Structures of the carbohydrate chains of membrane glycoproteins IIb and IIIa of human platelets

Human platelet membrane glycoproteins IIb (GPIIb) and IIIa (GPIIIa), which have been proposed to be subunits of a receptor for fibrinogen, were purified from Triton X-100-solubilized platelet membranes by affinity chromatography on a concanavalin A (Con A)-Sepharose column followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Compositional analyses of the purified glycoproteins showed that GPIIb and GPIIIa contain 15% and 18% carbohydrate by weight, respectively, which consists of galactose, mannose, glucosamine, fucose, and sialic acid. This suggested that these glycoproteins contained N-linked carbohydrate chains. The carbohydrate chains were released from each glycoprotein by hydrazinolysis and then fractionated by ion-exchange chromatography on a Mono Q column. From each glycoprotein, mono-, di-, and trisialylated and neutral oligosaccharide fractions were obtained. The structures of these oligosaccharides were investigated by means of compositional and methylation analyses and digestion by exoglycosidase, and their reactivities to immobilized lectins were also examined. The neutral oligosaccharides, which comprised about 14% of the total oligosaccharides released from GPIIb and about 52% of that from GPIIIa, were found to be of the high mannose-type, in that they contained 5 or 6 mannose residues. On the other hand, a major part of the acidic oligosaccharides was found to consist of typical bi- and triantennary complex-type sugar chains, and much smaller amounts of tetraantennary complex-type sugar chains, and complex-type sugar chains with a fucosyl residue at a N-acetylglucosamine residue in the peripheral portion or a bisecting N-acetylglucosamine at a beta-mannosyl residue in the core portion were also detected. In conclusion, we found that GPIIb contained mainly complex-type sugar chains, whereas high mannose-type sugar chains were the predominant carbohydrate units in GPIIIa, and that the detected differences in the carbohydrate moieties of GPIIb and GPIIIa were quantitative but not qualitative.     

C-terminal amino acid determination of the transmembrane subunits of the human platelet fibrinogen receptor, the GPIIb/IIIa complex

Glycoproteins IIb (GPIIb) and IIIa (GPIIIa) form the Ca2(+)-dependent GPIIb/IIIa complex, which acts as the fibrinogen receptor on activated platelets. GPIIb and GPIIIa are synthesized as single peptide chains. The GPIIb precursor is processed proteolytically to yield two disulphide-bonded chains, GPIIb alpha and GPIIb beta. The GPIIb/IIIa complex has two membrane attachment sites located at the C-termini of GPIIb beta and GPIIIa. The short cytoplasmic tails of GPIIb beta and/or GPIIIa become most likely associated to the cytoskeleton of activated platelets. In the present work the C-terminal amino acid residues of platelet GPIIb beta and GPIIIa have been analyzed by protein-chemical methods and compared with those predicted from cDNA analysis. We were able to confirm the positions of the C-termini in both glycoproteins and the identity of the C-terminus predicted for GPIIIa, i.e. threonine. However, glutamine, not glutamic acid as predicted for GPIIb beta from the human erythroleukemic cell line and megakaryocyte cells, was found to be the C-terminal amino acid of GPIIb beta. This indicates that the glutamic acid in the GPIIb precursor is posttranslationally modified to glutamine.     

Comparative study of the glycosylation of platelet glycoprotein GPIIb/IIIa and the vitronectin receptor. Differential processing of their beta-subunit.

The platelet glycoprotein GPIIb/IIIa and the vitronectin receptor (VNR) are alpha beta-heterodimeric proteins and share the same beta-subunit. By performing swainsonine treatment and digestion with endoglycosidase H (Endo H), we showed that the heavy chains of GPIIb and VNR alpha are glycosylated by complex-type oligosaccharide chains, and provided the first evidence for the presence of one complex carbohydrate residue on their light chains. The proteolytic cleavage of pro-GPIIb and the acquisition of Endo H-resistance are independent events occurring in the same Golgi compartment. We demonstrated the Endo H-sensitivity of GPIIIa and VNR beta in all cellular systems tested. In addition, this beta-subunit is differently glycosylated according to whether it is associated with GPIIb or VNR alpha, one carbohydrate chain being processed to the complex type on GPIIIa, but not on VNR beta.     

