The incoherence of the vesicle theory of protein secretion

The rates at which cells secrete peptides and proteins must on average equal their rate of synthesis. This basic equality has unanticipated and seemingly categorical negative consequences for the vesicle theory of protein secretion. This is because the transport mechanisms it proposes, such as the budding and fusion of small vesicles and secretion by exocytosis, are not capable of balancing forces. What follows is an account of the analysis that leads to this conclusion.     

Pancreatic acinar cell: New insights into the control of secretion

Pancreatic acinar cells secrete fluid and digestive enzymes. Both types of secretion are activated by a rise in intracellular calcium but how the stimulus-secretion cascade actually regulates secretory output is not well understood. It has long been known that the calcium response of acinar cells to physiological stimulation is complex. Dependent on the type and concentration of agonist, it consists of either local or global calcium increases as well as spreading waves of calcium across the cell. In the past it has been speculated that these different calcium signals drive different secretory responses. Now, recent employment of two-photon microscopy has enabled the simultaneous recording of both enzyme secretion and calcium signals and is beginning to resolve this issue. The data shows that local calcium responses exclusively drive fluid secretion. Where-as, global calcium responses drive both fluid and enzyme secretion. This differential control of secretory output is likely central to controlling the physiological responses of pancreatic acinar cells.     

Virulence protein export systems in Salmonella and Shigella: a new family or lost relatives?

A number of Gram-negative bacterial pathogens secrete 'virulence determinants' directly into the extracellular medium, where they interact with host cells to promote disease. The study of the secretion machinery used by these organisms to transport specific virulence determinants out to the cell surface and beyond is of growing importance in the field of bacterial pathogenesis. Elements of the secretion machinery are shared by several pathogens. These homologous elements may lead to a better understanding of how the machinery works, but the unique elements will tell us more about what distinguishes one bacterial pathogen from another.     

Paracrine effects of PAMP and adrenomedullin on the human adrenal H295R cell line: PAMP but not adrenomedullin stimulates DHEA secretion

It has previously been shown, by this laboratory and others, that adrenal cells actively secrete adrenomedullin. Here it is demonstrated that human adrenal cells also secrete the related peptide, proadrenomedullin N-terminal 20 peptide (PAMP). The actions of adrenomedullin and PAMP on adrenal steroid secretion were determined by measuring the aldosterone, cortisol and dehydroepiandrosterone (DHEA) content of cell culture medium after exposure of the human adrenal H295R cells to either PAMP or adrenomedullin. While PAMP was found to cause a dose-dependent increase in release of all the steroids into the medium, adrenomedullin only increased aldosterone and cortisol and had no effect on DHEA. These data suggest that both adrenomedullin and PAMP may be autocrine regulators of adrenal steroid secretion.     

HCO3- secretion in the esophageal submucosal glands

The mammalian esophagus has the capacity to secrete a HCO(3)(-) and mucin-rich fluid in the esophageal lumen. These secretions originate from the submucosal glands (SMG) and can contribute to esophageal protection against refluxed gastric acid. The cellular mechanisms by which glandular cells achieve these secretions are largely unknown. To study this phenomenon, we used the pH-stat technique to measure luminal alkali secretion in an isolated, perfused pig esophagus preparation. Immunohistochemistry was used to localize receptors and transporters involved in HCO(3)(-) transport. The SMG-bearing esophagus was found to have significant basal alkali secretion, predominantly HCO(3)(-), which averaged 0.21 +/- 0.04 microeq.h(-1).cm(-2). This basal secretion was doubled when stimulated by carbachol but abolished by HCO(3)(-) or Cl(-) removal. Basal- and carbachol-stimulated secretions were also blocked by serosal application of atropine, pirenzipine, DIDS, methazolamide, and ethoxzolamide. The membrane-impermeable carbonic anhydrase inhibitor benzolamide, applied to the serosal bath, partially inhibited basal HCO(3)(-) secretion and blocked the stimulation by carbachol. Immunohistochemistry using antibodies to M(1) cholinergic receptor or carbonic anhydrase-II enzyme showed intense labeling of duct cells and serous demilunes but no labeling of mucous cells. Labeling with an antibody to Na(+)-(HCO(3)(-))(n) (rat kidney NBC) was positive in ducts and serous cells, whereas labeling for Cl(-)/HCO(3)(-) exchanger (AE2) was positive in duct cells but less pronounced in serous cells. These data indicate that duct cells and serous demilunes of SMG play a role in HCO(3)(-) secretion, a process that involves M(1) cholinergic receptor stimulation. HCO(3)(-) transport in these cells is dependent on cytosolic and serosal membrane-bound carbonic anhydrase. HCO(3)(-) secretion is also dependent on serosal Cl(-) and is mediated by DIDS-sensitive transporters, possibly NBC and AE2.     

