typical signal sequence, with a predicted site of cleavage 20 amino acids after the start. This is followed by a very hydrophilic domain of 110 amino acids, which, by analogy with the cottonseed -globulin, is presumed to be cleaved off to leave a domain of approx. 47 kDa, very close to the observed size of the mature polypeptide. Like the hydrophilic domain of the cottonseed -globulin the cocoa hydrophilic domain is very rich in glutamine and charged residues (especially glutamate), and contains several Cys-X-X-X-Cys motifs. The cyanogen-bromide peptide common to the 47-kDa and 31-kDa polypeptides is very close to the proposed start of the mature domain, indicating that the 31-kDa polypeptide arises via further C-terminal processing. The polypeptide sequence is homologous to sequences of the vicilin class of storage proteins, previously found only in legumes and cotton. Most of these proteins have a mature polypeptide size of approx. 47 kDa, and are synthesised as precursors only slightly larger than this. Some, however, are larger polypeptides (e.g. -conglycinin from soybean is 72 kDa), usually due to an additional N-terminal domain. In cottonseed the situation appears to parallel that in cocoa in that the vicilin is synthesised as an approx. 70-kDa precursor and then processed to a 47-kDa (and in the case of cocoa also a 31-kDa) mature protein. In this context it is interesting that cotton is closer in evolutionary terms to cocoa than are the legumes, both cotton and cocoa being in the order Malvales.Abbreviations A absorbance - cDNA copy DNA - IgG immunoglobulin G - kb kilobase pairs - kDa kilodaltons - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulphate-polyacylamide gel electrophoresis The authors are very grateful to Dr R. Jennings of the Virology Department, Sheffield University Medical School, for help in raising antibodies.     

Fucosylation of membrane proteins in soybean cultured cells : effects of tunicamycin and swainsonine

Cultures of soybean cells incorporate [5,6-³H]-l-fucose into various cellular components including lipids and proteins. The membrane glyco-proteins were digested with pronase to produce glycopeptides, and the glycopeptides were isolated on columns of Biogel P-4. The major fucoselabeled glycopeptide sized as a Hexose_15-17-N-acetylglucosamine₂ (GlcNAc₂) on columns of Biogel P-4. Fucose incorporation was also examined in the presence of the processing inhibitor swainsonine, and the glycosylation inhibitor tunicamycin. In the presence of swainsonine, the incorporation of fucose was not reduced but the glycopeptides were smaller in size and migrated like Hexose_12-13-GlcNAc₂ structures. On the other hand, tunicamycin inhibited the incorporation of fucose into the glycopeptides by 70 to 80%, indicating that the l-fucose was present in N-linked oligosaccharides.     

Protein synthesis in tobacco pollen tubes: preferential synthesis of cell-wall 69-kDa and 66-kDa glycoproteins

One- and two-dimensional electrophoresis of Nicotiana tabacum pollen and pollen tube proteins confirmed that a new protein is preferentially synthesized during pollen germination and tube growth and becomes the most abundant protein in pollen tubes. Analysis of proteins extracted with sodium dodecyl sulfate (SDS) from different pollen tube fractions showed that it is the most abundant non-covalently bound wall protein, characterized by molecular mass of 69 kDa, pI between 7.9 and 8.2, and glycosylation with glucose and/or mannose. Amino acid analysis revealed relative abundance of serine, glutamic acid and glycine, but did not show the presence of hydroxyproline. According to all these characteristics, it cannot be classified as an extensin-like protein. Another prominent wall-bound glycoprotein has a molecular mass of 66 kDa and the same pI as the 69 kDa glycoprotein. These two glycoproteins are similar also in ConA binding, rate of synthesis, and rapid incorporation into pollen tube walls. Their synthesis is strongly reduced by tunicamycin and this inhibition results in the occurrence of new polypeptides in the range of 57–61 kDa. Tunicamycin also inhibited pollen tube growth. At 10 ng ml^-1 and 50 ng ml^-1 the inhibitor reduced pollen tube mass after 24 h of culture by 30% and 85%, respectively. This indicates that tobacco pollen presents a system highly sensitive to tunicamycin and that cotranslational N-linked glycosylation on the rough endoplasmic reticulum is required for 66 and 69 kDa glycoprotein formation and for pollen tube growth. Although other proteins appear during pollen germination and tube growth, the new proteins occur at low levels and seem to originate through modifications of preexisting polypeptides. In contrast to 69 and 66 kDa proteins, most proteins detected by [¹⁴C]amino acid incorporation and fluorography of gels were not revealed by Coomassie blue staining.     

