Autocatalytic activation of influenza hemagglutinin

Enveloped viruses contain surface proteins that mediate fusion between the viral and target cell membranes following an activating stimulus. Acidic pH induces the influenza virus fusion protein hemagglutinin (HA) via irreversible refolding of a trimeric conformational state leading to exposure of hydrophobic fusion peptides on each trimer subunit. Herein, we show that cells expressing fowl plague virus HA demonstrate discrete switching behavior with respect to the HA conformational change. Partially activated states do not exist at the scale of the cell, activation of HA leads to aggregation of cell surface trimers, and newly synthesized HA refold spontaneously in the presence of previously activated HA. These observations imply a feedback mechanism involving self-catalyzed refolding of HA and thus suggest a mechanism similar to the autocatalytic refolding and aggregation of prions.     

Intermonomer disulfide bonds impair the fusion activity of influenza virus hemagglutinin.

At a low pH, the influenza virus hemagglutinin (HA) undergoes conformational changes that promote membrane fusion. While the critical role of fusion peptide release from the trimer interface has been demonstrated previously, the role of globular head dissociation in the overall fusion mechanism remains unclear. To investigate this question, we have analyzed in detail the fusion activity and low pH-induced conformational changes of a mutant, Cys-HA, in which the globular head domains are locked together by engineered intermonomer disulfide bonds (L. Godley, J. Pfeifer, D. Steinhauer, B. Ely, G. Shaw, R. Kaufmann, E. Suchanek, C. Pabo, J. J. Skehel, D. C. Wiley, and S. Wharton, Cell 68:635-645, 1992). In this paper, we show that Cys-HA expressed on the cell surface is predominantly a disulfide-bonded trimer. Cell surface Cys-HA is impaired in its membrane fusion activity, as demonstrated by both content-mixing and lipid-mixing fusion assays. It is also impaired in its ability to change conformation at a low pH, as assessed by proteinase K sensitivity. The fusion activity and low pH-induced conformational changes of cell surface Cys-HA are, however, restored to nearly wild-type levels upon reduction of the intermonomer disulfide bonds. By using a set of conformation-specific monoclonal and anti-peptide antibodies, we found that purified Cys-HA trimers are impaired in changes that occur in the globular head domain interface. In addition, changes that occur at a great distance from the engineered intermonomer disulfide bonds, notably release of the fusion peptides, are also impaired. Our results are discussed with respect to current views of the fusion-active conformation of the HA trimer.     

Role of hemagglutinin surface density in the initial stages of influenza virus fusion: lack of evidence for cooperativity

Membrane fusion mediated by influenza virus hemagglutinin (HA) is believed to proceed via the cooperative action of multiple HA trimers. To determine the minimal number of HA trimers required to trigger fusion, and to assess the importance of cooperativity between these HA trimers, we have generated virosomes containing coreconstituted HAs derived from two strains of virus with different pH dependencies for fusion, X-47 (optimal fusion at pH 5.1; threshold at pH 5.6) and A/Shangdong (optimal fusion at pH 5.6; threshold at pH 6.0), and measured fusion of these virosomes with erythrocyte ghosts by a fluorescence lipid mixing assay. Virosomes with different X-47-to-A/Shangdong HA ratios, at a constant HA-to-lipid ratio, showed comparable ghost-binding activities, and the low-pH-induced conformational change of A/Shangdong HA did not affect the fusion activity of X-47 HA. The initial rate of fusion of these virosomes at pH 5.7 increased directly proportional to the surface density of A/Shangdong HA, and a single A/Shangdong trimer per virosome appeared to suffice to induce fusion. The reciprocal of the lag time before the onset of fusion was directly proportional to the surface density of fusion-competent HA. These results support the notion that there is no cooperativity between HA trimers during influenza virus fusion.     

