scholarly journals An antibody against secretogranin I (chromogranin B) is packaged into secretory granules.

1989 ◽  
Vol 109 (1) ◽  
pp. 17-34 ◽  
Author(s):  
P Rosa ◽  
U Weiss ◽  
R Pepperkok ◽  
W Ansorge ◽  
C Niehrs ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Secretory Granules ◽  
Intracellular Localization ◽  
Secretory Protein ◽  
Secretory Proteins ◽  
Chromogranin B ◽  
Double Labeling ◽  
Protein Protein Interaction ◽  
And Function ◽  
Secretogranin I

We have investigated the sorting and packaging of secretory proteins into secretory granules by an immunological approach. An mAb against secretogranin I (chromogranin B), a secretory protein costored with various peptide hormones and neuropeptides in secretory granules of many endocrine cells and neurons, was expressed by microinjection of its mRNA into the secretogranin I-producing cell line PC12. An mAb against the G protein of vesicular stomatitis virus--i.e., against an antigen not present in PC12 cells--was expressed as a control. The intracellular localization and the secretion of the antibodies was studied by double-labeling immunofluorescence using the conventional and the confocal microscope, as well as by pulse-chase experiments. The secretogranin I antibody, like the control antibody, was transported along the secretory pathway to the Golgi complex. However, in contrast to the control antibody, which was secreted via the constitutive pathway, the secretogranin I antibody formed an immunocomplex with secretogranin I, was packaged into secretory granules, and was released by regulated exocytosis. Our results show that a constitutive secretory protein, unaltered by genetic engineering, can be diverted to the regulated pathway of secretion by its protein-protein interaction with a regulated secretory protein. The data also provide the basis for immunologically studying the role of luminally exposed protein domains in the biogenesis and function of regulated secretory vesicles.

Interaction of calcium with porcine adrenal chromogranin A (secretory protein-I) and chromogranin B (secretogranin I)

1989 ◽  
Vol 257 (2) ◽  
pp. E247-E254 ◽  
Author(s):  
S. U. Gorr ◽  
J. Shioi ◽  
D. V. Cohn
Keyword(s):  
Chromogranin A ◽  
Secretory Granules ◽  
Extracellular Fluid ◽  
Secretory Protein ◽  
Secretory Proteins ◽  
Chromogranin B ◽  
Secretory Products ◽  
Adrenal Chromaffin ◽  
Secretogranin I

Secretory granules of endocrine cells contain one or more of the acidic secretory proteins chromogranin A (secretory protein-I), chromogranin B (secretogranin I), and secretogranin II (chromogranin C). It has been proposed that these proteins play a role in the packaging of secretory products. In the present study, lysates of purified porcine adrenal chromaffin granules containing chromogranins A and B and a putative chromogranin B fragment bound calcium and formed aggregates in the presence of 10–20 mM calcium at pH 5–6 and at 100 mM or less KCl, NaCl, or norepinephrine. The precipitates contained virtually all of the chromogranin B and the chromogranin B fragment and about one-third of the chromogranin A. The aggregates did not form or were dissociated at the pH and salt concentration of the extracellular fluid. Calcium precipitated purified chromogranin A and chromogranin B from pure solution to the same extent as from the granule lysates. Parathormone, added to the lysates, was incorporated in the precipitates, whereas the acidic secretory protein ovalbumin and norepinephrine were not. These findings suggest that secretory protein-I and secretogranin can exist in situ as aggregates that may include selected secretory products.

scholarly journals Efficient binding of regulated secretory protein aggregates to membrane phospholipids at acidic pH

1999 ◽  
Vol 338 (2) ◽  
pp. 289-294 ◽  
Author(s):  
Jean LAINÉ ◽  
Denis LeBEL
Keyword(s):  
Secretory Pathway ◽  
Membrane Lipids ◽  
Secretory Granules ◽  
Ph Dependence ◽  
Secretory Protein ◽  
Secretory Proteins ◽  
Zymogen Granule ◽  
Membrane Phospholipids ◽  
Aggregation Assay

