The position of the oligosaccharide side-chains of phytohemagglutinin and their accessibility to glycosidases determines their subsequent processing in the Golgi

Phytohemagglutinin (PHA), the glycoprotein lectin of Phaseolus vulgaris has two types of asparagine-linked oligosaccharides per polypeptide: a high-mannose chain with the formula (Man)8-9(GlcNAc)2 on Asn12 and a modified chain with fewer mannose residues and additional fucose and xylose residues on Asn60. Glycosylation of PHA is a cotranslational process, which occurs in the endoplasmic reticulum, and newly synthesized PHA has two high-mannose chains. Transport of PHA to the protein bodies via the Golgi complex is accompanied by the modification of one of the two high-mannose chains. Why is only one chain modified, while the other remains in the high-mannose configuration? By determining the effect of digestion with various glycosidases (alpha-mannosidase, endo-beta-N-acetylglucosaminidase H and endo-beta-N-acetylglucosaminidase F) on native and denatured PHA we obtained evidence consistent with the interpretation that the accessibility of oligosaccharide chains to modifying enzymes is of major importance in determining whether a high-mannose chain becomes modified or not. The high-mannose chain of mature undenatured PHA is only partially accessible to glycosidases, while PHA obtained from the endoplasmic reticulum has one high-mannose chain, which is readily accessible to alpha-mannosidase and endoglycosidases H and F. We show that this readily accessible chain is in the same position on the polypeptide (Asn60) as the modified oligosaccharide on mature PHA. Thus, accessibility of the oligosaccharide side-chains to processing enzymes in the Golgi determines whether a particular oligosaccharide side-chain is processed or not.     

Precursors of ricin and Ricinus communis agglutinin. Glycosylation and processing during synthesis and intracellular transport

During synthesis in vivo the castor bean lectin precursors initially appear in the endoplasmic reticulum as a group of core glycosylated polypeptides of relative molecular mass 64 000-68 000. Pretreatment of intact castor bean endosperm tissue with tunicamycin partially inhibits the cotranslational core glycosylation step and results in the accumulation of a single sized unglycosylated precursor polypeptide of relative molecular mass 59 000. The glycosylated precursors in the endoplasmic reticulum were enzymically converted to the 59 000-Mr form by incubation with endoglucosaminidase H. Intracellular transport of the glycosylated lectin precursors from the endoplasmic reticulum to a denser vesicle fraction was accompanied by modifications to the oligosaccharide moieties which conferred resistance to the action of endoglucosaminidase H. The post-translational addition of fucose to the carbohydrate chain was identified as one of the oligosaccharide modification steps. Fucose addition was catalysed by a glycosyltransferase associated with a smooth-surfaced membrane fraction which was distinct from the endoplasmic reticulum and which was tentatively identified as the Golgi apparatus. Glycosylation was not essential for intracellular transport of the lectin precursors: unglycosylated precursor synthesized in the presence of tunicamycin gave rise to unglycosylated lectin subunits in the protein bodies.     

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.     

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.     

Oligosaccharyltransferase: a complex multisubunit enzyme of the endoplasmic reticulum

The attachment of N-linked oligosaccharide chains to proteins is an important cotranslational process. These chains can, in some cases, serve to stabilize the protein, while in other cases they function as recognition elements. A key enzyme in the N-glycosylation process is oligosaccharyltransferase (OT). In yeast this enzyme, which is found in the endoplasmic reticulum, consists of nine different transmembrane protein subunits. Our general aim is to learn more about the functions of the multiple subunits of yeast OT and their mode of interaction with each other. Using a combination of biochemical and genetic techniques the subunit Ost1p has been shown to recognize Asn-X-Ser/Thr glycosylation sites. The principle tool used in the identification process was a benzophenone-based glycosylation site peptide that was shown to be crosslinked to Ost1p. Our current objective is to identify the domain in the primary structure that is involved in recognition of the glycosylation site sequence. By use of bifunctional crosslinkers, the possible interaction of Ost1p with other subunits of OT will be studied. This work and other studies on the OT subunits are concisely summarized.     

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.     

