Assembly of influenza hemagglutinin trimers and its role in intracellular transport

The hemagglutinin (HA) of influenza virus is a homotrimeric integral membrane glycoprotein. It is cotranslationally inserted into the endoplasmic reticulum as a precursor called HA0 and transported to the cell surface via the Golgi complex. We have, in this study, investigated the kinetics and cellular location of the assembly reaction that results in HA0 trimerization. Three independent criteria were used for determining the formation of quaternary structure: the appearance of an epitope recognized by trimer-specific monoclonal antibodies; the acquisition of trypsin resistance, a characteristic of trimers; and the formation of stable complexes which cosedimented with the mature HA0 trimer (9S20,w) in sucrose gradients containing Triton X-100. The results showed that oligomer formation is a posttranslational event, occurring with a half time of approximately 7.5 min after completion of synthesis. Assembly occurs in the endoplasmic reticulum, followed almost immediately by transport to the Golgi complex. A stabilization event in trimer structure occurs when HA0 leaves the Golgi complex or reaches the plasma membrane. Approximately 10% of the newly synthesized HA0 formed aberrant trimers which were not transported from the endoplasmic reticulum to the Golgi complex or the plasma membrane. Taken together the results suggested that formation of correctly folded quaternary structure constitutes a key event regulating the transport of the protein out of the endoplasmic reticulum. Further changes in subunit interactions occur as the trimers move along the secretory pathway.     

Monoclonal antibodies localize events in the folding, assembly, and intracellular transport of the influenza virus hemagglutinin glycoprotein

We used monoclonal antibodies that recognize monomeric and/or trimeric forms of the influenza virus hemagglutinin (HA) to study biosynthesis of this integral membrane protein in influenza virus-infected cells. We find the following: First, the globular head of the HA folds into its mature conformation in the endoplasmic reticulum prior to the assembly of HA monomers into trimers. Second, trimerization begins within 1 to 2 min following synthesis, with a half-time of approximately 5 min. Third, trimerization occurs only after the HA has been transported from the endoplasmic reticulum. Fourth, newly formed trimers are sensitive to acid-induced conformational alterations associated with viral fusion activity.     

The role of the endoplasmic reticulum in antigen processing. N-glycosylation of influenza hemagglutinin abrogates CD4+ cytotoxic T cell recognition of endogenously processed antigen.

Evidence is presented for an endogenous route of Ag processing for CD4+ T cell recognition of influenza hemagglutinin that requires obligatory traffic of de novo synthesized hemagglutinin across the lumen of the endoplasmic reticulum for processing in a cytosolic compartment. I-Ad-restricted T cell clones that recognize synthetic peptides corresponding to two distinct antigenic regions of the HA1 subunit, HA1 56-76 and HA1 177-199, are cytotoxic and, dependent on epitope specificity can recognize endogenously processed Ag and lyse class II+ target cells infected with a recombinant vaccinia-X31 HA virus. HA1 56-76 specific T cell clones fail to recognize (target cells infected with) influenza X31 viruses, containing a single residue change, HA1 63 Asp----Asn that introduces an oligosaccharide attachment site: Asp63Cys64Thr65. Recognition is restored, however, by tunicamycin treatment of mutant virus infected target cells. Inasmuch as N-glycosylation of nascent hemagglutinin polypeptides occurs in the lumen of the endoplasmic reticulum, this indicates a route of endogenous processing for hemagglutinin, requiring transport across the endoplasmic reticulum, which has been confirmed by the failure of CD4+ T cells to recognize a recombinant VACC-hemagglutinin virus in which the same single residue change, HA1 63 Asp----Asn has been introduced by site directed mutagenesis.     