Posttranslational processing of the prohormone-cleaving Kex2 protease in the Saccharomyces cerevisiae secretory pathway

The Kex2 protease of the yeast Saccharomyces cerevisiae is a prototypical eukaryotic prohormone-processing enzyme that cleaves precursors of secreted peptides at pairs of basic residues. Here we have established the pathway of posttranslational modification of Kex2 protein using immunoprecipitation of the biosynthetically pulse-labeled protein from a variety of wild-type and mutant yeast strains as the principal methodology. Kex2 protein is initially synthesized as a prepro-enzyme that undergoes cotranslational signal peptide cleavage and addition of Asn-linked core oligosaccharide and Ser/Thr-linked mannose in the ER. The earliest detectable species, I1 (approximately 129 kD), undergoes rapid amino-terminal proteolytic removal of a approximately 9-kD pro-segment yielding species I2 (approximately 120 kD) before arrival at the Golgi complex. Transport to the Golgi complex is marked by extensive elaboration of Ser/Thr-linked chains and minor modification of Asn-linked oligosaccharide. During the latter phase of its lifetime, Kex2 protein undergoes a gradual increase in apparent molecular weight. This final modification serves as a marker for association of Kex2 protease with a late compartment of the yeast Golgi complex in which it is concentrated about 27-fold relative to other secretory proteins.     

Biosynthesis and secretion of the rat core-specific lectin. Relationship of post-translational modification and assembly to attainment of carbohydrate binding activity

A soluble lectin, the core-specific lectin (CSL), is synthesized and secreted by rat hepatocytes and the rat hepatoma cell line, H-4-II-E. This lectin binds mannose and N-acetylglucosamine residues in the "core" region of Asn-linked oligosaccharides. Secretion of the CSL was found to occur over an extended period of time, greater than 4 h being required for secretion of 50% of the lectin (Brownell, M. D., Colley, K. J., and Baenziger, J. U. (1984) J. Biol. Chem. 259, 3925-3932). We have determined that following synthesis in the endoplasmic reticulum, the CSL is rapidly transported to the Golgi where it is retained for an extended period of time prior to secretion. The lectin undergoes two post-translational modifications within the Golgi: an increase from Mr 24,000 to 25,000 and a progressive decrease in pI with an accompanying increase in Mr to a final value of 26,000. The lectin is also assembled into high molecular weight complexes of 150-260 X 10(3) and acquires the ability to bind carbohydrate in the Golgi. In hepatoma cells, the 24,000-25,000 modification is completed 20 min after initiation of synthesis. Assembly of the CSL subunits into high molecular weight complexes, acquisition of carbohydrate binding activity, and the 25,000-26,000 modification occur between 20 and 80 min after initiation of synthesis. These events have slower kinetics in primary hepatocytes and this allowed us to determine that the sequence of these biosynthetic events is: the 24,000-25,000 modification, complex assembly, the 25,000-26,000 modification, and acquisition of carbohydrate binding activity. The 24,000-25,000 modification occurs prior to complex assembly. Complex assembly may occur prior to, or concomitant with, the 25,000-26,000 modification. Assembly into the oligomeric form and the 25,000-26,000 modification correlate with the attainment of carbohydrate binding activity. The kinetics of CSL modification and assembly cannot account for its retention within the Golgi. Interaction with Golgi components either through carbohydrate binding or another interaction, may act to selectively retain the lectin within the Golgi.     

Relationship between Golgi architecture and glycoprotein biosynthesis and transport in Chinese hamster ovary cells