Sugar-induced conformational change found in the HA-33/HA-17 trimer of the botulinum toxin complex

Large-sized botulinum toxin complex (L-TC) is formed by conjugation of neurotoxin, nontoxic nonhemagglutinin and hemagglutinin (HA) complex. The HA complex is formed by association of three HA-70 molecules and three HA-33/HA-17 trimers, comprised of a single HA-17 and two HA-33 proteins. The HA-33/HA-17 trimer isolated from serotype D L-TC has the ability to bind to and penetrate through the intestinal epithelial cell monolayer in a sialic acid-dependent manner, and thus it plays an important role in toxin delivery through the intestinal cell wall. In this study, we determined the solution structure of the HA-33/HA-17 trimer by using small-angle X-ray scattering (SAXS). The SAXS image of HA-33/HA-17 exhibited broadly similar appearance to the crystal image of the complex. On the other hand, in the presence of N-acetylneuraminic acid, glucose and galactose, the solution structure of the HA-33/HA-17 trimer was drastically altered compared to the structure in the absence of the sugars. Sugar-induced structural change of the HA-33/HA-17 trimer may contribute to cell binding and subsequent transport across the intestinal cell layer.     

ELPylated haemagglutinins produced in tobacco plants induce potentially neutralizing antibodies against H5N1 viruses in mice

Reducing the cost of vaccine production is a key priority for veterinary research, and the possibility of heterologously expressing antigen in plants provides a particularly attractive means of achieving this. Here, we report the expression of the avian influenza virus haemagglutinin (AIV HA) in tobacco, both as a monomer and as a trimer in its native and its ELPylated form. We firstly presented evidence to produce stabilized trimers of soluble HA in plants. ELPylation of these trimers does not influence the trimerization. Strong expression enhancement in planta caused by ELPylation was demonstrated for trimerized H5‐ELP. ELPylated trimers could be purified by a membrane‐based inverse transition cycling procedure with the potential of successful scale‐up. The trimeric form of AIV HA was found to enhance the HA‐specific immune response compared with the monomeric form. Plant‐derived AIV HA trimers elicited potentially neutralizing antibodies interacting with both homologous virus‐like particles from plants and heterologous inactivated AIV. ELPylation did not influence the functionality and the antigenicity of the stabilized H5 trimers. These data allow further developments including scale‐up of production, purification and virus challenge experiments with the final goal to achieve suitable technologies for efficient avian flu vaccine production.     

Cell‐free production of trimeric influenza hemagglutinin head domain proteins as vaccine antigens

In order to effectively combat pandemic influenza threats, there is a need for more rapid and robust vaccine production methods. In this article, we demonstrate E. coli‐based cell‐free protein synthesis (CFPS) as a method to rapidly produce domains from the protein hemagglutinin (HA), which is present on the surface of the influenza virus. The portion of the HA coding sequence for the “head” domain from the 2009 pandemic H1N1 strain was first optimized for E. coli expression. The protein domain was then produced in CFPS reactions and purified in soluble form first as a monomer and then as a trimer by a C‐terminal addition of the T4 bacteriophage foldon domain. Production of soluble trimeric HA head domain was enhanced by introducing stabilizing amino acid mutations to the construct in order to avoid aggregation. Trimerization was verified using size exclusion HPLC, and the stabilized HA head domain trimer was more effectively recognized by antibodies from pandemic H1N1 influenza vaccine recipients than was the monomer and also bound to sialic acids more strongly, indicating that the trimers are correctly formed and could be potentially effective as vaccines. Biotechnol. Bioeng. 2012; 109: 2962–2969. © 2012 Wiley Periodicals, Inc.     

