Characterization and subcellular localization of vicilin and phytohemagglutinin,the two major reserve proteins of Phaseolus vulgaris L.

Extracts of bean (Phaseolus vulgaris L. cv. Greensleeves) cotyledons contained two abundant proteins: vicilin and phytohemagglutinin. Vicilin, a 6.9 S protein fraction at neutral pH, associated to an 18.0 S form at pH 4.5 and had 3 non-identical subunits with molecular weights (MW) of 52,000, 49,000 and 46,000. Phytohemagglutinin, a 6.4 S protein fraction, had 2 non-identical subunits with MW of 34,000 and 36,000. Phytohemagglutinin could be separated by isoelectrofocusing into a mitogenic and non-erythroagglutinating protein with a single subunit of MW=34,000, and a mitogenic and erythroagglutinating protein fraction which contained both subunits. Vicilin is apparently identical with the so called glycoprotein II (A. Pusztai and W.B. Watt, Biochim. Biophys. Acta 365, 57–71, 1970) and with globulin G1 (R.C. McLeester, T.C. Hall, S.M. Sun, F.A. Bliss, Phytochem. 2, 85; 1973), while phytohemagglutinin is identical with globulin G2 (McLeester et al., 1973). Since vicilin and phytohemagglutinin are internationally used names there is no need to introduce new names to describe P. vulgaris reserve proteins. Both proteins are catabolized in the course of seedling growth and are located in the protein bodies, indicating that they are reserve proteins. Vicilin isolated in its 18.0 S form from the cotyledons of young seedlings contains substantial quantities of smaller polypeptides, in addition the 3 original ones. We suggest that the presence of these small polypeptides represents partial breakdown of the vicilin prior to its complete catabolism.     

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.     

Apparent Inhibition of beta-Fructosidase Secretion by Tunicamycin May Be Explained by Breakdown of the Unglycosylated Protein during Secretion

Suspension-cultured carrot (Daucus carota) cells synthesize and secrete β-fructosidase, a glycoprotein with asparagine-linked glycans. Treatment of the cells with tunicamycin completely inhibits the apparent secretion of β-fructosidase as measured by the accumulation of the radioactive protein in the cell wall or the culture medium. In the past, such a result has been interpreted as an inhibition of secretion by tunicamycin, but we suggest another explanation based on the following results. In the presence of tunicamycin, unglycosylated β-fructosidase is synthesized and is associated with an endoplasmic-reticulum-rich microsomal fraction. Pulse-chase experiments show that the unglycosylated β-fructosidase does not remain in the cells and appears to be secreted in the same way as glycosylated β-fructosidase; however, no radioactive, unglycosylated β-fructosidase accumulates extracellularly (cell wall or medium). Protoplasts obtained from carrot cells secrete β-fructosidase protein and activity, and treatment of the protoplasts with tunicamycin results in the synthesis of unglycosylated β-fructosidase. In the presence of tunicamycin, there is no accumulation of β-fructosidase activity or unglycosylated β-fructosidase polypeptide in the protoplast incubation medium. These results are consistent with the interpretation that the glycans of β-fructosidase are necessary for its stability, and that in these suspension-cultured cells, the unglycosylated enzyme is degraded during the last stage(s) of secretion, or immediately after its arrival in the wall.     

UDP-GlcNAc:Glycoprotein GlcNAc-Transferase is Located in the Golgi Apparatus of Developing Bean Cotyledons

The transport and accumulation of phytohemagglutinin in developing bean (Phaseolus vulgaris L.) cotyledons is accompanied by the transient presence of N-acetylglucosamine (GlcNAc) residues on the oligosaccharide sidechains of this glycoprotein. These peripheral GlcNAc residues can be distinguished from those in the chitobiose portion of the oligosaccharide sidechains by their sensitivity to removal by the exoglycosidase β-N-acetylglucosaminidase. GlcNAc residues sensitive to removal by β-N-acetylglucosaminidase are present not only on phytohemagglutinin, but also on other newly synthesized proteins. The enzyme UDPGlcNAc:glycoprotein GlcNAc-transferase which transfers GlcNAc residues to glycoproteins was first described by Davies and Delmer (Plant Physiol 1981 68: 284-291). The data presented here show that this enzyme is associated with the Golgi complex of developing cotyledons.     