Some regulated secretory proteins are thought to be targeted to secretory granules through an acidic-dependent aggregation in the trans-Golgi network. In this report we use pancreatic zymogens, a paradigm of regulated proteins, to test this hypothesis, because they qualitatively aggregate upon acidification in vitro. Pig zymogens were found to start to aggregate significantly at pH ∼ 6.0, a pH slightly lower than that at which rat zymogens aggregate, but still compatible with the pH of the cell-sorting compartments. When pig zymogen granule membranes were mixed with the zymogens in the aggregation assay, membranes that normally floated on 1 M sucrose were observed to be pelleted by the aggregating zymogens. Rat membranes were pelleted by pig zymogens and vice versa. Igs, typical constitutively secreted proteins, which needed chemical cross-linking to serve as an aggregated protein control, pelleted membranes almost independently of pH. Corresponding cross-linked zymogen-binding ability and pH dependence was unaffected by the chemical modification. Membranes treated with sodium carbonate, pH 11, or with protease K, were still pelleted by zymogens, suggesting that the aggregated zymogens bound to membrane lipids. This hypothesis was confirmed by the efficient pelleting of unilamellar vesicles composed of granule membrane lipids. Vesicles composed of single classes of phospholipids were also pelleted, but with various efficacies. We conclude that pancreatic zymogen aggregates, formed under the acidic conditions of the secretory pathway sorting compartments, have the capacity to bind firmly to membranes through their phospholipid constituents.

scholarly journals Distinct molecular mechanisms for protein sorting within immature secretory granules of pancreatic beta-cells.

1994 ◽  
Vol 126 (1) ◽  
pp. 77-86 ◽  
Author(s):  
R Kuliawat ◽  
P Arvan
Keyword(s):  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Secretory Pathway ◽  
Molecular Mechanisms ◽  
Secretory Granules ◽  
Protein Sorting ◽  
Secretory Protein ◽  
Secretory Proteins ◽  
Lysosomal Hydrolases ◽  
Storage Granules

In the beta-cells of pancreatic islets, insulin is stored as the predominant protein within storage granules that undergo regulated exocytosis in response to glucose. By pulse-chase analysis of radiolabeled protein condensation in beta-cells, the formation of insoluble aggregates of regulated secretory protein lags behind the conversion of proinsulin to insulin. Condensation occurs within immature granules (IGs), accounting for passive protein sorting as demonstrated by constitutive-like secretion of newly synthesized C-peptide in stoichiometric excess of insulin (Kuliawat, R., and P. Arvan. J. Cell Biol. 1992. 118:521-529). Experimental manipulation of condensation conditions in vivo reveals a direct relationship between sorting of regulated secretory protein and polymer assembly within IGs. By contrast, entry from the trans-Golgi network into IGs does not appear especially selective for regulated secretory proteins. Specifically, in normal islets, lysosomal enzyme precursors enter the stimulus-dependent secretory pathway with comparable efficiency to that of proinsulin. However, within 2 h after synthesis (the same period during which proinsulin processing occurs), newly synthesized hydrolases are fairly efficiently relocated out of the stimulus-dependent pathway. In tunicamycin-treated islets, while entry of new lysosomal enzymes into the regulated secretory pathway continues unperturbed, exit of nonglycosylated hydrolases from this pathway does not occur. Consequently, the ultimate targeting of nonglycosylated hydrolases in beta-cells is to storage granules rather than lysosomes. These results implicate a post-Golgi mechanism for the active removal of lysosomal hydrolases away from condensed granule contents during the storage process for regulated secretory proteins.

Prohormone transport through the secretory pathway of neuroendocrine cells

2000 ◽  
Vol 78 (3) ◽  
pp. 289-298 ◽  
Author(s):  
Roland P Kuiper ◽  
Gerard JM Martens
Keyword(s):  
Secretory Pathway ◽  
Potential Role ◽  
Protein Transport ◽  
Secretory Granules ◽  
Secretory Protein ◽  
Neuroendocrine Cells ◽  
Secretory Proteins ◽  
Regulated Secretory Pathway ◽  
Made In

En route through the secretory pathway of neuroendocrine cells, prohormones pass a series of membrane-bounded compartments. During this transport, the prohormones are sorted to secretory granules and proteolytically cleaved to bioactive peptides. Recently, progress has been made in a number of aspects concerning secretory protein transport and sorting, particularly with respect to transport events in the early regions of the secretory pathway. In this review we will deal with some of these aspects, including: i) selective exit from the endoplasmic reticulum via COPII-coated vesicles and the potential role of p24 putative cargo receptors in this process, ii) cisternal maturation as an alternative model for protein transport through the Golgi complex, and iii) the mechanisms that may be involved in the sorting of regulated secretory proteins to secretory granules. Although much remains to be learned, interesting new insights into the functioning of the secretory pathway have been obtained.Key words: regulated secretory pathway, p24 family, vesicular transport, POMC, protein sorting, secretory granule, Xenopus laevis.