Immunocytochemical Localization of Wheat Storage Proteins in Endosperm Cells 30 Days After Anthesis

Antisera against seven different wheat (Triticum aestivum L.)storage protein subfractions were characterized using (1) ELISAwith gliadins and low- and high-molecular weight glutenin subunitsand (2) electrophoresis (SDS-PAGE and acidic buffer PAGE) andimmunoblotting. The specificities of these antisera (polyclonalantibodies) and 13 monoclonal antibodies covered various patternsof reactivity with alpha-, beta-, gamma- and omega-gliadinsand low- and high-molecular weight glutenins. The antisera andantibodies were applied to ultrathin sections of wheat endospermtissue, from kernels fixed 30 d after anthesis, and were detectedby secondary antibodies tagged with either 5 or 15 nm gold particlesusing transmission electron microscopy. Labelling was denserwhen the small gold particles were used but irrespective ofgold particle size, labelling of polyclonal antisera predominatedwhen the endosperm cells were subjected to both mono- and polyclonalantibodies. Each of the antisera and monoclonal antibodies thatlabelled the protein bodies, labelled them more or less uniformly.This indicates that only one kind of protein body, containingall gliadin and glutenin subfractions, exists during this stageof grain development. Electron-dense globular inclusions foundin many protein bodies were not labelled. Label was also foundon protein-like material present in the lumen of the rough endoplasmicreticulum and on vesicles of the Golgi apparatus. Thus concentrationof storage proteins takes place both at the site of synthesis,the lumen of the rough endoplasmic reticulum, and at the siteof processing and transport, the vesicles of the Golgi apparatus.Fusions between these proteinaceous materials give rise to largerprotein bodies and ultimately to the protein matrix. Key words: Wheat, immunocytochemistry, protein bodies, rough endoplasmic reticulum, Golgi apparatus     

Oligomerization of the human cytomegalovirus major envelope glycoprotein complex gB (gp55-116).

The disulfide-linked glycoprotein B (gB; gp55-116) complex of human cytomegalovirus represents the most abundant and immunogenic component of the virion envelope. We have studied the oligomerization and transport of this molecule, using a series of murine monoclonal antibodies. Our results indicated that oligomerization of this molecule occurred shortly after its synthesis, with a half-time of maximal formation of approximately 25 min. The oligomeric form had an estimated mass of 340,000 Da and likely consisted of a homodimer of the gp55-116 complex. By using a conformation-specific monoclonal antibody, postoligomerization folding of this molecule was demonstrated. This event exhibited an unusually prolonged half-maximal time of approximately 160 min. Both oligomerization and folding occurred in the endoplasmic reticulum. Oligomerization and folding occurred in the absence of carbohydrate modifications, although likely at lower efficiency. Finally, the oligomeric and folded forms were shown to be transported to the surface of infected cells and infectious virions.     

The molecular chaperone calnexin associates with the vacuolar H(+)-ATPase from oat seedlings.

Acidification of endomembrane compartments by the vacuolar-type H(+)-ATPase (V-ATPase) is central to many cellular processes in eukaryotes, including osmoregulation and protein sorting. The V-ATPase complex consists of a peripheral sector (V1) and a membrane integral sector (V0); however, it is unclear how the multimeric enzyme is assembled. A 64-kD polypeptide that had copurified with oat V-ATPase subunits has been identified as calnexin, an integral protein on the endoplasmic reticulum. To determine whether calnexin interacted physically with the V-ATPase, microsomal membranes were Triton X-100 solubilized, and the protein-protein interaction was analyzed by coimmunoprecipitation. Monoclonal antibodies against calnexin precipitated both calnexin and V-ATPase subunits, including A and B and those of 44, 42, 36, 16, and 13 kD. A monoclonal antibody against subunit A precipitated the entire V-ATPase complex as well as calnexin and BiP, an endoplasmic reticulum lumen chaperone. The results support our hypothesis that both calnexin and BiP act as molecular chaperones in the folding and assembly of newly synthesized V1V0-ATPases at the endoplasmic reticulum.     

Membrane glycoprotein folding, oligomerization and intracellular transport: effects of dithiothreitol in living cells.

Using influenza hemagglutinin (HA0) and vesicular stomatitis virus G protein as model proteins, we have analyzed the effects of dithiothreitol (DTT) on conformational maturation and transport of glycoproteins in the secretory pathway of living cells. While DTT caused reduction of folding intermediates and misfolded proteins in the endoplasmic reticulum (ER), it did not affect molecules that had already acquired a mature trimeric conformation, whether present in the ER or elsewhere. The conversion to DTT resistance was therefore a pre-Golgi event. Reduction of folding intermediates was dependent on the intactness of the ER and on metabolic energy, suggesting cooperativity between DTT and ER folding factors. DTT did not inhibit most cellular functions, including ATP synthesis and protein transport within the secretory pathway. The results established DTT as an effective tool for analyzing the folding and compartmental distribution of proteins with disulfide bonds.     