Crosslinking of microsomal proteins identifies P-9000, a protein that is co-transported with phaseolin and phytohemagglutinin in bean cotyledons

Developing cotyledons of the common bean, Phaseolus vulgaris L., transport within their secretory system (endoplasmic reticulum and Golgi apparatus) the abundant vacuolar proteins, phaseolin and phytohemagglutinin. To identify proteins that may play a role in vacuolar targeting, we treated cotyledon microsomal fractions with a bifunctional crosslinking reagent, dithiobis(succinimidyl propionate), isolated protein complexes with antibodies to phaseolin and phytohemagglutinin, and analysed the polypeptides by sodium dodecylsulfate polyacrylamide gel electrophoresis. This allowed us to identify a protein of M_r=9000 (P-9000) that was crosslinked to both phaseolin and phytohemagglutinin. P-900 is abundantly present in the endoplasmic reticulum. The aminoterminus of P-9000 shows extensive sequence identity with the amino-terminus of PA1 (M_r=11 000), a cysteine-rich albumin whose processing products accumulate in the vacuoles of pea (Pisum sativum L.) cotyledons. Like PA1, P-9000 is synthesized as a pre-proprotein that is posttranslationally processed into smaller polypeptides. The possible functions of P-9000 are discussed.Abbreviations DSP dithiobis(succinimidyl propionate) - EDTA ethylenediaminetetraacetic acid - ER endoplasmic reticulum - kDa kilodalton - M_r relative molecular mass - PHA phytohemagglutinin - SDS sodium dodecylsulfate - PAGE polyacrylamide gel electrophoresis     

Localization of phytohemagglutinin in the embryonic axis of Phaseolus vulgaris with ultra-thin cryosections embedded in plastic after indirect immunolabeling

We have examined the properties and subcellular localization of phytohemagglutinin (PHA), the major lectin of the common bean (Phaseolus vulgaris.), in the axis cells of nearly mature and imbibed mature seeds. On a protein basis the axis contained about 15% as much PHA as the cotyledons. Localization of PHA was done with an indirect immunolabeling method (rabbit antibodies against PHA, followed by colloidal gold particles coated with goat antibodies against rabbit immunoglobulins) on ultra-thin cryosections which were embedded in plastic on the grids after the immunolabeling procedure. The embedding greatly improved the visualization of the subcellular structures. The small (4 nm) collodial gold particles, localized with the electron microscope, were found exclusively over small vacuoles or protein bodies in all the cell types examined (cortical parenchyma cells, vascular-bundle cells, epidermal cells). The matrix of these vacuoles-protein bodies appears considerably less dense than that of the protein bodies in the cotyledons, but the results confirm that in all parts of the embryo PHA is localized in similar structures.Abbreviations IgG immunoglobulin G - M_r relative molecular weight - PBS phosphate-buffered saline - PHA phytohemagglutinin - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Secretion of phytohemagglutinin by monkey COS cells

The entire coding region of a gene, which encodes a polypeptide of phytohemagglutinin (PHA-L), obtained from a library of genomic DNA of the common bean Phaseolus vulgaris cv. Greensleeves, was introduced into the SV40 expression vector pJC119. Monkey COS1 cells were transfected with the recombinant clone and the synthesis, glycosylation, and transport of PHA-L studied and compared with the normal processes in bean cotyledons. In the bean, phytohemagglutinin is synthesized on the rough endoplasmic reticulum and transported via the Golgi complex to protein bodies, vacuole-like organelles. Phytohemagglutinin was synthesized and glycosylated at the ER and processed in the Golgi apparatus of the transfected COS1 cells. After passing the Golgi apparatus, PHA-L was slowly secreted into the culture medium (half-time of 3-6 h), a result indicating that the signals for targeting proteins beyond the Golgi apparatus in plant cells are different from those in animal cells. PHA, which is stored in protein bodies in the plant cells, is secreted by animal cells. Tunicamycin inhibited both glycosylation and secretion of PHA by the COS1 cells, a finding indicating an essential role of the oligosaccharides for transport of PHA in these cells in contrast to the situation found in bean cotyledons. PHA, secreted into the culture medium, was partially sensitive to endo H, a result indicating the presence of one high-mannose and one complex oligosaccharide chain, a situation identical to that in beans.     