What is pea legumin — Is it glycosylated?

Since there is some question as to whether or not legumin is glycosylated, this storage protein was isolated by various procedures from developing cotyledons of Pisum sativum L. supplied with [¹⁴C]-labeled glucosamine and analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Legumin isolated by the classical method of Danielsson [(1949) Biochem. J. 44, 387–400] a procedure in which globulins extracted with a buffered salt solution are precipitated with ammonium sulfate (70% saturation) and legumin separated from vicilin by isoelectric precipitation, was labeled. The glucosamine incorporated into legumin was associated with low-molecular-weight polypeptides. In contrast, legumin isolated by the method of Casey [(1979) Biochem. J. 177, 509–520], a procedure where legumin is prepared by zonal isoelectric precipitation from globulins precipitated with 40–70% ammonium sulfate, was not labeled. However, the globulin fraction precipitated with 40% ammonium sulfate was labeled and the radioactive glucosamine was associated with low-molecular-weight polypeptides. Legumin isolated from protein bodies [Thomson et al. (1978) Aust. J. Plant Physiol. 5, 263–279] was not extensively labeled. However, the saltinsoluble fraction of protein body extracts was labeled and the radioactivity was associated with low-molecular-weight polypeptides. These results indicate that protein bodies contain a glycoprotein of low-molecular-weight that co-purifies with legumin isolated by the method of Danielsson but that is discarded when isolation methods developed more recently are used.     

Glycoprotein Metabolism in the Cotyledons of Pisum sativum during Development and Germination

The glycoprotein nature of legumin and vicilin, the reserve globulins in the cotyledons of Pisum sativum was studied. Legumin from mature seed was found to contain 1% neutral sugars (mannose and glucose) and 0.1% amino sugar (glucosamine), whereas vicilin contained 0.3% neutral sugar (mannose) and 0.2% amino sugar (glucosamine). On the basis of the incorporation of ¹⁴C-labeled glucosamine, it appeared that not all of the component subunits of the reserve proteins are glycosylated to the same extent. In addition, it has been established that glycosylation occurs after peptide synthesis. During seed development there was a change in neutral sugars and amino sugar ratio in vicilin. During germination, the neutral sugars and the amino sugar content of the glycoproteins declined. These findings are discussed in relation to the synthesis and degradation of the glycosyl component of the glycoproteins.     

Synthesis and role of cell surface glycoproteins in preimplantation mouse development

Cell surface glycoproteins apparently influence cell interactions, morphogenesis and the course of cellular differentiation. We have therefore investigated the biosynthesis of glycoproteins in 8-cell mouse embryos by using 1.0 μg tunicamycin/ml, a specific inhibitor of glycosylation of N-glycosidically linked glycoproteins. The antibiotic had little effect on the incorporation of leucine into polypeptides, but the incorporation of glucosamine and mannose was inhibited by about 60% with a marked reduction in their incorporation into the majority of the glycopeptides as analysed on polyacrylamide gels. The binding of concanavalin A (conA) and peanut lectin to the embryonic cell surface was also markedly diminished by tunicamycin. However, the binding of peanut lectin to isolated blastomeres displayed a polar distribution with predominant binding to the outer apical surface in all cases, despite a marked reduction in microvilli. Hence tunicamycin has no substantial effect on the molecular distribution of at least some cell surface antigens. Analysis of iodinated cell surface components showed that two components of mol, wt <68 000 and >165 000 were inhibited by tunicamycin. Whereas embryos in the control group underwent compaction and blastulation, those in the experimental group remained uncompacted, although cleavage continued to about the 32-cell stage. However, some embryos initially underwent partial compaction but later decompacted in the presence of tunicamycin; numerous adherens and possibly a few gap junctions were also detected between blastomeres. We suggest that a number of cell surface antigens including N-glycosidically linked glycoproteins may be engaged sequentially during compaction.     