The plant vacuolar protein, phytohemagglutinin, is transported to the vacuole of transgenic yeast

Phytohemagglutinin (PHA), the major seed lectin of the common bean, Phaseolus vulgaris, accumulates in the parenchyma cells of the cotyledons. It has been previously shown that PHA is cotranslationally inserted into the endoplasmic reticulum with cleavage of the NH2-terminal signal peptide. Two N-linked oligosaccharide side chains are added, one of which is modified to a complex type in the Golgi apparatus. PHA is then deposited in membrane-bound protein storage vacuoles which are biochemically and functionally equivalent to the vacuoles of yeast cells and the lysosomes of animal cells. We wished to determine whether yeast cells would recognize the vacuolar sorting determinant of PHA and target the protein to the yeast vacuole. We have expressed the gene for leukoagglutinating PHA (PHA-L) in yeast under control of the yeast acid phosphatase (PHO5) promoter. Under control of this promoter, PHA-L accumulates to 0.1% of the total yeast protein. PHA-L produced in yeast is glycosylated as expected for a yeast vacuolar glycoprotein. Cell fractionation studies show that PHA-L is efficiently transported to the yeast vacuole. This is the first demonstration that vacuolar targeting information is recognized between two highly divergent species. A small proportion of yeast PHA-L is secreted which may be due to inefficient recognition of the vacuolar sorting signal because of the presence of an uncleaved signal peptide on a subset of the PHA-L polypeptides. This system can now be used to identify the vacuolar sorting determinant of a plant vacuolar protein.     

Proteomics reveals N-linked glycoprotein diversity in Caenorhabditis elegans and suggests an atypical translocation mechanism for integral membrane proteins

Protein glycosylation is one of the most common post-translational modifications in eukaryotes and affects various aspects of protein structure and function. To facilitate studies of protein glycosylation, we paired glycosylation site-specific stable isotope tagging of lectin affinity-captured N-linked glycopeptides with mass spectrometry and determined 1,465 N-glycosylated sites on 829 proteins expressed in Caenorhabditis elegans. The analysis shows the diversity of protein glycosylation in eukaryotes in terms of glycosylation sites and oligosaccharide structures attached to polypeptide chains and suggests the substrate specificity of oligosaccharyltransferase, a single multienzyme complex in C. elegans that incorporates an oligosaccharide moiety en bloc to newly synthesized polypeptides. In addition, topological analysis of 257 N-glycosylated proteins containing a putative single transmembrane segment that were identified based on the relative positions of glycosylation sites and transmembrane segments suggests that an atypical non-cotranslational mechanism translocates large N-terminal segments from the cytosol to the endoplasmic reticulum lumen in the absence of signal sequence function.     

Post-translational glycosylation of coronavirus glycoprotein E1: inhibition by monensin.

The intracellular sites of biosynthesis of the structural proteins of murine hepatitis virus A59 have been analyzed using cell fractionation techniques. The nucleocapsid protein N is synthesized on free polysomes, whereas the envelope glycoproteins E1 and E2 are translated on the rough endoplasmic reticulum (RER). Glycoprotein E2 present in the RER contains N-glycosidically linked oligosaccharides of the mannose-rich type, supporting the concept that glycosylation of this protein is initiated at the co-translational level. In contrast, O-glycosylation of E1 occurs after transfer of the protein to smooth intracellular membranes. Monensin does not interfere with virus budding from the membranes of the endoplasmic reticulum, but it inhibits virus release and fusion of infected cells. The oligosaccharide side chains of E2 obtained under these conditions are resistant to endoglycosidase H and lack fucose suggesting that transport of this glycoprotein is inhibited between the trans Golgi cisternae and the cell surface. Glycoprotein E1 synthesized in the presence of monensin is completely carbohydrate-free. This observation suggests that the intracellular transport of this glycoprotein is also blocked by monensin.     

Differential Asparagine-Linked Glycosylation of Voltage-Gated K+ Channels in Mammalian Brain and in Transfected Cells

Glycosylation of ion channel proteins dramatically impacts channel function. Here we characterize the asparagine (N)-linked glycosylation of voltage-gated K⁺ channel α subunits in rat brain and transfected cells. We find that in brain Kv1.1, Kv1.2 and Kv1.4, which have a single consensus glycosylation site in the first extracellular interhelical domain, are N-glycosylated with sialic acid-rich oligosaccharide chains. Kv2.1, which has a consensus site in the second extracellular interhelical domain, is not N-glycosylated. This pattern of glycosylation is consistent between brain and transfected cells, providing compelling support for recent models relating oligosaccharide addition to the location of sites on polytopic membrane proteins. The extent of processing of N-linked chains on Kv1.1 and Kv1.2 but not Kv1.4 channels expressed in transfected cells differs from that seen for native brain channels, reflecting the different efficiencies of transport of K⁺ channel polypeptides from the endoplasmic reticulum to the Golgi apparatus. These data show that addition of sialic acid-rich N-linked oligosaccharide chains differs among highly related K⁺ channel α subunits, and given the established role of sialic acid in modulating channel function, provide evidence for differential glycosylation contributing to diversity of K⁺ channel function in mammalian brain. Received: 17 December 1998/Accepted: 20 January 1999     