硝态氮对黄瓜子叶谷氨酰胺合成酶和谷氨酸脱氢酶活性的影响

在硝态氮存在或缺乏的条件下,测定了黄瓜(Cucumis sativus L．)种子萌发和子叶发育过程中子叶可溶性蛋白质含量以及谷氨酰胺合成酶(GS)和谷氨酸脱氢酶(NAD(H)-GDH)活性的变化。在子叶发育初期,无论外源氮存在与否,每对子叶可溶性蛋白质含量和GS、NADH—GDH、NAD^ -GDH活性随发育上升。在外源氮存在下,第4d后,可溶性蛋白质含量虽有所下降,但基本保持恒定;第6d后,GS和NADH—GDH活性逐渐降低,NAD^ -GDH却相反增高。但在无外源氮条件下,于第4d后,可溶性蛋白质水平以及GS、NADH—GDH和NAD^ -GDH活性都逐渐降低。在子叶发育的整个过程中,外源氮对GS和NAD^ -GDH活性有促进作用,尤其是在子叶发育的后期对NAD^ -GDH活性的促进更为明显。     

Mannose analog 1-deoxymannojirimycin inhibits the Golgi-mediated processing of bean storage glycoproteins

The asparagine-linked oligosaccharide chains of glycoproteins can be processed to form a wide variety of structures. The Golgi complex is the main compartment involved in this processing. In mammalian cells the first enzyme acting along the Golgi processing pathway is mannosidase I, whose action is a prerequisite for any further processing and which is inhibited by the mannose analog 1-deoxymannojirimycin (dMM). To have insights into the processing pathway in plant cells, we have studied the in vivo effect of dMM on the processing of the bean (Phaseolus vulgaris) storage proteins phaseolin and phytohemagglutinin, two well characterized plant glycoproteins. Cotyledons obtained from developing seeds were labeled with radioactive leucine, glucosamine, or fucose in the presence or absence of dMM. Treatment with dMM fully inhibited the acquisition of resistance to endo-β-N-acetylglucosaminidase H by phaseolin and phytohemagglutinin and the incorporation of fucose into protein. Furthermore, the apparent molecular weight of the polypeptides of phaseolin and phytohemagglutinin synthesized in dMM-treated cotyledons was consistent with the exclusive presence of oligommanose oligosaccharide chains which had not been processed in the Golgi complex. The inhibition of processing did not prevent exit from the Golgi complex, and most probably the storage proteins were correctly targeted to the protein bodies as indicated by the post-translational polypeptide cleavage of phaseolin. These results indicate that the action of a mannosidase is the first obligatory step of Golgi-mediated processing also in a plant cell and, together with data obtained in other laboratories on the in vitro specificity of glycosidases and glycosyltransferases present in the Golgi complex of plant cells, support the hypothesis that the key early reactions in Golgi-mediated processing are similar if not identical in plants and mammals.     

Synthesis of Lectin-Like Protein in Developing Cotyledons of Normal and Phytohemagglutinin-Deficient Phaseolus vulgaris

The genome of the common bean Phaseolus vulgaris contains a small gene family that encodes lectin and lectin-like proteins (phytohemagglutinin, arcelin, and others). One of these phytohemagglutinin-like genes was cloned by L. M. Hoffman et al. ([1982] Nucleic Acids Res 10: 7819-7828), but its product in bean cells has never been identified. We identified the product of this gene, referred to as lectin-like protein (LLP), as an abundant polypeptide synthesized on the endoplasmic reticulum (ER) of developing bean cotyledons. The gene product was first identified in extracts of Xenopus oocytes injected with either cotyledonary bean RNA or LLP-mRNA obtained by hybrid-selection with an LLP cDNA clone. A tryptic map of this protein was identical with a tryptic map of a polypeptide with the same SDS-PAGE mobility detectable in the ER of bean cotyledons pulse-labeled with either [³H]glucosamine or [³H]amino acids, both in a normal and in a phytohemagglutinin-deficient cultivar (cultivars Greensleeves and Pinto UI 111). Greensleeves LLP has M_r 40,000 and most probably has four asparagine-linked glycans. Pinto UI 111 LLP has M_r 38,500. Unlike phytohemagglutinin which is a tetramer, LLP appears to be a monomer by gel filtration analysis. Incorporation of [³H]amino acids indicates that synthesis of LLP accounts for about 3% of the proteins synthesized on the ER, a level similar to that of phytohemagglutinin.     