Ligand-dependent regulation of intracellular protein transport: effect of vitamin a on the secretion of the retinol-binding protein

As a model of ligand-dependent protein secretion the biosynthesis, intracellular transport, and release of the retinol-binding protein (RBP) were studied in primary cultures of rat hepatocytes pulse-labeled with [35S]methionine. After various periods of chase RBP was isolated by immunoprecipitation and identified by SDS PAGE. Both normal and vitamin A-deficient hepatocytes synthesized RBP. The normal cells secreted the pulse-labeled RBP within 2 h. RBP synthesized by deficient cells was not secreted, and intracellular degradation of the protein appeared to be slow. Deficient cells could be induced to secrete RBP on the addition of retinol to the culture medium. This occurred also after protein synthesis had been blocked by cycloheximide. Since retinol induces the secretion of RBP, accumulated in the endoplasmic reticulum (ER), it seems reasonable to conclude that the transport of RBP from the ER to the Golgi complex is regulated by retinol.     

Physiology of the ECL cells.

The enterochromaffin-like (ECL) cells of the oxyntic mucosa (fundus) of the stomach produce, store and secrete histamine, chromogranin A-derived peptides such as pancreastatin, and an unanticipated but as yet unidentified peptide hormone. The cells are stimulated by gastrin and pituitary adenylate cyclase activating peptide and suppressed by somatostatin and galanin. Choline esters and histamine seem to be without effect on ECL cell secretion. The existence of a gastrin-ECL cell axis not only explains how gastrin stimulates acid secretion but also may help to explore the functional significance of the ECL cells with respect to the nature and bioactivity of its peptide hormone. From the results of studies of gastrectomized/fundectomized and gastrin-treated rats, it has been speculated that the anticipated ECL-cell peptide hormone acts on bone metabolism.     

Secretion of surfactant by primary cultures of alveolar type II cells isolated from rats

Pulmonary surfactant conventionally is prepared from material obtained by endobronchial lavage. Although it has been assumed that the components of surfactant are secreted by alveolar type II cells, direct proof of this assumption has not been available. Furthermore, it is possible that the final material obtained by lavage has been modified after secretion or altered during the isolation procedure. It has been shown previously that type II cells, after 1 day in primary culture, secrete saturated phosphatidylcholine, one of the lipid components of surfactant. Because saturated phosphatidylcholine is not unique to surfactant and because type II cells in culture lose differentiated characteristics over the first several days in culture, it has not previously been established how closely the secretory products of cultures of type II cells resemble surfactant as obtained by endobronchial lavage. We therefore studied the morphologic, physical and chemical characteristics of the material that type II cells secrete under basal conditions and after stimulation with terbutaline or 12-O-tetradecanoyl-13-phorbol acetate. The secreted material resembled surfactant obtained by lavage; it was similar morphologically to the lamellar material and tubular myelin seen in the fluid-filled alveoli of fetal rats, it lowered surface tension to 5 mN per meter, and it contained the 72000 dalton apolipoprotein of surfactant (as measured by the 'rocket' immunoelectrophoresis technique). When cells were incubated for 22 h with [1-(14)C]acetate, the distribution of radioactivity in the secreted material was very similar to the phospholipid composition of rat surfactant. We conclude that the material secreted by alveolar type II cells after 1 day in primary culture is similar to surfactant obtained by endobronchial lavage.     