A Scj1p homolog and folding catalysts present in dog pancreas microsomes

Dog pancreas microsomes represent the key components of the established model system for the analysis of protein transport into the mammalian endoplasmic reticulum. More recently, these microsomes were also employed in cell-free systems which address questions related to protein folding and protein degradation in the mammalian endoplasmic reticulum. In order to get at a complete picture of these undoubtedly related processes in the in vitro system we need to know all the proteins we are dealing with, and their respective stoichiometries. Here we give a progress report on our attempts to identify and to quantify the soluble molecular chaperones and folding catalysts which are present in the lumen of dog pancreas microsomes. Eventually, we will need to know how the in vitro system compares with the situation in intact pancreatic cells as well as in other cells.     

Effect of anti-ER antibodies within the ER lumen of living cells

We describe the production and partial characterization of 12 monoclonal antibodies raised against a preparation of endoplasmic reticulum membranes obtained from Xenopus laevis liver. Four of the antibodies cross-react with liver melanocytes; two of the antibodies recognize extracellular antigens, whilst the remaining six recognize antigens present in hepatocytes. The concentrations of these latter antigens increase markedly in livers stimulated by estrogen. Western blotting analysis revealed that the six anti-hepatocyte monoclonal antibodies recognize at least five different antigens whose molecular weights are 14K, 18K, 19K, 43K, and 125K. The possible functional involvement of the various antigens in the secretory pathway was investigated using Xenopus oocytes as a surrogate secretory system. The mRNAs coding for the monoclonal antibodies were injected into oocytes and the resulting immunoglobulin chains were translated and assembled into active anti-ER antibodies inside the lumen of the ER. The effect on secretion was then observed. Our data indicate that the binding of antibodies to most antigens of the endoplasmic reticulum membrane may result in a blockage of secretion.     

Mutations in the cytoplasmic domain of the influenza virus hemagglutinin affect different stages of intracellular transport

Mutations have been introduced into the cloned DNA sequences coding for influenza virus hemagglutinin (HA), and the resulting mutant genes have been expressed in simian cells by the use of SV40-HA recombinant viral vectors. In this study we analyzed the effect of specific alterations in the cytoplasmic domain of the HA molecule on its rate of biosynthesis and transport, cellular localization, and biological activity. Several of the mutants displayed abnormalities in the pathway of transport from the endoplasmic reticulum to the cell surface. One mutant HA remained within the endoplasmic reticulum; others were delayed in reaching the Golgi apparatus after core glycosylation had been completed in the endoplasmic reticulum, but then progressed at a normal rate from the Golgi apparatus to the cell surface; another was delayed in transport from the Golgi apparatus to the plasma membrane. However, two mutants were indistinguishable from wild-type HA in their rate of movement from the endoplasmic reticulum through the Golgi apparatus to the cell surface. We conclude that changes in the cytoplasmic domain can powerfully influence the rate of intracellular transport and the efficiency with which HA reaches the cell surface. Nevertheless, absolute conservation of this region of the molecule is not required for maturation and efficient expression of a biologically active HA on the surface of infected cells.     

Calcium is required for folding of newly made subunits of the asialoglycoprotein receptor within the endoplasmic reticulum.

By resolving immunoprecipitates on nonreducing sodium dodecyl sulfate gels, we have detected several disulfide-bonded intermediates in folding within the endoplasmic reticulum of newly made H1 subunits of the asialoglycoprotein receptor. H1 in the endoplasmic reticulum (ER) can be partially unfolded by treatment of cells with dithiothreitol, but H1 in Golgi or post-Golgi organelles is resistant to such unfolding. This defines a late step in H1 folding that occurs just prior to exit from the ER. Depletion of calcium from the endoplasmic reticulum, either by treatment with A23187 or thapsigargin, has no effect on folding or secretion of newly made albumin, but totally blocks H1 maturation from the ER. No ER intermediates in H1 folding are formed in cells treated with A23187 or thapsigargin, indicating that at least an early step in H1 folding requires a high Ca2+ concentration in the ER lumen. As judged by cross-linking experiments, formation of H1 dimers and trimers occurs immediately after biosynthesis of the peptide chain, before monomer folding, and occurs normally in cells in which ER Ca2+ is reduced and where the monomer never folds properly. Calcium is essential for the asialoglycoprotein receptor to bind galactose, and our results suggest that Ca2+ is also essential for the receptor polypeptides to fold in the ER.     