Disruption of disulfide bonds exhibits differential effects on trafficking of regulated secretory proteins

1999 ◽  
Vol 277 (1) ◽  
pp. C121-C131 ◽  
Author(s):  
Sven-Ulrik Gorr ◽  
Xue Fen Huang ◽  
Darrin J. Cowley ◽  
Regina Kuliawat ◽  
Peter Arvan
Keyword(s):  
Pancreatic Islets ◽  
Disulfide Bond ◽  
Protein Trafficking ◽  
Disulfide Bonds ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Secretory Proteins ◽  
Cell Type ◽  
Chromogranin B ◽  
Disulfide Reduction

For several secretory proteins, it has been hypothesized that disulfide-bonded loop structures are required for sorting to secretory granules. To explore this hypothesis, we employed dithiothreitol (DTT) treatment in live pancreatic islets, as well as in PC-12 and GH4C1cells. In islets, disulfide reduction in the distal secretory pathway did not increase constitutive or constitutive-like secretion of proinsulin (or insulin). In PC-12 cells, DTT treatment caused a dramatic increase in unstimulated secretion of newly synthesized chromogranin B (CgB), presumably as a consequence of reducing the single conserved chromogranin disulfide bond (E. Chanat, U. Weiss, W. B. Huttner, and S. A. Tooze. EMBO J.12: 2159–2168, 1993). However, in GH4C1cells that also synthesize CgB endogenously, DTT treatment reduced newly synthesized prolactin and blocked its export, whereas newly synthesized CgB was routed normally to secretory granules. Moreover, on transient expression in GH4C1cells, CgA and a CgA mutant lacking the conserved disulfide bond showed comparable multimeric aggregation properties and targeting to secretory granules, as measured by stimulated secretion assays. Thus the conformational perturbation of regulated secretory proteins caused by disulfide disruption leads to consequences in protein trafficking that are both protein and cell type dependent.

scholarly journals Isoproterenol increases sorting of parotid gland cargo proteins to the basolateral pathway

2007 ◽  
Vol 293 (2) ◽  
pp. C558-C565 ◽  
Author(s):  
Srirangapatnam G. Venkatesh ◽  
Jinlian Tan ◽  
Sven-Ulrik Gorr ◽  
Douglas S. Darling
Keyword(s):  
Secretory Pathway ◽  
Secretory Granules ◽  
Immunoblot Analysis ◽  
Secretory Protein ◽  
Secretory Proteins ◽  
Clear Understanding ◽  
Regulated Secretion ◽  
Cargo Proteins ◽  
Parotid Secretory Protein ◽  
Endogenous Proteins

Exocrine cells have an essential function of sorting secreted proteins into the correct secretory pathway. A clear understanding of sorting in salivary glands would contribute to the correct targeting of therapeutic transgenes. The present work investigated whether there is a change in the relative proportions of basic proline-rich protein (PRP) and acidic PRPs in secretory granules in response to chronic isoproterenol treatment, and whether this alters the sorting of endogenous cargo proteins. Immunoblot analysis of secretory granules from rat parotids found a large increase of basic PRP over acidic PRPs in response to chronic isoproterenol treatment. Pulse chase experiments demonstrated that isoproterenol also decreased regulated secretion of newly synthesized secretory proteins, including PRPs, amylase and parotid secretory protein. This decreased efficiency of the apical regulated pathway may be mediated by alkalization of the secretory granules since it was reversed by treatment with mild acid. We also investigated changes in secretion through the basolateral (endocrine) pathways. A significant increase in parotid secretory protein and salivary amylase was detected in sera of isoproterenol-treated animals, suggesting increased routing of the regulated secretory proteins to the basolateral pathway. These studies demonstrate that shifts of endogenous proteins can modulate regulated secretion and sorting of cargo proteins.

Regulated and constitutive secretion of distinct molecular forms of acetylcholinesterase from PC12 cells

1993 ◽  
Vol 106 (3) ◽  
pp. 731-740 ◽  
Author(s):  
E.S. Schweitzer
Keyword(s):  
Pc12 Cells ◽  
Secretory Pathway ◽  
Intracellular Localization ◽  
Synaptic Function ◽  
Molecular Forms ◽  
Secretory Proteins ◽  
Secretory Pathways ◽  
Regulated Secretion ◽  
Constitutive Secretion ◽  
A Cell