Acid and base secretion in the Calu-3 model of human serous cells

Submucosal glands are the primary source of airway mucus, a critical component of lung innate defenses. Airway glands are defective in cystic fibrosis (CF), showing a complete absence of secretion to vasoactive intestinal peptide or forskolin, which increase intracellular cAMP concentration. This defect is attributed to gland serous cells, which express the cystic fibrosis transmembrane conductance regulator. Calu-3 cells, which mimic many features of serous cells, secrete Cl(-) and HCO(3)(-), with HCO(3)(-) secretion predominating for forskolin stimulation and Cl(-) secretion predominating for stimuli that open basolateral K(+) channels to hyperpolarize the cells. We used pH stat and ion substitution experiments to clarify the mechanisms and consequences of these two modes of secretion. We confirm that Calu-3 cells secrete primarily HCO(3)(-) in response to forskolin. Unexpectedly, HCO(3)(-) secretion continued in response to K(+) channel openers, with Cl(-) secretion being added to it. Secretion of HCO(3)(-) from hyperpolarized cells occurs via the conversion of CO(2) to HCO(3)(-) and is reduced by approximately 50% with acetazolamide. A gap between the base equivalent current and short-circuit current was observed in all experiments and was traced to secretion of H(+) via a ouabain-sensitive, K(+)-dependent process (possibly H(+)-K(+)-ATPase), which partially neutralized the secreted HCO(3)(-). The conjoint secretion of HCO(3)(-) and H(+) may help explain the puzzling finding that mucus secreted from normal and CF glands has the same acidic pH as does mucus from glands stimulated with forskolin or ACh. It may also help explain how human airway glands produce mucus that is hypotonic.     

Vesicular transport in Histoplasma capsulatum: an effective mechanism for trans-cell wall transfer of proteins and lipids in ascomycetes

Vesicular secretion of macromolecules has recently been described in the basidiomycete Cryptococcus neoformans , raising the question as to whether ascomycetes similarly utilize vesicles for transport. In the present study, we examine whether the clinically important ascomycete Histoplasma capsulatum produce vesicles and utilized these structures to secrete macromolecules. Transmission electron microscopy (TEM) shows transcellular secretion of vesicles by yeast cells. Proteomic and lipidomic analyses of vesicles isolated from culture supernatants reveal a rich collection of macromolecules involved in diverse processes, including metabolism, cell recycling, signalling and virulence. The results demonstrate that H. capsulatum can utilize a trans-cell wall vesicular transport secretory mechanism to promote virulence. Additionally, TEM of supernatants collected from Candida albicans , Candida parapsilosis , Sporothrix schenckii and Saccharomyces cerevisiae documents that vesicles are similarly produced by additional ascomycetes. The vesicles from H. capsulatum react with immune serum from patients with histoplasmosis, providing an association of the vesicular products with pathogenesis. The findings support the proposal that vesicular secretion is a general mechanism in fungi for the transport of macromolecules related to virulence and that this process could be a target for novel therapeutics.     