We have investigated the effect of colcemid-induced disassembly of microtubules, which is accompanied by retraction of the endoplasmic reticulum and fragmentation of the Golgi apparatus, on glycoprotein biosynthesis and transport in Chinese hamster ovary (CHO) cells. CHO cells were metabolically radiolabeled with [6- 3H]galactose or [2- 3H]mannose in the presence of either 0.1% dimethyl sulfoxide or 10 microM colcemid in dimethyl sulfoxide. The fine structure of glycoprotein asparagine-linked oligosaccharide structures synthesized in the presence or absence of colcemid was analyzed by lectin affinity chromatography, ion exchange chromatography, and methylation analysis using radiolabeled glycopeptides prepared by Pronase digestion. The fractionation patterns of [3H]mannose- and [3H]galactose-labeled glycopeptides on immobilized lectins indicated that processing to complex N-linked chains and poly-N-acetyllactosamine modification were similar in control and colcemid-treated cells. In addition, colcemid treatment did not alter the extent of sialylation or the linkage position of sialic acid residues to galactose. Using a trypsin release protocol, it was also found that the transport of newly synthesized glycoproteins to the cell surface was not affected by colcemid. These results demonstrate that the morphologically altered ER and Golgi apparatus in colcemid-treated CHO cells are completely functional with respect to the rate and fidelity of protein asparagine-linked glycosylation. Furthermore, movement of newly synthesized glycoproteins to and through the ER and Golgi apparatus and their transport to the cell surface in nonpolarized cells appears to be microtubule-independent.     

Biogenesis of acetylcholinesterase molecular forms in muscle. Evidence for a rapidly turning over, catalytically inactive precursor pool

Tissue-cultured chicken embryo muscle cells synthesize several molecular forms of acetylcholinesterase (AChE) which differ in oligomeric structure and fate as membrane-bound or secreted molecules. Using irreversible inhibitors to inactivate AChE molecules we show that muscle cells rapidly synthesize and assemble catalytically active oligomers which transit an obligatory pathway through the Golgi apparatus. These oligomers acquire complex oligosaccharides and are ultimately localized on the cell surface or secreted into the medium. Immunoprecipitation of isotopically labeled AChE shows that the oligomers are assembled shortly after synthesis from two allelic polypeptide chains. About two-thirds of the newly synthesized molecules are assembled into dimers and tetramers, and once assembled these forms do not interconvert. Comparison of newly synthesized catalytically active AChE molecules with isotopically labeled ones indicates that a large fraction of the immature molecules are catalytically inactive. Pulse-chase studies measuring both catalytic activity and isotopic labeling indicate that only the catalytically active oligomers are further processed by the cell, whereas inactive molecules are rapidly degraded intracellularly by an as yet unknown mechanism. Approximately 70-80% of the newly synthesized AChE molecules are degraded in this manner and do not transit the Golgi apparatus. These studies indicate that muscle cells synthesize an excess of this important synaptic component over that which is necessary for maintaining normal levels of this protein. In addition, these studies indicate the existence of an intracellular route of protein degradation which may function as a post-translational regulatory step in the control of exportable proteins.     

Golgi apparatus immunolocalization of endomannosidase suggests post-endoplasmic reticulum glucose trimming: implications for quality control

Trimming of N-linked oligosaccharides by endoplasmic reticulum (ER) glucosidase II is implicated in quality control of protein folding. An alternate glucosidase II-independent deglucosylation pathway exists, in which endo-alpha-mannosidase cleaves internally the glucose-substituted mannose residue of oligosaccharides. By immunogold labeling, we detected most endomannosidase in cis/medial Golgi cisternae (83.8% of immunogold labeling) and less in the intermediate compartment (15.1%), but none in the trans-Golgi apparatus and ER, including its transitional elements. This dual localization became more pronounced under 15 degrees C conditions indicative of two endomannosidase locations. Under experimental conditions when the intermediate compartment marker p58 was retained in peripheral sites, endomannosidase was redistributed to the Golgi apparatus. Double immunogold labeling established a mutually exclusive distribution of endomannosidase and glucosidase II, whereas calreticulin was observed in endomannosidase-reactive sites (17.3% in intermediate compartment, 5.7% in Golgi apparatus) in addition to the ER (77%). Our results demonstrate that glucose trimming of N-linked oligosaccharides is not limited to the ER and that protein deglucosylation by endomannosidase in the Golgi apparatus and intermediate compartment additionally ensures that processing to mature oligosaccharides can continue. Thus, endomannosidase localization suggests that a quality control of N-glycosylation exists in the Golgi apparatus.     