N-Linked Glycosylation of the α-Amino-3-Hydroxy-5-Methylisoxazole-4-Propionate(AMPA)-Selective Glutamate Receptor Channel α2 Subunit Is Essential for the Acquisition of Ligand-Binding Activity

Abstract: The N-linked glycosylation of the α2 subunit of the mouse α-amino-3-hydroxy-5-methylisoxazole-4-propionate(AMPA)-selective glutamate receptor (GluR) channel was characterized. The receptor subunit protein has five putative N -glycosylation sites. The recombinant receptor proteins were identified by [³⁵S]methionine/[³⁵S]cysteine metabolic labeling, western blot analysis, immunocytochemical detection, and [³H]AMPA binding experiments when expressed in insect Spodoptera frugiperda cells using a baculovirus system. The effect of tunicamycin on the metabolic labeling and immunoblots suggested that the two products, a major protein species of ∼102 kDa and a minor species of ∼98 kDa, correspond to glycosylated and unglycosylated forms, respectively, which was also supported by the enzymic deglycosylation experiments. Immunofluorescence staining of tunicamycin-treated cells expressing only the unglycosylated form differed little from that of tunicamycin-nontreated cells expressing both glycosylated and unglycosylated forms. The lack of AMPA-binding activity of the unglycosylated form expressed in the presence of tunicamycin suggested that N -glycosylation is required, directly or indirectly, for functional expression in insect cells for ligand binding. These results demonstrate that occupancy of at least one N -glycosylation site is required for the formation and maintenance of the GluRα2 subunit protein in an active conformation for ligand binding. Possible roles of N -glycosylation of GluRα2 subunit protein are discussed.     

Growth and osmoregulation in Pisum sativum subhooks as affected by gibberellic acid and cotyledon excision

Growth stimulation by gibberellic acid (GA) of the Alaska pea ( Pisum sativum L.) subhook was observed within 6 h after its application; the stimulation being larger in cuttings with cotyledons than in decotylized ones. The osmotic potential in the subhook increased as it grew, the rate of its increase being faster in cuttings without than in cuttings with cotyledons. GA had no effect on the change in the osmotic potential until 8 h after GA application, but afterwards it suppressed the increase in cuttings with cotyledons. This GA effect was not observed in decotylized cuttings. Changes in the osmotic potential were well correlated with changes in the concentration of soluble sugars, but not with changes in amino acids and K⁺, Soluble sugars accumulated in the subhook of cuttings with and without cotyledons in proportion to growth, irrespective of the presence or absence of GA. Cotyledon excision suppressed sugar accumulation, and GA promoted it in cuttings with cotyledons but not in decotylized ones. These results suggest that GA stimulates the translocation of sugars from the cotyledons to the subhook and, thereby, maintains the osmotic potential low, resulting in enhanced growth.     

Xenopus oocytes can synthesise but do not secrete the Z variant of human alpha 1-antitrypsin

Human liver mRNA was prepared from a patient homozygous for alpha 1-antitrypsin deficiency (PiZZ) and from a normal subject (PiMM). Both liver RNAs were microinjected into Xenopus oocytes and alpha 1-antitrypsin identified by immunoprecipitation. The normal M variant of alpha 1-antitrypsin is synthesised and secreted by Xenopus oocytes, the abnormal Z protein is not secreted and an intracellular form accumulates in the oocytes. In the presence of tunicamycin an unglycosylated form of M alpha 1-antitrypsin appears in the incubation medium but no corresponding unglycosylated version of the Z protein is secreted.     

Glycosylation increases potassium channel stability and surface expression in mammalian cells.