Pulmonary surfactant phosphatidylcholine transport bypasses the brefeldin A sensitive compartment of alveolar type II cells

Brefeldin A (BFA) causes disassembly of the Golgi apparatus and blocks protein transport to this organelle from the endoplasmic reticulum. However, there still remains considerable ambiguity regarding the involvement of the Golgi apparatus in glycerolipid transport pathways. We examined the effects of BFA upon the intracellular translocation of phosphatidylcholine in alveolar type II cells, that synthesize, transport, store and secrete large amounts of phospholipid for regulated exocytosis. BFA at concentrations as high as 10 microg/ml failed to alter the assembly of phosphatidylcholine into lamellar bodies, the specialized storage organelles for pulmonary surfactant. The same concentration of BFA was also ineffective at altering the secretion of newly synthesized phosphatidylcholine from alveolar type II cells. In contrast, concentrations of the drug of 2.5 microg/ml completely arrested newly synthesized lysozyme secretion from the same cells, indicating that BFA readily blocked protein transport processes in alveolar type II cells. The disassembly of the Golgi apparatus in alveolar type II cells following BFA treatment was also demonstrated by showing the redistribution of the resident Golgi protein MG-160 to the endoplasmic reticulum. These results indicate that intracellular transport of phosphatidylcholine along the secretory pathway in alveolar type II cells proceeds via a BFA insensitive route and does not require a functional Golgi apparatus.     

Comparison of the Sec and Tat secretion pathways for heterologous protein production by Streptomyces lividans

Streptomyces is an interesting host for the secretory production of recombinant proteins because of its natural ability to secrete high levels of active proteins into the culture broth and the availability of extensive fermentation knowledge. In bacterial expression systems, heterologous protein secretion has, so far, almost exclusively been investigated using signal peptides that direct the secretion to the Sec pathway. In this study, we assessed the possibility of the Streptomyces lividans twin-arginine translocation (Tat) pathway to secrete the human proteins tumor necrosis factor (TNF) alpha and interleukin (IL) 10 by fusing the coding sequences of mature hTNFalpha and hIL10 to the twin-arginine signal peptides of S. lividans xylanase C (XlnC) and Streptomyces antibioticus tyrosinase. Both proteins were secreted and this secretion was blocked in the DeltatatB and DeltatatC single mutants, indicating that the transport of hTNFalpha and hIL10 could be directed through the Tat pathway. Secretion levels of hTNFalpha and hIL10, however, were lower for Tat-dependent than for Sec-dependent transport using the Sec-dependent signal peptide of the Streptomyces venezuelae subtilisin inhibitor. Surprisingly, Sec-dependent transport was enhanced in the tatB deletion strain. This was especially interesting in the case of hIL10, where Sec-dependent transport of hIL10 was at least 15 times higher in the DeltatatB mutant than in the wild-type strain.     

Metabolite secretion in microorganisms: the theory of metabolic overflow put to the test

Introduction

Microbial cells secrete many metabolites during growth, including important intermediates of the central carbon metabolism. This has not been taken into account by researchers when modeling microbial metabolism for metabolic engineering and systems biology studies.

Materials and Methods

The uptake of metabolites by microorganisms is well studied, but our knowledge of how and why they secrete different intracellular compounds is poor. The secretion of metabolites by microbial cells has traditionally been regarded as a consequence of intracellular metabolic overflow.

Conclusions

Here, we provide evidence based on time-series metabolomics data that microbial cells eliminate some metabolites in response to environmental cues, independent of metabolic overflow. Moreover, we review the different mechanisms of metabolite secretion and explore how this knowledge can benefit metabolic modeling and engineering.

     

Secretory function of autophagy in innate immune cells

Eukaryotic cells utilize two main secretory pathways to transport proteins to the extracellular space. Proteins with a leader signal sequence often undergo co‐translational transport into the endoplasmic reticulum (ER), and then to the Golgi apparatus before they reach their destination. This pathway is called the conventional secretory pathway. Proteins without signal peptides can bypass this ER‐Golgi system and are secreted by a variety of mechanisms collectively called the unconventional secretory pathway. The molecular mechanisms of unconventional secretion are emerging. Autophagy is a conserved bulk degradation mechanism that regulates many intracellular functions. Recent evidence implicates autophagy in the secretory pathway. This review focuses on potential secretory roles of autophagy and how they could modulate the functions of innate immune cells that secrete a wide range of mediators in response to environmental and biological stimuli. We provide a brief overview of the secretory pathways, enumerate the potential mechanistic themes by which autophagy interacts with these pathways and describe their relevance in the context of innate immune cell function.     