Assembly of cell-wall glycoproteins of Chlamydomonas reinhardii: Oligosaccharides are added in medial and trans Golgi compartments

A series of monoclonal antibodies and a polyclonal antiserum have been used to investigate the localisation and pathway of biosynthesis of the cell-wall hydroxyproline-rich glycoprotein 2BII in the alga Chlamydomonas reinhardii. Glyco-protein precursors were detected within the endoplasmic reticulum using a polyclonal antiserum raised to the deglycosylated 2BII. Monoclonal antibodies which are known to recognise different carbohydrate epitopes of 2BII were found to label two distinct regions of the Golgi stack. The immunolabelling results demonstrate that there is compartmentation of protein synthesis and glycosylation steps for these O-glycosidically linked glycoproteins. Newly synthesised glycoproteins are transported from the Golgi apparatus to the cell surface via two distinct routes. They then undergo assembly into a cell wall, the inner wall layer being formed first and probably functionaing as a template within which the outer crystalline wall layers are assembled.Abbreviations DGP deglycosylated glycoprotein - ER endoplasmic reticulum - MAC monoclonal antibody centre - M _r relative molecular mass     

Intracellular transport of membrane glycoproteins: two closely related histocompatibility antigens differ in their rates of transit to the cell surface

The intracellular transport of two closely related membrane glycoproteins was studied in the murine B cell lymphoma line, AKTB-1b. Using pulse-chase radiolabeling, the kinetics of appearance of the class I histocompatibility antigens, H-2Kk and H-2Dk, at the cell surface were compared and found to be remarkably different. Newly synthesized H-2Kk is transported rapidly such that all radiolabeled molecules reach the surface within 1 h. In contrast, the H-2Dk antigen is transported slowly with a half-time of 4-5 h. The rates of surface appearance for the two antigens closely resemble the rates at which their Asn-linked oligosaccharides mature from endoglucosaminidase H (endo H)-sensitive to endo H-resistant forms, a process that occurs in the Golgi apparatus. This suggests that the rate-limiting step in the transport of H-2Dk to the cell surface occurs before the formation of endo H-resistant oligosaccharides in the Golgi apparatus. Subcellular fractionation experiments confirmed this conclusion by identifying the endoplasmic reticulum (ER) as the site where the H-2Dk antigen accumulates. The retention of this glycoprotein in the ER does not appear to be due to a lack of solubility or an inability of the H-2Dk heavy chain to associate with beta 2-microglobulin. Our data is inconsistent with a passive membrane flow mechanism for the intracellular transport of membrane glycoproteins. Rather, it suggests that one or more receptors localized to the ER membrane may mediate the selective transport of membrane glycoproteins out of the ER to the Golgi apparatus. The fact that H-2Kk and H-2Dk are highly homologous (greater than or equal to 80%) indicates that this process can be strongly influenced by limited alterations in protein structure.     

The biosynthesis, processing, and secretion of laminin by human choriocarcinoma cells

Laminin, a glycoprotein component of basal laminae, is synthesized and secreted in culture by a human malignant cell line (JAR) derived from gestational choriocarcinoma. Biosynthetically labeled human laminin subunits A (Mr approximately 400,000) and B (Mr = 200,000 doublet) are glycoslyated with asparagine-linked high mannose oligosaccharides that are processed to complex oligosaccharides before the laminin molecule is externalized by the cell. The rate-limiting step in the processing of the asparagine-linked glycans of laminin is at the point of action of alpha-mannosidase I since the principal laminin forms that accumulate in JAR cells contain Man9GlcNAc2 and Man8GlcNAc2 oligosaccharide units. The combination of subunits to form the disulfide-linked laminin molecule (Mr approximately 950,000) occurs rapidly within the cell at a time when the subunits contain these high mannose oligosaccharides. The production of laminin is limited by the availability of the A subunit such that excess B subunit forms accumulate intracellularly as uncombined B and a disulfide-linked B dimer. Pulse-chase kinetic studies establish these B forms as intermediates in the assembly of the laminin molecule. The fully assembled laminin undergoes further oligosaccharide processing and translocation to the cell surface, but uncombined B and B dimer are neither processed nor secreted to any significant extent. Therefore, laminin subunit combination appears to be a prerequisite for intracellular translocation, processing, and secretion. The mature laminin that contains complex oligosaccharides does not accumulate intracellularly but is rapidly externalized upon completion, either secreted into the culture medium (25%) or associated with the cell surface (75%) as determined by susceptibility to degradation by trypsin. About one-third of the laminin molecules secreted or shed by JAR cells into the chase medium contain a smaller A subunit form that appears to have been modified by limited proteolytic cleavage. The putative proteolytic event is closely timed to the release of the laminin into the culture medium.     