Different roles of individual N-linked oligosaccharide chains in folding, assembly, and transport of the simian virus 5 hemagglutinin-neuraminidase.

The role of N-linked glycosylation in protein maturation and transport has been studied by using the simian virus 5 hemagglutinin-neuraminidase (HN) protein, a model class II integral membrane glycoprotein. The sites of N-linked glycosylation on HN were identified by eliminating each of the potential sites for N-linked glycosylation by oligonucleotide-directed mutagenesis on a cDNA clone. Expression of the mutant HN proteins in eucaryotic cells indicated that four sites are used in the HN glycoprotein for the addition of N-linked oligosaccharide chains. These functional glycosylation sites were systematically eliminated in various combinations from HN to form a panel of mutants in which the roles of individual carbohydrate chains and groups of carbohydrate chains could be analyzed. Alterations in the normal glycosylation pattern resulted in the impairment of HN protein folding and assembly which, in turn, affected the intracellular transport of HN. The severity of the consequences on HN maturation depended on both the number of deleted carbohydrate sites and their position in the HN molecule. Analysis of the reactivity pattern of HN conformation-specific monoclonal antibodies with the mutant HN proteins indicated that one specific carbohydrate chain plays a major role in promoting the correct folding of HN. Another carbohydrate chain, which is not essential for the initial folding of HN was found to play a role in preventing the aggregation of HN oligomers. The HN molecules which were misfolded, owing to their altered glycosylation pattern, were retained in the endoplasmic reticulum. Double-label immunofluorescence experiments indicate that misfolded HN and folded HN are segregated in the same cell. Misfolded HN forms disulfide-linked aggregates and is stably associated with the resident endoplasmic reticulum protein, GRP78-BiP, whereas wild-type HN forms a specific and transient complex with GRP78-BiP during its folding process.     

Protein folding and translocation across the endoplasmic reticulum membrane (Review)

Proteins destined for secretion are translocated across or inserted into the endoplasmic reticulum membrane whereupon they fold and assemble to their native state before their subsequent transport to the Golgi apparatus. Proteins that fail to fold correctly are translocated back across the endoplasmic reticulum membrane to the cytosol where they become substrates for the cytosolic degradative machinery. Central to translocation is a protein pore in the membrane called the translocon that allows passage of proteins in and out of the endoplasmic reticulum. It is clear that the conformation of the polypeptide chain influences the translocation process and that there is a temporal relationship between modification of the chain, translocation and folding. This review will consider when and how the polypeptide chain folds, and how this might influence translocation into and out of the ER; and discuss how protein folding might affect post-translational modification of the polypeptide chain following translocation into the ER lumen.     

The effects of foreign transmembrane domains on the biosynthesis of the influenza virus hemagglutinin

Eleven chimeric proteins were created in which the transmembrane, the cytoplasmic, or both topological domains of the influenza virus hemagglutinin (HA) were replaced with those from five other glycoproteins. All of the chimeric HAs reached the cell surface but appeared to differ in the degree to which they were stably folded. Comparisons of the rates of folding, passage into the Golgi, and arrival at the plasma membrane of wild-type HA and the chimeric proteins suggest that formation of a stable HA trimer is not an absolute requirement for export from the endoplasmic reticulum. In addition, there appear to be at least two steps at which the rate of transport can be altered during exocytosis, one occurring before and the other after the trimming of oligosaccharides by Golgi mannosidases. Certain of the chimeras differed from HA in their ability to pass through each of these steps. Replacement of the HA transmembrane domain with the analogous sequences from other proteins affected folding and transport of the chimeric HAs in ways that suggest that the HA transmembrane sequences form a specific structure in the membrane that differs from that formed by analogous sequences from the other proteins.     