Clioquinol targets zinc to lysosomes in human cancer cells

A panel of 52 murine monoclonal antibodies was found to recognize antigenic determinants that had been conserved among all major genetic subgroups of the H5N1 avian influenza virus prevalent since 1997. We screened a phage display library for peptides recognized by one such antibody (8H5). We analysed the specificity of 8H5 for reactive peptides presented as fusion proteins of HBc (hepatitis B core protein) and HEV (hepatitis E virus) structural protein, p239. This was then related to the specificity of the native HA (haemagglutinin) molecule by virtue of the capacity of fusion proteins to compete for 8H5 binding with different strains of H5N1 virus and the reactivity of antisera generated against fusion proteins to bind native HA molecules, and to inhibit haemagglutination and arrest infection by the virus. Nine reactive peptides of different amino acid sequences were identified, six of which were also reactive with the antibody in association with HBc and four were in association with p239. Binding occurred with the dimeric form of the four p239-fusion proteins and one of the HBc-fusion proteins, but not with the monomeric form. The HBc-fusion proteins blocked 8H5 binding with four strains of H5N1 influenza virus. Mouse antisera generated against fusion proteins bound to HA molecules, but did not inhibit haemagglutination or arrest H5N1 infection. Our findings indicate that 8H5 recognizes discontinuous sites presented by secondary and possibly higher structural orders of the peptides in spatially favourable positions for binding with the antibody, and that the peptides partially mimic the native 8H5 epitopes on the H5N1 virus.     

Comparative efficacy of neutralizing antibodies elicited by recombinant hemagglutinin proteins from avian H5N1 influenza virus

Although the human transmission of avian H5N1 virus remains low, the prevalence of this highly pathogenic infection in avian species underscores the need for a preventive vaccine that can be made without eggs. Here, we systematically analyze various forms of recombinant hemagglutinin (HA) protein for their potential efficacy as vaccines. Monomeric, trimeric, and oligomeric H5N1 HA proteins were expressed and purified from either insect or mammalian cells. The immunogenicity of different recombinant HA proteins was evaluated by measuring the neutralizing antibody response. Neutralizing antibodies to H5N1 HA were readily generated in mice immunized with the recombinant HA proteins, but they varied in potency depending on their multimeric nature and cell source. Among the HA proteins, a high-molecular-weight oligomer elicited the strongest antibody response, followed by the trimer; the monomer showed minimal efficacy. The coexpression of another viral surface protein, neuraminidase, did not affect the immunogenicity of the HA oligomer, as expected from the immunogenicity of trimers produced from insect cells. As anticipated, HA expressed in mammalian cells without NA retained the terminal sialic acid residues and failed to bind alpha2,3-linked sialic acid receptors. Taken together, these results suggest that recombinant HA proteins as individual or oligomeric trimers can elicit potent neutralizing antibody responses to avian H5N1 influenza viruses.     

Reversible Association of the Hemagglutinin Subcomplex,HA-33/HA-17 Trimer,with the Botulinum Toxin Complex

Botulinum neurotoxin (BoNT) associates with nontoxic proteins, either a nontoxic nonhemagglutinin (NTNHA) or the complex of NTNHA and hemagglutinin (HA), to form M- or L-toxin complexes (TCs). Single BoNT and NTNHA molecules are associated and form M-TC. A trimer of the 70-kDa HA protein (HA-70) attaches to the M-TC to form M-TC/HA-70. Further, 1–3 arm-like 33- and 17-kDa HA molecules (HA-33/HA-17 trimer), consisting of 1 HA-17 protein and 2 HA-33 proteins, can attach to the M-TC/HA-70 complex, yielding 1-, 2-, and 3-arm L-TC. In this study, the purified 1- and 2-arm L-TCs spontaneously converted into another L-TC species after acquiring the HA-33/HA-17 trimer from other TCs during long-term storage and freezing/thawing. Transmission electron microscopy analysis provided evidence of the formation of detached HA-33/HA-17 trimers in the purified TC preparation. These findings provide evidence of reversible association/dissociation of the M-TC/HA-70 complex with the HA-33/HA-17 trimers, as well as dynamic conversion of the quaternary structure of botulinum TC in culture.     

Configuration of influenza hemagglutinin fusion peptide monomers and oligomers in membranes

The 20 N-terminal residues of the HA2 subunit of influenza hemagglutinin (HA), known as the fusion peptide, play a crucial role in membrane fusion. Molecular dynamics simulations with implicit solvation are employed here to study the structure and orientation of the fusion peptide in membranes. As a monomer the α-helical peptide adopts a shallow, slightly tilted orientation along the lipid tail-head group interface. The average angle of the peptide with respect to membrane plane is 12.4 °. We find that the kinked structure proposed on the basis of NMR data is not stable in our model because of the high energy cost related to the membrane insertion of polar groups. Because hemagglutinin-mediated membrane fusion is promoted by low pH, we examined the effect of protonation of the Glu and Asp residues. The configurations of the protonated peptides were slightly deeper in the membrane but at similar angles. Finally, because HA is a trimer, we modeled helical fusion peptide trimers. We find that oligomerization affects the insertion depth of the peptide and its orientation with respect to the membrane: a trimer exhibits equally favorable configurations in which some or all of the helices in the bundle insert obliquely deep into the membrane.     