Substrate Specificities of N-Acetylglucosaminyl-, Fucosyl-, and Xylosyltransferases that Modify Glycoproteins in the Golgi Apparatus of Bean Cotyledons

As part of their posttranslational maturation process, newly synthesized glycoproteins that contain N-linked oligosaccharide side chains pass through the Golgi apparatus, where some of their oligosaccharides become modified by carbohydrate processing reactions. In this paper, we report the presence of Golgi-localized enzymes in plant cells (Phaseolus vulgaris cotyledons) that transfer GlcNAc, fucosyl, and xylosyl residues to the oligosaccharide side chains of glycoproteins. All three enzyme activities are involved in the transformation of high mannose side chains into complex glycans. As judged by acceptor specificity studies, at least two GlcNAc residues can be added to the nonreducing side of high mannose oligosaccharides, which have been trimmed by α-mannosidase(s). A Man₅(GlcNAc)₂-peptide serves as the acceptor for the first GlcNAc added. The second GlcNAc can be added only after the prior removal of two additional mannose residues, ultimately yielding (GlcNAc)₂Man₃(GlcNAc)₂-peptide. Fucosyltransferase can transfer fucose to GlcNAcMan₅(GlcNAc)₂Asn, GlcNAcMan₃(GlcNAc)₂Asn, and (GlcNAc)₂Man₃(GlcNAc)₂Asn; xylosyltransferase exhibits significant activity toward the latter two substrates only. These results suggest an overlapping sequence of oligosaccharide modification in the Golgi apparatus that, in regard to GlcNAc and fucose additions, is analogous to pathways of oligosaccharide processing reported for animal cells. To our knowledge, this is the first report characterizing a xylosyltransferase involved in N-linked oligosaccharide modification, an activity that is apparently absent in most animal cells.     

Biosynthesis and processing of platelet GPIIb-IIIa in human megakaryocytes

Platelet membrane glycoprotein IIb-IIIa forms a calcium-dependent heterodimer and constitutes the fibrinogen receptor on stimulated platelets. GPIIb is a two-chain protein containing disulfide-linked alpha and beta subunits. GPIIIa is a single chain protein. These proteins are synthesized in the bone marrow by megakaryocytes, but the study of their synthesis has been hampered by the difficulty in obtaining enriched population of megakaryocytes in large numbers. To examine the biosynthesis and processing of GPIIb-IIIa, purified human megakaryocytes were isolated from liquid cultures of cryopreserved leukocytes stem cell concentrates from patients with chronic myelogenous leukemia. Immunoprecipitation of [35S]methionine pulse-chase-labeled cell extracts by antibodies specific for the alpha or beta subunits of GPIIb indicated that GPIIb was derived from a precursor of Mr 130,000 that contains the alpha and beta subunits. This precursor was converted to GPIIb with a half-life of 4-5 h. No precursor form of GPIIIa was detected. The glycosylation of GPIIb-IIIa was examined in megakaryocytes by metabolic labeling in the presence of tunicamycin, monensin, or treatment with endoglycosidase H. The polypeptide backbones of the GPIIb and the GPIIIa have molecular masses of 120 and 90 kD, respectively. High-mannose oligosaccharides are added to these polypeptide backbones co-translationally. The GPIIb precursor is then processed with conversion of high-mannose to complex type carbohydrates yielding the mature subunits GPIIb alpha (Mr 116,000) and GPIIb beta (Mr 25,000). No posttranslational processing of GPIIIa was detected.     