N-linked glycosylation is not required for the cell surface expression of functional Shaker potassium channels in Xenopus oocytes (Santacruz-Toloza, L., Huang, Y., John, S. A., and Papazian, D. M. (1994) Biochemistry 33, 5607-5613). We have now investigated whether glycosylation increases the stability, cell surface expression, and proper folding of Shaker protein expressed in mammalian cells. The turnover rates of wild-type protein and an unglycosylated mutant (N259Q,N263Q) were compared in pulse-chase experiments. The wild-type protein was stable, showing little degradation after 48 h. In contrast, the unglycosylated mutant was rapidly degraded (t(1/2) = approximately 18 h). Lactacystin slowed the degradation of the mutant protein, implicating cytoplasmic proteasomes in its turnover. Rapid lactacystin-sensitive degradation could be conferred on wild-type Shaker by a glycosylation inhibitor. Expression of the unglycosylated mutant on the cell surface, assessed using immunofluorescence microscopy and biotinylation, was dramatically reduced compared with wild type. Folding and assembly were analyzed by oxidizing intersubunit disulfide bonds, which provides a fortuitous hallmark of the native structure. Surprisingly, formation of disulfide-bonded adducts was quantitatively similar in the wild-type and unglycosylated mutant proteins. Our results indicate that glycosylation increases the stability and cell surface expression of Shaker protein but has little effect on acquisition of the native structure.     

Selective shift in the metabolic pathways of excised tomato cotyledons in response to mannitol induced water stress

Excised tomato cotyledons subjected to mannitol induced water stress had more total sugars than normal cotyledons during injury but less total sugars than normal cotyledons in the adaptive phase. During water stress injury, protein and UNA synthesis were reduced, activities of beta-fructofuranosidase and phosphofructokinase were reduced but activities of proteases, ribonuclease, hexokinase and glucose-6-phosphate dehydrogenase were increased. Additions of kinetin reversed the water stress effects on enzyme activities. It is concluded that during water stress injury, there was a fundamental reduction in cytokinin activities leading to the selective shift in the enzyme populations.     

Physical and biological properties of an epsilon-heavy chain mRNA and a kappa-light chain mRNA coding for rat immunoglobulin E

The mRNA coding for epsilon-heavy chain and kappa-light chain have been highly enriched from a rat IgE-producing myeloma, IR-162. Based on denaturing gel analyses, the 20 S epsilon-heavy chain mRNA has an estimated molecular weight of 850,000, equivalent to about 2500 nucleotides. The 14S rat kappa-light chain mRNA has an estimated molecular weight of 410,000, equivalent to about 1200 nucleotides. Only about two-thirds of the length of these mature cytoplasmic rat mRNA code for protein. The 20 S mRNA stimulates the in vitro synthesis of a single major serologically related protein which is large enough to be epsilon-heavy chain. It is unglycosylated and has an apparent molecular weight of about 62,000. The in vivo unglycosylated epsilon-heavy chain, obtained in the presence of tunicamycin, has an apparent molecular weight of about 59,000, compared with about 76,000 for the glycosylated heavy chain of the secreted rat IgE. Therefore, the in vitro synthesized epsilon-heavy chain protein is about Mr = 3000 larger than the in vivo unglycosylated epsilon-heavy chain, equivalent to about 25 extra amino acids. This is consistent with the synthesis of an epsilon-heavy chain putative precursor. Likewise, the 14 S mRNA stimulates the in vitro synthesis of a single putative precursor protein, which is serologically related to kappa chain, is unglycosylated, and is about an extra 20 amino acids. This is the first report on the physical and biological properties of an epsilon-heavy chain mRNA, as well as any rat immunoglobulin mRNA.     

Transport and processing of the glycosylated precursor of Concanavalin A in jack-bean