GPI- and transmembrane-anchored influenza hemagglutinin differ in structure and receptor binding activity

We investigated the influence of a glycosylphosphatidylinositol (GPI) anchor on the ectodomain of the influenza hemagglutinin (HA) by replacing the wild type (wt) transmembrane and cytoplasmic domains with a GPI lipid anchor. GPI-anchored HA (GPI-HA) was transported to the cell surface with equal efficiency and at the same rate as wt-HA. Like wt-HA, cell surface GPI-HA, and its ectodomain released with the enzyme PI-phospholipase C (PI-PLC), were 9S trimers. Compared to wt-HA, the GPI-HA ectodomain underwent additional terminal oligosaccharide modifications; some of these occurred near the receptor binding pocket and completely inhibited the ability of GPI-HA to bind erythrocytes. Growth of GPI-HA-expressing cells in the presence of the mannosidase I inhibitor deoxymannojirimycin (dMM) abrogated the differences in carbohydrate modification and restored the ability of GPI-HA to bind erythrocytes. The ectodomain of GPI-HA produced from cells grown in the presence or absence of dMM underwent characteristic low pH-induced conformational changes (it released its fusion peptides and became hydrophobic and proteinase sensitive) but at 0.2 and 0.4 pH units higher than wt-HA, respectively. These results demonstrate that although GPI-HA forms a stable trimer with characteristics of the wt, its structure is altered such that its receptor binding activity is abolished. Our results show that transmembrane and GPI-anchored forms of the same ectodomain can exhibit functionally important differences in structure at a great distance from the bilayer.     

A mechanism of protein-mediated fusion: coupling between refolding of the influenza hemagglutinin and lipid rearrangements.

Although membrane fusion mediated by influenza virus hemagglutinin (HA) is the best characterized example of ubiquitous protein-mediated fusion, it is still not known how the low-pH-induced refolding of HA trimers causes fusion. This refolding involves 1) repositioning of the hydrophobic N-terminal sequence of the HA2 subunit of HA ("fusion peptide"), and 2) the recruitment of additional residues to the alpha-helical coiled coil of a rigid central rod of the trimer. We propose here a mechanism by which these conformational changes can cause local bending of the viral membrane, priming it for fusion. In this model fusion is triggered by incorporation of fusion peptides into viral membrane. Refolding of a central rod exerts forces that pull the fusion peptides, tending to bend the membrane around HA trimer into a saddle-like shape. Elastic energy drives self-assembly of these HA-containing membrane elements in the plane of the membrane into a ring-like cluster. Bulging of the viral membrane within such cluster yields a dimple growing toward the bound target membrane. Bending stresses in the lipidic top of the dimple facilitate membrane fusion. We analyze the energetics of this proposed sequence of membrane rearrangements, and demonstrate that this simple mechanism may explain some of the known phenomenological features of fusion.     

Configuration of influenza hemagglutinin fusion peptide monomers and oligomers in membranes

The 20 N-terminal residues of the HA2 subunit of influenza hemagglutinin (HA), known as the fusion peptide, play a crucial role in membrane fusion. Molecular dynamics simulations with implicit solvation are employed here to study the structure and orientation of the fusion peptide in membranes. As a monomer the alpha-helical peptide adopts a shallow, slightly tilted orientation along the lipid tail-head group interface. The average angle of the peptide with respect to membrane plane is 12.4 degrees . We find that the kinked structure proposed on the basis of NMR data is not stable in our model because of the high energy cost related to the membrane insertion of polar groups. Because hemagglutinin-mediated membrane fusion is promoted by low pH, we examined the effect of protonation of the Glu and Asp residues. The configurations of the protonated peptides were slightly deeper in the membrane but at similar angles. Finally, because HA is a trimer, we modeled helical fusion peptide trimers. We find that oligomerization affects the insertion depth of the peptide and its orientation with respect to the membrane: a trimer exhibits equally favorable configurations in which some or all of the helices in the bundle insert obliquely deep into the membrane.     