Mannose processing is an important determinant in the assembly of phosphorylated high mannose-type oligosaccharides

Phosphorylation of the high mannose-type oligosaccharides attached to newly synthesized acid hydrolases occurs in two sequential steps within the endoplasmic reticulum and the Golgi apparatus, and the products generated at the two sites differ with respect to the location of the phosphorylated mannose residue. To investigate the mechanism of this two-step phosphorylation, biosynthesis of the Man-6-P recognition marker was studied in class E Thy-1- and J774 cells metabolically labeled with [2-3H]mannose. Class E Thy-1- cells produce truncated high mannose oligosaccharides that lack 4 mannose residues from the alpha 1,6-branch of the core beta-linked mannose residue; three of the missing residues are potential phosphorylation sites. Acid hydrolases produced by these mutant cells were phosphorylated on the alpha 1,3-branch of the truncated oligosaccharide even when transport to the Golgi apparatus was inhibited. J774 cells produce normal high mannose oligosaccharides, but they secrete a large percentage of their newly synthesized acid hydrolases. The secreted enzymes contained primarily diphosphorylated units in which a phosphate was positioned to both the alpha 1,3- and alpha 1,6-branches of the core beta-linked mannose. J774 cells treated with deoxymannojirimycin continued to phosphorylate and to secrete acid hydrolases. The secreted hydrolases, however, contained only monophosphorylated oligosaccharides in which the phosphate was restricted to the alpha 1,6-branch. These results indicate that mannose residues within high mannose oligosaccharides impose constraints on the phosphorylation of their composite structures. We conclude that the two-step phosphorylation occurs as a result of a common phosphotransferase at both the pre-Golgi and Golgi locations and a change in the conformation of the oligosaccharides attached to the acid hydrolases through the action of Golgi-associated alpha-mannosidase I.     

Posttranslational association of immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting and secreting hybridomas

A rat monoclonal antibody specific for immunoglobulin (Ig) heavy chain binding protein (BiP) has allowed the examination of the association of BiP with assembling Ig precursors in mouse B lymphocyte-derived cell lines. The anti-BiP monoclonal antibody immunoprecipitates BiP along with noncovalently associated Ig heavy chains. BiP is a component of the endoplasmic reticulum and binds free intracellular heavy chains in nonsecreting pre-B (mu+, L-) cell lines or incompletely assembled Ig precursors in (H+, L+) secreting hybridomas and myelomas. In the absence of light chain synthesis, heavy chains remain associated with BiP and are not secreted. The association of BiP with assembling Ig molecules in secreting hybridomas is transient and is restricted to the incompletely assembled molecules which are found in the endoplasmic reticulum. BiP loses affinity and disassociates with Ig molecules when polymerization with light chain is complete. We propose that the association of BiP with Ig heavy chain precursors is a novel posttranslational processing event occurring in the endoplasmic reticulum. The Ig heavy chains associated with BiP are not efficiently transported from the endoplasmic reticulum to the Golgi apparatus. Therefore, BiP may prevent the premature escape and eventual secretion of incompletely assembled Ig molecules.     

The manganese cation disrupts membrane dynamics along the secretory pathway

The endoplasmic reticulum and Golgi apparatus play key roles in regulating the folding, assembly, and transport of newly synthesized proteins along the secretory pathway. We find that the divalent cation manganese disrupts the Golgi apparatus and endoplasmic reticulum (ER). The Golgi apparatus is fragmented into smaller dispersed structures upon manganese treatment. Golgi residents, such as TGN46, beta1,4-galactosyltransferase, giantin, and GM130, are still segregated and partitioned correctly into smaller stacked fragments in manganese-treated cells. The mesh-like ER network is substantially affected and peripheral ER elements are collapsed. These effects are consistent with manganese-mediated inhibition of motor proteins that link membrane organelles along the secretory pathway to the cytoskeleton. This divalent cation thus represents a new tool for studying protein secretion and membrane dynamics along the secretory pathway.     

Study of the endoproteolytic cleavage of platelet glycoprotein IIb using oligonucleotide-mediated mutagenesis.