Secretion of rat hepatic lipase is blocked by inhibition of oligosaccharide processing at the stage of glucosidase I

Rat hepatic lipase is a glycoprotein bearing two N-linked oligosaccharide chains. The importance of glycosylation in the secretion of hepatic lipase was studied using freshly isolated rat hepatocytes. Various inhibitors of oligosaccharide synthesis and processing were used at concentrations that selectively interfere with protein glycosylation. Secretion of hepatic lipase activity was abolished by tunicamycin, castanospermine, and N-methyldeoxynojirimycin. No evidence was found by ELISA or Western blotting for secretion of inactive protein. Inhibition of secretion became apparent after a 30-min lag, corresponding to the time of intracellular transport of pre-existing protein. Simultaneously, intracellular hepatic lipase activity ws depleted. Secretion of hepatic lipase protein and activity was not affected by deoxymannojirimycin and swainsonine. Upon SDS-polyacrylamide gel electrophoresis, hepatic lipase secretion by deoxymannojirimycin- or swainsonine-treated cells showed an apparent Mr of 53 kDa and 55 kDa, respectively, which was distinct from hepatic lipase secreted by untreated cells (Mr = 58 kDa). We conclude that glycosylation and subsequent oligosaccharide processing play a permissive role in the secretion of hepatic lipase. As secretion is prevented by the glucosidase inhibitors castanospermine and N-methyldeoxynojirimycin, but not by inhibitors of subsequent oligosaccharide trimming, the removal of glucose residues from the high-mannose oligosaccharide intermediate in the rough endoplasmic reticulum appears the determining step.     

Structural requirements for protein N-glycosylation. Influence of acceptor peptides on cotranslational glycosylation of yeast invertase and site-directed mutagenesis around a sequon sequence

To understand better the structural requirements of the protein moiety important for N-glycosylation, we have examined the influence of proline residues with respect to their position around the consensus sequence (or sequon) Asn-Xaa-Ser/Thr. In the first part of the paper, experiments are described using a cell-free translation/glycosylation system from reticulocytes supplemented with dog pancreas microsomes to test the ability of potential acceptor peptides to interfere with glycosylation of nascent yeast invertase chains. It was found that peptides, being acceptors for oligosaccharide transferase in vitro, inhibit cotranslational glycosylation, whereas nonacceptors have no effect. Acceptor peptides do not abolish translocation of nascent chains into the endoplasmic reticulum. Results obtained with proline-containing peptides are compatible with the notion that a proline residue in an N-terminal position of a potential glycosylation site does not interfere with glycosylation, whereas in the position Xaa or at the C-terminal of the sequon, proline prevents and does not favour oligosaccharide transfer, respectively. This statement was further substantiated by in vivo studies using site-directed mutagenesis to introduce a proline residue at the C-terminal of a selected glycosylation site of invertase. Expression of this mutation in three different systems, in yeast cells, frog oocytes and by cell-free translation/glycosylation in reticulocytes supplemented with dog pancreas microsomes, leads to an inhibition of glycosylation with both qualitative and quantitative differences. This may indicate that host specific factors also contribute to glycosylation.     

A modeling framework for the study of protein glycosylation

The key step in the asparagine-linked glycosylation of secretory proteins is the transfer of oligosaccharide from a dolichol precursor to the polypeptide at an Asp-X-Ser/Thr (NXS/T) consensus sequence. It is often the case, both in cultured cells and in vivo, that this reaction does not occur for every molecule of a given protein. Thus, the cell may create two protein populations, one bearing and one lacking oligosaccharide, for each potential glycosylation site. We present a structured kinetic modeling framework of the initial glycosylation event based on a balance of available glycosylation sites through the region of endoplasmic reticulum lumen proximal to the membrane. Oligosaccharyltransferase, a multimeric protein complex, catalyzes the sugar transfer. This enzyme is integral to the endoplasmic reticulum membrane, and it is thought to act cotranslationally. The nascent polypeptide may also fold in such a way as to prevent glycosylation from occurring. The net result is a potentially complex spatial and temporal relationship among translation, glycosylation, and other cotranslational events. Model results predict how fractional glycosylation site occupancy may depend on protein synthesis rate, oligosaccharyldolichol availability, and mRNA elongation rate. Although we are currently unable to quantitatively compare predicted to experimentally obtained fractional site occupancy, we are able to determine qualitative trends which may be confirmed experimentally. (c) 1996 John Wiley & Sons, Inc.     