Protein folding and translocation across the endoplasmic reticulum membrane

Proteins destined for secretion are translocated across or inserted into the endoplasmic reticulum membrane whereupon they fold and assemble to their native state before their subsequent transport to the Golgi apparatus. Proteins that fail to fold correctly are translocated back across the endoplasmic reticulum membrane to the cytosol where they become substrates for the cytosolic degradative machinery. Central to translocation is a protein pore in the membrane called the translocon that allows passage of proteins in and out of the endoplasmic reticulum. It is clear that the conformation of the polypeptide chain influences the translocation process and that there is a temporal relationship between modification of the chain, translocation and folding. This review will consider when and how the polypeptide chain folds, and how this might influence translocation into and out of the ER; and discuss how protein folding might affect post-translational modification of the polypeptide chain following translocation into the ER lumen.     

FAD transport and FAD-dependent protein thiol oxidation in rat liver microsomes

The transport of FAD and its effect on disulfide bond formation was investigated in rat liver microsomal vesicles. By measuring the intravesicular FAD-accessible space, we observed that FAD permeates across the microsomal membrane and accumulates in the lumen. Rapid filtration experiments also demonstrated the uptake and efflux of the compound, which could be inhibited by atractyloside and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. FAD entering the lumen promoted the oxidation of protein thiols and increased the intraluminal oxidation of glucose-6-phosphate. These findings support the notion that, similar to yeast, free FAD may have a decisive role in the mechanism of oxidative protein folding in the endoplasmic reticulum lumen of mammalian cells.     

Role of conserved glycosylation sites in maturation and transport of influenza A virus hemagglutinin.

The role of three N-linked glycans which are conserved among various hemagglutinin (HA) subtypes of influenza A viruses was investigated by eliminating the conserved glycosylation (cg) sites at asparagine residues 12 (cg1), 28 (cg2), and 478 (cg3) by site-directed mutagenesis. An additional mutant was constructed by eliminating the cg3 site and introducing a novel site 4 amino acids away, at position 482. Expression of the altered HA proteins in eukaryotic cells by a panel of recombinant vaccinia viruses revealed that rates and efficiency of intracellular transport of HA are dependent upon both the number of conserved N-linked oligosaccharides and their respective positions on the polypeptide backbone. Glycosylation at two of the three sites was sufficient for maintenance of transport of the HA protein. Conserved glycosylation at either the cg1 or cg2 site alone also promoted efficient transport of HA. However, the rates of transport of these mutants were significantly reduced compared with the wild-type protein or single-site mutants of HA. The transport of HA proteins lacking all three conserved sites or both amino-terminally located sites was temperature sensitive, implying that a polypeptide folding step had been affected. Analysis of trimer assembly by these mutants indicated that the presence of a single oligosaccharide in the stem domain of the HA molecule plays an important role in preventing aggregation of molecules in the endoplasmic reticulum, possibly by maintaining the hydrophilic properties of this domain. The conformational change observed after loss of all three conserved oligosaccharides also resulted in exposure of a normally mannose-rich oligosaccharide at the tip of the large stem helix that allowed its conversion to a complex type of structure. Evidence was also obtained suggesting that carbohydrate-carbohydrate interactions between neighboring oligosaccharides at positions 12 and 28 influence the accessibility of the cg2 oligosaccharide for processing enzymes. We also showed that terminal glycosylation of the cg3 oligosaccharide is site specific, since shifting of this site 4 amino acids away, to position 482, yielded an oligosaccharide that was arrested in the mannose-rich form. In conclusion, carbohydrates at conserved positions not only act synergistically by promoting and stabilizing a conformation compatible with transport, they also enhance trimerization and/or folding rates of the HA protein.     

Stress on redox

Redox imbalance in the endoplasmic reticulum lumen is the most frequent cause of endoplasmic reticulum stress and consequent apoptosis. The mechanism involves the impairment of oxidative protein folding, the accumulation of unfolded/misfolded proteins in the lumen and the initiation of the unfolded protein response. The participation of several redox systems (glutathione, ascorbate, FAD, tocopherol, vitamin K) has been demonstrated in the process. Recent findings have attracted attention to the possible mechanistic role of luminal pyridine nucleotides in the endoplasmic reticulum stress. The aim of this minireview is to summarize the luminal redox systems and the redox sensing mechanisms of the endoplasmic reticulum.