Elicitor induction of the synthesis of a novel lectin-like arabinosylated hydroxyproline-rich glycoprotein in suspension cultures of Phaseolus vulgaris L.

A novel lectin-like glycoprotein which accumulates in response to fungal elicitor action has been characterised in endomembranes from suspension cultures of French bean (Phaseolus vulgaris L.). The lectin, which has specificity towards N-acetylglucosamine oligomers, consists of a polypeptide of apparent molecular weight (M_r) 31 000 which is rich in glycine and contains 6.7% hydroxyproline O-linked to arabinose-containing oligosaccharides to give a glycoprotein of M_r 42500. A dual-labelling technique has been used to identify changes in the synthesis of the glycoprotein in cells exposed to fungal elicitor molecules. Thus, incorporation of [¹⁴C]proline into membranes in vivo and of [1^-3H]arabinose from uridine 5-diphosphate [1^-3H]arabinose in vitro and analysis by isoelectric focussing-polyacrylamide gel electrophoresis gave absolute correspondence of the labelled isoform of the glycoprotein. Having established the absence of contaminating polypeptides, subsequent analysis of microsomal fractions bysodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the peak of sythesis of the M_r-42500 glycoprotein occurred 4 h after the addition of fungal elicitor. The changes in the level of incorporation into the glycoprotein monomers were concomitant with increases in the activity of prolyl hydroxylase (EC 1.14.11.2)Incorporation of [¹⁴C]proline and its subsequent post-translational modification to hydroxyproline in microsomal polypeptides was followed by rapid transfer into the wall with an average t _1/2 of about 7 min. The M_r-42500 glycoprotein was rapidly transferred out of the endomembrane fraction with a t _1/2 of 2 min and could be detected in wall fractions where it became progressively less extractable. The glycoprotein, which clearly differs from bean extensin, accounts for up to 40% of the hydroxyproline newly exported in response to elicitor action. The lectin, which resembles those found in the Solanaceae and which is coinduced with enzymes of phytoalexin synthesis, may play some role in disease resistance.Abbreviations HRGP hydroxyproline-rich glycoprotein - IEF isoelectric focussing - M_r apparent molecular weight - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate     

Analysis of storage proteins in normal and aborted seeds from embryo-lethal mutants of Arabidopsis thaliana

The major storage proteins isolated from wild-type seeds of Arabidopsis thaliana (L.) Heynh., strain Columbia, were studied by sucrose gradient centrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Both the hypocotyl and cotyledons of mature embryos contained abundant 12 S (cruciferin) and 2 S (arabin) proteins that appeared similar in size and subunit composition to the cruciferin (12 S) and napin (1.7 S) seed-storage proteins of Brassica napus. The 12 S protein from Arabidopsis was resolved by SDS-PAGE into two groups of subunits with approximate relative molecular weights of 22–23 kDa (kilodalton) and 30–34 kDa. These polypeptides accumulated late in embryo development, disappeared early in germination, and were not detected in other vegetative or reproductive tissues. Accumulation of the 12 S proteins in aborted seeds from nine embryo-lethal mutants with different patterns of abnormal development was studied to determine the extent of cellular differentiation in arrested embryos from each mutant line. Abundant 12 S proteins were found in arrested embryos from two mutants with late lethal phases, but not in seven other mutants with lethal phases ranging from the globular to the cotyledon stages of embryo development. These results indicate that the accumulation of seed-storage proteins in wild-type embryos of Arabidopsis is closely tied to morphogenetic changes that occur during embryo development. Embryo-lethal mutants may therefore be useful in future studies on the developmental regulation of storage-protein synthesis.Abbreviations kDa kilodalton - M_r relative molecular weight - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Biosynthesis of glycoconjugates in mitochondrial outer membranes