Gene Expression and Synthesis of Phytohemagglutinin in the Embryonic Axes of Developing Phaseolus vulgaris Seeds

Phytohemagglutinin (PHA), the major seed lectin of the common bean (Phaseolus vulgaris), is found largely in the cotyledons, but is also present in the embryonic axis. At mid-maturation, the percentage of total protein synthesis which is directed towards making PHA is 5 to 10 times greater in the cotyledons than in the axes. This lower rate of synthesis in the axes is correlated with a lower abundance of mRNA for PHA, as determined by dot blot hybridization using a cDNA clone for PHA. Manen and Pusztai (Planta 1982 155: 328-334) have claimed on the basis of immunocytochemical evidence that, in the axis, PHA is found in the cytosol although it is present in protein bodies in the cotyledons. In the cotyledons, PHA is synthesized on rough endoplasmic reticulum, and its transport to the protein bodies via the Golgi complex is associated with specific posttranslational processing steps (Vitale and Chrispeels, J Cell Biol 1984 In press). A cytosolic localization of axis PHA would be an indication of a different site of synthesis and transport pathway. The results presented here indicate that the site of synthesis of PHA and the posttranslational modifications of PHA are the same in the axes as in the cotyledons. Since in the cotyledons these modifications take place in the endoplasmic reticulum, the Golgi, and the protein bodies, it appears that the transport pathway and the site of accumulation of PHA in the axes is similar to that in the cotyledons. On the basis of our evidence, we suggest that the subcellular localization of PHA in the axes should be reexamined.     

Partial characterization and purification of the glycosylation site recognition component of oligosaccharyltransferase

Oligosaccharyltransferase, the enzyme catalyzing the co-translational transfer of oligosaccharide from dolichyl-PP-GlcNAc2Man9Glc3 to -Asn-X-Ser/Thr- sequences in nascent polypeptide chains, was studied in hen oviduct microsomes using the active site-directed photoaffinity probe 125I-labeled N alpha-3-(4-hydroxyphenylpropionyl)-Asn-Lys(N epsilon-p-azidobenzoyl)-Thr-NH2. Several lines of evidence established that the tripeptide probe interacted with a 57-kDa protein of the endoplasmic reticulum that was subsequently glycosylated and converted to a 60-kDa form. The 57-kDa protein, isolated by two-dimensional gel electrophoresis, was used as immunogen to prepare polyclonal antisera. The specificity of the antibody was established on the basis of its ability to 1) recognize the 57-kDa protein by immunoblotting and 2) immunoprecipitate the photolabeled protein. The antibody also recognized photolabeled protein from different tissues and organisms. The 57-kDa protein isolated by immunoprecipitation retained its ability to interact with the photoaffinity probe but was inactive in catalyzing glycosylation of peptides. This result suggests that the 57-kDa protein is the component of oligosaccharyltransferase that recognizes the glycosylation site in polypeptides. These results are discussed in terms of possible models for the structure of oligosaccharyltransferase in the endoplasmic reticulum.     

Role of glycosylation in secretion of yeast acid phosphatase

The minimal glycosylation requirement for acid phosphatase secretion and activity was investigated using tunicamycin, an inhibitor of protein glycosylation, and a yeast mutant defective in the synthesis of oligosaccharide outer chains. The results obtained show that outer chain addition is not essential for secretion of active enzyme and that only 4 core chains, out of 8 normally attached to a protein subunit, are sufficient for enzyme transport to the periplasmic space. Enzyme forms with less than 4 chains were retained in membranes of endoplasmic reticulum. Secreted underglycosylated enzyme forms are partially or completely inactive.     

Biosynthesis and processing of legumin-like storage proteins in Lupinus angustifolius (lupin).