Concanavalin A (ConA) is a tetrameric lectin which is synthesized in the developing cotyledons of jack bean (Canavalia ensiformis L.) as a glycosylated precursor, pro-concanavalin A (pro-ConA). The processing of pro-ConA involves the excision of a small glycopeptide from the center of the pro-ConA molecule, and the ligation of the two polypeptides. In this paper, we show that pro-ConA is associated with the endoplasmic reticulum/Golgi fraction of the cells, and that the processing of pro-ConA occurs in the protein bodies. Processing is a complex process and different intermediate-sized polypeptides appear at different times during cotyledon development. The ConA-related polypeptides which accumulate during seed development may be the products of alternate processing events or breakdown products of ConA, rather than precursors of ConA. When glycosylation is prevented by tunicamycin, there is very little transport of pro-ConA out of the endoplasmic reticulum/Golgi system to the protein bodies; the unglycosylated pro-ConA which is transported is slowly processed. Tunicamycin does not prevent the transport of canavalin (a protein which is not glycosylated) or the transport and processing of the small amounts of glycosylated pro-ConA synthesized in the presence of the drug. This is, to our knowledge, the first demonstration that the transport of a glycoprotein in plant cells is dependent on the presence of the glycan.Abbreviations ConA concanavalin A - ER endoplasmic reticulum - GlcN glucosamine - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis supported by a grant from NATO     

Genetic tests of the roles of the embryonic ureases of soybean

We assayed the in vivo activity of the ureases of soybean (Glycine max) embryos by genetically eliminating the abundant embryo-specific urease, the ubiquitous urease, or a background urease. Mutant embryos accumulated urea (250-fold over progenitor) only when lacking all three ureases and only when developed on plants lacking the ubiquitous urease. Thus, embryo urea is generated in maternal tissue where its accumulation is not mitigated by the background urease. However, the background urease can hydrolyze virtually all urea delivered to the developing embryo. Radicles of 2-day-old germinants accumulated urea in the presence or absence of the embryo-specific urease (2 micromoles per gram dry weight radicle). However, mutants lacking the ubiquitous urease exhibited increased accumulation of urea (to 4-5 micromoles urea per gram dry weight radicle). Thus, the ubiquitous and not the embryo-specific urease hydrolyzes urea generated during germination. In the absence of both of these ureases, the background urease activity (4% of ubiquitous urease) may hydrolyze most of the urea generated. A pleiotropic mutant lacking all urease accumulated 34 micromoles urea per gram dry weight radicle (increasing 2.5-fold at 3 days after germination). Urea (20 millimolar) was toxic to in vitro-cultured cotyledons which contained active embryo-specific urease. Cotyledons lacking the embryo-specific urease accumulated more protein when grown with urea than with no nitrogen source. Among cotyledons lacking the embryo-specific urease, fresh weight increases were virtually unchanged whether grown on urea or on no nitrogen and whether in the presence or absence of the ubiquitous urease. However, elimination of the ubiquitous urease reduced protein deposition on urea-N, and elimination of both the ubiquitous and background ureases further reduced urea-derived protein. The evidence is consistent with the lack of a role in urea hydrolysis for the embryo-specific urease in developing embryos or germinating seeds. Because the embryo-specific urease is deleterious to cotyledons cultured in vitro on urea-N, its role may be to hydrolyze urea in wounded or infected embryos, creating a hostile environment for pest or pathogen. While the ubiquitous urease is operative in leaves and in seedlings, all or most of its function can be assumed by the background urease in embryos and in seedlings.     

Glycosylation of a membrane protein is restricted to the growing polypeptide chain but is not necessary for insertion as a transmembrane protein.

The membrane glycoprotein of vesicular stomatitis virus (VSV), synthesized in vitro in the presence of pancreatic microsomes, is glycosylated in two distinct steps while its polypeptide chain is nascent (Rothman and Lodish, 1977). We show here that unglycosylated glycoprotein, which accumulates in vivo following treatment of cells with tunicamycin and in vitro as a result of translation in the presence of detergent-treated microsomal membranes, is inserted normally as a transmembrane protein. This means that glycosylation, while normally occurring concurrently with insertion, is not required for insertion. Our experiments also show that the two steps in glycosylation correspond to the sequential transfer of preformed “core” oligosaccharides of typical structure to two Asn residues in the growing chain. The accumulation of unglycosylated glycoprotein in vitro is due to the fact that the completed transmembrane polypeptide cannot be glycosylated. The detergent treatment of microsomes impairs their rate of glycosylation so that chains are frequently completed before they can be glycosylated. This provides a simple explanation for certain types of heterogeneity often found in glycoproteins. We believe that the detergent treatment procedure results in the solubilization of the microsomal membrane followed by reconstitution. This is a prerequisite for the eventual purification of the membrane proteins and lipids involved in insertion and glycosylation of this model membrane protein.