The results reported in this paper show two distinct ways for the incorporation ofN-acetylglucosamine into mitochondrial outer membranes. The first one is the glycosylation of dolichol acceptors, which is indicated by the inhibition of the synthesis of these products by the inhibitors of the dolichol intermediates (tunicamycin and GDP). The second one is the incorporation ofN-acetylglucosamine into protein acceptors directly from UDP-N-acetylglucosamine. This second way of glycosylation is only localized in mitochondria outer membranes.The existence of a direct route forN-glycoprotein biosynthesis has been based on the following evidence. First, the synthesis of theN-acetylglucosaminylated protein acceptors was not inhibited by tunicamycin or GDP. Second, the addition of exogenous dolichol-phosphate did not change the rate of biosynthesis of glycosylated protein material. Third, the sequential incorporation ofN-acetylglucosamine and mannose from their nucleotide derivatives in the presence of GDP and tunicamycin led to the synthesis of glycosylated protein material which entirely bound to Concanavalin A-Sepharose. The oligosaccharide moiety of the glycosylated protein material resulting from the direct transfer of sugars from their nucleotide derivatives to the protein acceptor is of theN-glycan type. On sodium dodecylsulphate polyacrylamide gel electrophoresis, this glycosylated material migrated as a marker protein with a molecular weight between 45 000 and 63 000. HPLC chromatofocusing analysis revealed that the fraction studied was anionic. The oligosaccharide moiety of the glycoprotein material can only be elongated by the incorporation ofN-acetylglucosamine and galactose from their nucleotide derivatives.     

The effect of tunicamycin on Leishmania brasiliensis. Glycosylation and the cell surface components

Culture conditions of Leishmania cells were developed to allow the study of the effect of tunicamycin (TM) on glycosylation and on the cell surface components. Leishmania incorporate [¹⁴C]-mannose and [³⁵S]-methionine in vitro. The incorporation of [¹⁴C]-mannose is linear for 150 min and is inhibited by TM (2 g/ml) in a time dependent effect which reaches a plateau of 45% inhibition at 36 h. Under the same experimental conditions [³⁵S]-methionine incorporation into protein is slightly affected. This is reflected by an almost identical polypeptide pattern for TM treated and non-treated cells when analyzed on SDS-PAGE. On the contrary, strong differences were detected on the labeled compounds analyzed on SDS-PAGE followed by autoradiography when the precursor used was [¹⁴C]-mannose. A shift in the electrophoretic mobility of most of the glycopeptides synthesized in the presence of TM was observed, which is also reflected in the structure of the main Leishmania cell surface components.The findings are discussed in the light of biological implications.     

Effects of cholera toxin and actinomycin on synthesis of [35S]methionine-labeled proteins during progesterone-induced maturation of Xenopus laevis oocytes

The incorporation of [³⁵S]methionine into polypeptides during progesterone-induced meiotic maturation of Xenopus laevis oocytes was studied by two-dimensional polyacrylamide gel electrophoresis. Five modifications were consistently observed: two polypeptides of an approximate molecular weight of 150K daltons and pI 5 were new proteins, two represent increased incorporation and one was decreased incorporation. Cholera toxin inhibited the appearance of the modifications induced by progesterone. Actinomycin and enucleation did not significantly alter the modifications. These data indicate that a good correlation exists between the modifications in protein synthesis induced by progesterone and the resumption of meiotic cell division.     

Plant tyrosine decarboxylase can be strongly inhibited by l-α-aminooxy-β-phenylpropionate

The effect of tunicamycin, an inhibitor of N-glycosylation of proteins, on growth and on synthesis of DNA and protein was studied in suspension cultures from Nicotiana tabacum and Catharanthus rosea. In the presence of 0.1–1 g · ml^-1 tunicamycin, cell division and DNA synthesis stopped in cells which had been proliferating logarithmically, but protein formation continued. Cytophotometric determination of the nuclear DNA content in Catharanthus cells showed that a cell-cycle arrest had occurred in G1 phase. Metabolic labelling of cells with the glycoprotein precursors glucosamine or mannose was inhibited, too. The results indicate that one or more glycoproteins are needed for the cell to pass through the G1 phase, as was recently postulated for animal and yeast cells.Abbreviations TCA trichloroacetic acid - TM tunicamycin