Synthesis, secretion and post-translational proteolysis of the storage proteins in cotyledons of Lupinus angustifolius L. (lupin) have been examined in vivo and in vitro by using a combination of pulse-chase experiments with [3H]- or [35S]-labelled amino acids, subcellular fractionation and cell-free translation from poly(A)+ (polyadenylylated) RNA or membrane-bound polyribosomes. Related polypeptides were identified by immunoprecipitation, separation on sodium dodecyl sulphate/polyacrylamide gels and fluorography. The synthesis and processing of two proteins were compared. Conglutin alpha, the 11 S protein, was found as a family of precursor polypeptides of Mr 68000-88000 when translated from poly(A)+ RNA under conditions where signal segments were not cleaved, and Mr 64000-85000 both when sequestered into the endoplasmic reticulum and when accumulated in the protein bodies. Pulse-chase labelling showed that cotyledons from early stages of development were completely incapable of further proteolysis of these precursors. Nevertheless, in the same juvenile cotyledons, the precursors of the minor storage protein conglutin gamma, two polypeptides with Mr 50000-51000, were proteolytically cleaved to mature subunits of Mr 32000 and 17000 within 2 h. Further cleavage of the precursors of conglutin alpha into families of mature subunits of Mr 21000-24000 and 42000-62000 was detected in more mature cotyledons. A model is proposed which suggests that the mature subunits are produced by a single proteolytic cleavage of each of the three major precursors of conglutin alpha and also suggests that a close similarity exists between these subunits and those of other legumin-like proteins. The enzyme responsible for this cleavage, which appears at a specific stage in the middle of cotyledonary development, seems to be an integral part of the programmed developmental sequence in these pods.     

Role of endoplasmic reticulum in biosynthesis of oat globulin precursors

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

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

     

Regulation of processing of a plant glycoprotein in the Golgi complex: A comparative study usingXenopus oocytes

The synthesis of phytohemagglutinin (PHA), the major seed lectin ofPhaseolus vulgaris, was investigated inXenopus oocytes injected with RNA isolated from developing bean cotyledons. As is the case for normal PHA, oocyte-synthesized PHA polypeptides were found to contain two asparagine-linked oligosaccharide chains, one of which was of the high-mannose type and the other one of the Golgi-modified type, being largely resistant to endo--N-acetylglucosaminidase H digestion and containing fucose. The modified oligosaccharide chain of oocyte-synthesized PHA appeared to be much larger and more heterogeneous with respect to the modified chain normally present on PHA. When the oocytes were injected with purified mRNA for PHA, isolated by hybrid-selection using a PHA complementary-DNA clone, the results were the same as those obtained by injecting total cotyledonary RNA. On the whole, these results indicate that plant glycoproteins are directed to the Golgi complex even when synthesized in an animal cell, and that correct sorting of the oligosaccharide chains to be processed is independent of the cell-type in which protein synthesis occurs. The form of processing is however cell-type specific.Abbreviations endo H endo H--N-acetylglucosaminidase H - ER endoplasmic reticulum - PHA phytohemagglutinin - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Biosynthesis and processing of a variant surface glycoprotein from Trypanosoma brucei brucei

Iowa trypanosome antigen type (IaTat) 1.2 variant surface glycoprotein (VSG) is synthesized in vitro as a Mr 54,000 preprotein that contains a 31-amino-acid signal peptide. Translation of mRNA in the presence of either dog pancreas or trypanosome microsomal membranes results in cotranslational cleavage of the signal peptide and addition of core oligosaccharide side chains to the protein. Analysis of these products on sodium dodecyl sulfate (SDS)-gels indicates that removal of the signal peptide (Mr 3200) is almost exactly compensated for by an increase in molecular weight due to carbohydrate addition. Pulse-chase experiments in cultures of isolated trypanosomes indicate that two IaTat 1.2 VSG species (Mr 58,000 and 60,000) occur in vivo. When glycosylation is inhibited by incubation of trypanosomes with tunicamycin, a single Mr 50,000 polypeptide is immunoprecipitated. The multiple protein species, therefore, arise from heterogeneity in carbohydrate side chains whose synthesis and transfer to the protein are tunicamycin sensitive. Sequence analysis verified that both species of VSG contain identical amino-terminal sequences. Further post-translational processing of IaTat 1.2 VSG includes addition of phosphate and myristic acid residues, both of which have been shown to be located in the immunologically cross-reactive determinant at the carboxyl terminus of the protein. Exposure of this attachment site requires post-translational proteolytic removal of a 17-amino-acid peptide from the carboxyl terminus of an intermediate form of VSG.