How a cytokine is chaperoned through the secretory pathway by complexing with its own receptor: lessons from interleukin-15 (IL-15)/IL-15 receptor alpha

While it is well appreciated that receptors for secreted cytokines transmit ligand-induced signals, little is known about additional roles for cytokine receptor components in the control of ligand transport and secretion. Here, we show that interleukin-15 (IL-15) translocation into the endoplasmic reticulum occurs independently of the presence of IL-15 receptor α (IL-15Rα). Subsequently, however, IL-15 is transported through the Golgi apparatus only in association with IL-15Rα and then is secreted. This intracellular IL-15/IL-15Rα complex already is formed in the endoplasmic reticulum and, thus, enables the further trafficking of complexed IL-15 through the secretory pathway. Just transfecting IL-15Rα in cells, which transcribe but normally do not secrete IL-15, suffices to induce IL-15 secretion. Thus, we provide the first evidence of how a cytokine is chaperoned through the secretory pathway by complexing with its own high-affinity receptor and show that IL-15/IL-15Rα offers an excellent model system for the further exploration of this novel mechanism for the control of cytokine secretion.     

Immune-related intestinal chloride secretion. III. Acute and chronic effects of mast cell mediators on chloride secretion by a human colonic epithelial cell line.

Excessive fluid and electrolyte secretion, resulting symptomatically in diarrhea, has been associated with mast cell activation in a variety of experimental and clinical settings. The present study has used a human colonic epithelial cell line to examine mechanisms underlying this phenomenon. Acute addition of mixed mast cell mediators (as a lysate of rat basophilic leukemia cells) to epithelial cells led to prompt and sustained chloride secretion. The response was partially inhibitable by an antihistaminic drug and an adenosine antagonist, suggesting that histamine, adenosine, and possibly other mediators are responsible for producing the acute effect. Supernatants from immunologically activated rat basophilic leukemia cells had similar effects. Chronic exposure of epithelial cells to the lysate mediator preparation, followed by washing, had no effect on their basal electrical or electrolyte-transporting properties. However, the chloride secretory response of the cells to subsequent addition of vasoactive intestinal peptide, carbachol, and heat-stable enterotoxin of Escherichia coli was significantly enhanced, whereas responses to an adenosine agonist or PGE1 were unaffected. This study has, therefore, demonstrated two ways in which mast cell mediators can directly influence intestinal epithelial cells to secrete more chloride and, hence, to enhance fluid secretion in the gut. The findings may be of relevance to our understanding of inflammatory diarrhea and may aid the development of novel therapies for this disorder.     

Mechanisms and regulation of ion transport in the renal collecting duct

1. In the present paper the ion transport function of the renal mammalian collecting duct and its regulation is briefly reviewed. 2. The epithelium is characterized by different cell types: principal cells, intercalated cells, type A, and intercalated cells, type B. 3. Using microelectrodes and various microscopic techniques active Na+ absorption as well as K+ secretion has been localized to the principal cells, while Cl- absorption was found to proceed largely, though not exclusively, through the tight junctions between cells. 4. Intercalated cells of type A, which prevail in the outer medullary collecting duct, secrete H+ and intercalated cells of type B, which are most frequent in the late cortical collecting duct, secrete HCO3-. 5. This specialization of different cells in transporting individual ions provides the basis for the efficient adaptive regulation of urinary ion excretion.     

Yop fusions to tightly folded protein domains and their effects on Yersinia enterocolitica type III secretion

Yersinia enterocolitica organisms secrete Yop proteins via the type III pathway. Translational fusion of yop genes to ubiquitin or dihydrofolate reductase results in hybrid proteins that cannot be secreted. The folding of hybrids prevents their own transport, but it does not hinder the type III secretion of other Yops.