Heptapeptide ligands against receptor-binding sites of influenza hemagglutinin toward anti-influenza therapy

The initial attachment of influenza virus to cells is the binding of hemagglutinin (HA) to the sialyloligosaccharide receptor; therefore, the small molecules that inhibit the sugar–protein interaction are promising as HA inhibitors to prevent the infection. We herein demonstrate that sialic acid-mimic heptapeptides are identified through a selection from a primary library against influenza virus HA. In order to obtain lead peptides, an affinity selection from a phage-displayed random heptapeptide library was performed with the HAs of the H1 and H3 strains, and two kinds of the HA-binding peptides were identified. The binding of the peptides to HAs was inhibited in the presence of sialic acid, and plaque assays indicated that the corresponding N-stearoyl peptide strongly inhibited infections by the A/Aichi/2/68 (H3N2) strain of the virus. Alanine scanning of the peptides indicated that arginine and proline were responsible for binding. The affinities of several mutant peptides with single-amino-acid substitutions against H3 HA were determined, and corresponding docking studies were performed. A Spearman analysis revealed a correlation between the affinity of the peptides and the docking study. These results provide a practicable method to design of peptide-based HA inhibitors that are promising as anti-influenza drugs.     

Inhibition of Influenza A Virus Infection In Vitro by Peptides Designed In Silico

Influenza A viruses are enveloped, segmented negative single-stranded RNA viruses, capable of causing severe human respiratory infections. Currently, only two types of drugs are used to treat influenza A infections, the M2 H⁺ ion channel blockers (amantadine and rimantadine) and the neuraminidase inhibitors (NAI) (oseltamivir and zanamivir). Moreover, the emergence of drug-resistant influenza A virus strains has emphasized the need to develop new antiviral agents to complement or replace the existing drugs. Influenza A virus has on the surface a glycoprotein named hemagglutinin (HA) which due to its important role in the initial stage of infection: receptor binding and fusion activities of viral and endosomal membranes, is a potential target for new antiviral drugs. In this work we designed nine peptides using several bioinformatics tools. These peptides were derived from the HA1 and HA2 subunits of influenza A HA with the aim to inhibit influenza A virus infection. The peptides were synthetized and their antiviral activity was tested in vitro against several influenza A viral strains: Puerto Rico/916/34 (H1N1), (H1N1)pdm09, swine (H1N1) and avian (H5N2). We found these peptides were able to inhibit the influenza A viral strains tested, without showing any cytotoxic effect. By docking studies we found evidence that all the peptides were capable to bind to the viral HA, principally to important regions on the viral HA stalk, thus could prevent the HA conformational changes required to carry out its membranes fusion activity.     

Acid-induced changes in thermal stability and fusion activity of influenza hemagglutinin

The conformational and thermal stability of full-length hemagglutinin (HA) of influenza virus (strain X31) has been investigated using a combination of differential scanning calorimetry (DSC), analytical ultracentrifugation, fluorescence, and circular dichroism (CD) spectroscopy as a function of pH. HA sediments as a rosette comprised of 5-6 trimers (31-35 S) over the pH range of 7.4-5.4. The DSC profile of HA in the native state at pH 7.4 is characterized by a single cooperative endotherm with a transition temperature (Tm) of 66 degrees C and unfolding enthalpy (DeltaH(cal)) of 800 kcal x (mol of trimer)(-1). Upon acidification to pH 5.4, there is a significant decrease in the transition temperature (from 66 to 45 degrees C), unfolding enthalpy [from 800 to 260 kcal x (mol of trimer)(-1)], and DeltaH(cal)/DeltaH(vH) ratio (from 3.0 to approximately 1.3). Whereas the far- and near-UV ellipticities are maintained over this pH range, there is an acid-induced increase in surface hydrophobicity and decrease in intrinsic tryptophanyl fluorescence. The major contribution to the DSC endotherm arises from unfolding HA1 domains. The relationship between acid-induced changes in thermal stability and the fusion activity of HA has been examined by evaluating the kinetics and extent of fusion of influenza virus with erythrocytes over the temperature and pH range of the DSC measurements. Surprisingly, X31 influenza virus retains its fusion activity at acidic pH and temperatures significantly below the unfolding transition of HA. This finding is consistent with the notion that the fusion activity of influenza virus may involve structural changes of only a small fraction of HA molecules.     