The precursor of platelet membrane glycoprotein IIb (GPIIb) undergoes endoproteolytic cleavage into heavy and light chains post-translation. Endoproteolysis occurs within a 17-amino acid stretch of the precursor that contains 4 arginine residues, 3 in dibasic sequences [Lys-Arg (855-856) and Arg-Arg (858-859)] and a single arginine at 871. To determine the site of GPIIb cleavage and its role in the function of the glycoprotein IIb/IIIa heterodimer, we mutated arginine 856, the di-arginine sequence 858-859, and arginine 871 and coexpressed the mutants with glycoprotein IIIa (GPIIIa) in COS-1 cells. Each GPIIb mutant formed recombinant GPIIb-IIIa heterodimers, but mutants lacking arginine at 856 or 858-859 failed to undergo cleavage. Nevertheless, heterodimers containing the uncleaved GPIIb were expressed on the cell surface. Because endoproteolysis most often occurs after arginines in dibasic sequences, we next expressed GPIIb mutants containing lysine at 856 or aspartic acid at 855 with GPIIIa. Both mutants were cleaved and surface-expressed, indicating that the dibasic sequence at 858-859, but not at 855-856, is required for GPIIb cleavage. Lastly, we tested the function of GPIIb-IIIa containing uncleaved GPIIb by measuring adhesion of transfected cells to immobilized fibrinogen. We found no difference in the adhesion of cells expressing either wild-type or mutant GPIIb, indicating GPIIb-IIIa heterodimers containing uncleaved GPIIb maintain their ability to interact with fibrinogen.     

Giving protein traffic the green light

Most proteins that are secreted or expressed on a cell surface are synthesized on membrane polysomes and enter the endoplasmic reticulum (ER) as unfolded polypeptide chains. A complex series of interactions with resident enzymes and molecular chaperones ensure that these proteins are folded and assembled to achieve their correct tertiary structures before being transported to the Golgi and along the secretory pathway. However, the mechanism by which properly folded molecules are sorted from incompletely or improperly folded proteins and from the resident proteins that guide this process remains unclear.     

Regulation of nicotinic receptor trafficking by the transmembrane Golgi protein UNC-50

Nicotinic acetylcholine receptors (AChRs) are pentameric ligand-gated ion channels that mediate fast synaptic transmission at the neuromuscular junction (NMJ). After assembly in the endoplasmic reticulum (ER), AChRs must be transported to the plasma membrane through the secretory apparatus. Little is known about specific molecules that mediate this transport. Here we identify a gene that is required for subtype-specific trafficking of assembled nicotinic AChRs in Caenorhabditis elegans. unc-50 encodes an evolutionarily conserved integral membrane protein that localizes to the Golgi apparatus. In the absence of UNC-50, a subset of AChRs present in body-wall muscle are sorted to the lysosomal system and degraded. However, the trafficking of a second AChR type and of GABA ionotropic receptors expressed in the same muscle cells is not affected in unc-50 mutants. These results suggest that, in addition to ER quality control, assembled AChRs are sorted within the Golgi system by a mechanism that controls the amount of cell-surface AChRs in a subtype-specific way.     

Trans-Golgi Network—An Intersection of Trafficking Cell Wall Components

The cell wall, a crucial cell compartment, is composed of a network of polysaccharides and proteins, providing structural support and protection from external stimuli. While the cell wall structure and biosynthesis have been extensively studied, very little is known about the transport of polysaccharides and other components into the developing cell wall. This review focuses on endomembrane trafficking pathways involved in cell wall deposition. Cellulose synthase complexes are assembled in the Golgi, and are transported in vesicles to the plasma membrane. Non-cellulosic polysaccharides are synthesized in the Golgi apparatus, whereas cellulose is produced by enzyme complexes at the plasma membrane. Polysaccharides and enzymes that are involved in cell wall modification and assembly are transported by distinct vesicle types to their destinations; however, the precise mechanisms involved in selection, sorting and delivery remain to be identified. The endomembrane system orchestrates the delivery of Golgi-derived and possibly endocytic vesicles carrying cell wall and cell membrane components to the newly-formed cell plate. However, the nature of these vesicles, their membrane compositions, and the timing of their delivery are largely unknown. Emerging technologies such as chemical genomics and proteomics are promising avenues to gain insight into the trafficking of cell wall components.