PC12 cells secrete the enzyme acetylcholinesterase (AChE) while at rest, and increase the overall rate of this secretion 2-fold upon depolarization. This behavior is different from the release of other markers by the constitutive or regulated secretory pathways in PC12 cells. Both the resting and stimulated release of AChE are unchanged after treatment with a membrane-impermeable esterase inhibitor, demonstrating that it represents true secretion and not shedding from the cell surface. The stimulation release of AChE is Ca(2+)-dependent, while the unstimulated release is not. Analysis of the molecular forms of AChE secreted by PC12 cells indicates that the release of AChE actually involves two concurrent but independent secretory processes, and that the G4 form of the enzyme is secreted constitutively, while both the G2 and G4 forms are secreted in a regulated manner, presumably from regulated secretory vesicles. Compared with other regulated secretory proteins, a much smaller fraction of cellular AChE is secreted, and the intracellular localization of this enzyme differs from that of other regulated secretory proteins. The demonstration that a cell line that exhibits regulated secretion of acetylcholine (ACh) is also capable of regulated secretion of AChE provides additional evidence for the existence of multiple regulated secretory pathways within a single cell. Moreover, there appears to be a selective packaging of different molecular forms of AChE into the regulated versus the constitutive secretory pathway. Both the specificity of sorting of AChE and the regulation of its secretion suggest that AChE may play a more dynamic role in synaptic function than has been recognized previously.

Colocalization of chaperone Cpn60, proinsulin and convertase PC1 within immature secretory granules of insulin-secreting cells suggests a role for Cpn60 in insulin processing

2000 ◽  
Vol 113 (11) ◽  
pp. 2075-2083 ◽  
Author(s):  
A.E. Arias ◽  
C.S. Velez-Granell ◽  
G. Mayer ◽  
M. Bendayan
Keyword(s):  
Islet Cell ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Double Labeling ◽  
In Vitro Binding ◽  
Cell Extracts ◽  
Vitro Binding ◽  
Preferential Binding ◽  
Insulin Secreting Cells

Many of the mechanisms that control insulin processing and packaging by interaction with different elements along the secretory pathway remain poorly understood. We have investigated the possibility that Cpn60, a member of the heat shock protein family, may be present in rat insulin-secreting cells, participating in the proinsulin-insulin maturation process. Immunofluorescence and high resolution immunocytochemical studies revealed the presence of the Cpn60 protein all along the insulin secretory pathway, being particularly abundant over the proinsulin-containing immature secretory granules. Double-labeling experiments showed associations between Cpn60 and proinsulin, as well as between Cpn60 and PC1 convertase, with a preferential binding to proinsulin. These findings paralleled those of coimmunoprecipitation studies showing the Cpn60 chaperone and the mature form of the PC1 convertase in proinsulin immunoprecipitates, as well as the PC1 in Cpn60 immunoprecipitates from total islet cell extracts. In vitro binding of Cpn60 to proinsulin, insulin and glucagon was also documented. Cpn60, significantly abundant in proinsulin-containing secretory granules where conversion of proinsulin to insulin takes place, and the colocalization of the chaperone with proinsulin and PC1 convertase suggest that the Cpn60 protein may play a role directing precise molecular interactions during insulin processing and/or packaging.

scholarly journals Sorting and storage during secretory granule biogenesis: looking backward and looking forward

1998 ◽  
Vol 332 (3) ◽  
pp. 593-610 ◽  
Author(s):  
Peter ARVAN ◽  
David CASTLE
Keyword(s):  
Secretory Pathway ◽  
Molecular Mechanisms ◽  
Secretory Granules ◽  
Secretory Proteins ◽  
Membrane Composition ◽  
Granule Formation ◽  
Granule Membrane ◽  
Regulated Secretory Pathway ◽  
Secretory Products

Secretory granules are specialized intracellular organelles that serve as a storage pool for selected secretory products. The exocytosis of secretory granules is markedly amplified under physiologically stimulated conditions. While granules have been recognized as post-Golgi carriers for almost 40 years, the molecular mechanisms involved in their formation from the trans-Golgi network are only beginning to be defined. This review summarizes and evaluates current information about how secretory proteins are thought to be sorted for the regulated secretory pathway and how these activities are positioned with respect to other post-Golgi sorting events that must occur in parallel. In the first half of the review, the emerging role of immature secretory granules in protein sorting is highlighted. The second half of the review summarizes what is known about the composition of granule membranes. The numerous similarities and relatively limited differences identified between granule membranes and other vesicular carriers that convey products to and from the plasmalemma, serve as a basis for examining how granule membrane composition might be established and how its unique functions interface with general post-Golgi membrane traffic. Studies of granule formation in vitro offer additional new insights, but also important challenges for future efforts to understand how regulated secretory pathways are constructed and maintained.

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