Stochastic Fusion Simulations and Experiments Suggest Passive and Active Roles of Hemagglutinin during Membrane Fusion

Influenza enters the host cell cytoplasm by fusing the viral and host membrane together. Fusion is mediated by hemagglutinin (HA) trimers that undergo conformational change when acidified in the endosome. It is currently debated how many HA trimers, w, and how many conformationally changed HA trimers, q, are minimally required for fusion. Conclusions vary because there are three common approaches for determining w and q from fusion data. One approach correlates the fusion rate with the fraction of fusogenic HA trimers and leads to the conclusion that one HA trimer is required for fusion. A second approach correlates the fusion rate with the total concentration of fusogenic HA trimers and indicates that more than one HA trimer is required. A third approach applies statistical models to fusion rate data obtained at a single HA density to establish w or q and suggests that more than one HA trimer is required. In this work, all three approaches are investigated through stochastic fusion simulations and experiments to elucidate the roles of HA and its ability to bend the target membrane during fusion. We find that the apparent discrepancies among the results from the various approaches may be resolved if nonfusogenic HA participates in fusion through interactions with a fusogenic HA. Our results, based on H3 and H1 serotypes, suggest that three adjacent HA trimers and one conformationally changed HA trimer are minimally required to induce membrane fusion (w = 3 and q = 1).     

Stochastic Fusion Simulations and Experiments Suggest Passive and Active Roles of Hemagglutinin during Membrane Fusion

Influenza enters the host cell cytoplasm by fusing the viral and host membrane together. Fusion is mediated by hemagglutinin (HA) trimers that undergo conformational change when acidified in the endosome. It is currently debated how many HA trimers, w, and how many conformationally changed HA trimers, q, are minimally required for fusion. Conclusions vary because there are three common approaches for determining w and q from fusion data. One approach correlates the fusion rate with the fraction of fusogenic HA trimers and leads to the conclusion that one HA trimer is required for fusion. A second approach correlates the fusion rate with the total concentration of fusogenic HA trimers and indicates that more than one HA trimer is required. A third approach applies statistical models to fusion rate data obtained at a single HA density to establish w or q and suggests that more than one HA trimer is required. In this work, all three approaches are investigated through stochastic fusion simulations and experiments to elucidate the roles of HA and its ability to bend the target membrane during fusion. We find that the apparent discrepancies among the results from the various approaches may be resolved if nonfusogenic HA participates in fusion through interactions with a fusogenic HA. Our results, based on H3 and H1 serotypes, suggest that three adjacent HA trimers and one conformationally changed HA trimer are minimally required to induce membrane fusion (w = 3 and q = 1).     

Introduction of intersubunit disulfide bonds in the membrane-distal region of the influenza hemagglutinin abolishes membrane fusion activity.

Influenza virus hemagglutinin (HA) mediates viral entry into cells by a low pH-induced membrane fusion event in endosomes. A number of structural changes occur throughout the length of HA at the pH of fusion. To probe their significance and their necessity for fusion activity, we have prepared a site-directed mutant HA containing novel intersubunit disulfide bonds designed to cross-link covalently the membrane-distal domains of the trimer. These mutations inhibited the low pH-induced conformational changes and prevented HA-mediated membrane fusion; conditions that reduced the novel disulfide bonds restored membrane fusion activity. We conclude that structural rearrangements in the membrane distal region of the HA are required for membrane fusion activity.     

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.