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.

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.

Proteins are secreted from heterogeneous prestored sources in the exocrine pancreas

1987 ◽  
Vol 252 (6) ◽  
pp. G768-G775
Author(s):  
P. E. Miller ◽  
J. W. Adelson
Keyword(s):  
Digestive Enzymes ◽  
Exocrine Pancreas ◽  
Secretory Proteins ◽  
Pancreatic Ducts ◽  
Regulated Secretion ◽  
Correlation And Regression Analysis ◽  
Label Method ◽  
A Cell ◽  
Partial Depletion

Recent studies demonstrating nonparallel regulated secretion of prestored digestive enzymes in tightly linked groups consistent with the exocytosis mechanism led us to predict that digestive enzymes would be found to be secreted from heterogeneous sources within the exocrine pancreas (J. W. Adelson, and P.E. Miller, Science Wash. DC 228: 993-996, 1985). We explored whether the gland was heterogeneous with respect to its sources of prestored secretory proteins with a double isotopic label method not dependent on activity of secreted digestive enzymes. Rabbit pancreatic proteins were double labeled in vivo by injection of each animal with chemically identical but isotopically distinct mixtures of 3H- and 14C-labeled amino acids, which were administered separately or together on consecutive days after partial depletion of prestored proteins by administration of cholecystokinin (CCK), methacholine chloride, or saline in a protocol in which order of both isotope and secretagogue administration was varied. Three days after labeling, proteins were recovered by collection from cannulated pancreatic ducts of anesthetized animals after stimulation with alternating increasing doses of CCK and methacholine chloride. Pooled secretory data were analyzed to determine whether secretagogue pretreatment resulted in specific and heterogeneous sequestration of proteins after synthesis; data after final secretory stimulation with methacholine chloride and CCK were individually analyzed to determine whether presequestered proteins were mobilized from heterogeneous compartments during secretion. Correlation and regression analysis of isotopic outputs and variance analysis of specific radioactivities of secreted proteins showed sequestration into and secretion from heterogeneous pools of secretory proteins, directly confirming out hypothesis. These results provide a cell biological mechanism explaining regulated nonparallel secretion of digestive enzymes.

scholarly journals Differential Expression and Processing of Chromogranin A and Secretogranin II in Relation to the Secretory Status of Endocrine Cells

Endocrinology ◽  
2006 ◽  
Vol 147 (3) ◽  
pp. 1408-1418 ◽  
Author(s):  
J. R. Peinado ◽  
R. Vazquez-Martinez ◽  
D. Cruz-García ◽  
A. Ruiz-Navarro ◽  
Y. Anouar ◽  
...  
Keyword(s):  
Chromogranin A ◽  
Secretory Pathway ◽  
Secretory Activity ◽  
Biologically Active ◽  
Molecular Forms ◽  
Secretory Proteins ◽  
Intermediate Lobe ◽  
Secretory Rate ◽  
Secretogranin Ii ◽  
Storage Cells

Chromogranin A (CgA) and secretogranin II (SgII) are neuroendocrine secretory proteins that participate in regulation of the secretory pathway and also serve as precursors of biologically active peptides. To investigate whether there is a relationship between the expression, distribution, and processing of CgA and SgII and the degree of secretory activity, we employed two melanotrope subpopulations of the pituitary intermediate lobe that exhibit opposite secretory phenotypes. Thus, although one of the melanotrope subtypes shows high secretory activity, the other exhibits characteristics of a hormone storage phenotype. Our data show that SgII expression levels were higher in secretory melanotropes, whereas CgA expression showed similar rates in both cell subsets. The use of various antibodies revealed the presence of the unprocessed proteins as well as three CgA-derived peptides (67, 45, and 30 kDa) and six SgII-derived peptides (81, 66, 55, 37, 32, and 30 kDa) in both subpopulations. However, the smallest molecular forms of both granins predominated in secretory melanotropes, whereas the largest SgII- and CgA-immunoreactive peptides were more abundant in storage melanotropes, which is suggestive of a more extensive processing of granins in the secretory subset. Confocal microscopy studies showed that CgA immunoreactivity was higher in storage cells, but SgII immunoreactivity was higher in secretory melanotropes. Taken together, our results indicate that SgII and CgA are differentially regulated in melanotrope subpopulations. Thus, SgII expression is strongly related to the secretory activity of melanotrope cells, whereas CgA expression may not be related to secretory rate, but, rather, to hormone storage in this endocrine cell type.

Perturbation of regulated secretion in the pancreatic acinar cell line, AR42J

1992 ◽  
Vol 262 (2) ◽  
pp. G257-G266
Author(s):  
E. Sachs ◽  
J. D. Jamieson
Keyword(s):  
Secretory Pathway ◽  
Secretory Granules ◽  
Pancreatic Acinar Cell ◽  
Secretory Proteins ◽  
Regulated Secretion ◽  
Golgi Transport ◽  
Test Conditions ◽  
Intracellular Compartments ◽  
Regulated Secretory Pathway ◽  
Acidic Compartments

The regulated secretory pathway comprises accelerated discharge of proteins in response to hormonal stimuli, their presence in secretory granules (SG), and a long intracellular residence time. Dexamethasone induction of AR42J results in an increase in granule content and responsiveness to cholecystokinin (CCK). We studied the effects of conditions implicated in sorting of secretory proteins into the regulated pathway using [35S]methionine pulse-chase protocols that examine transport of secretory proteins from the rough endoplasmic reticulum (RER)----SG and specifically from the Golgi complex (GC)----SG. The latter uses a chase at 20 degrees C to allow accumulation of labeled proteins in the trans-Golgi, followed by a shift to 37 degrees C that initiates their transport to SG under test conditions. Quantitation of CCK-8-stimulated discharge of prestored amylase and of newly synthesized labeled proteins that have entered SG during the chase enables us to examine the effect of perturbants over selected parts of the pathway. The effects of acidic intracellular compartments, the cytoskeleton, protein synthesis, ATP, and temperature on pre- and post-Golgi entry of proteins into the regulated pathway were studied. NH4Cl, monensin, Na azide, incubation at 20 degrees C, and pertussis toxin retarded RER----SG transport without affecting amylase discharge. Only incubation with 20 mM NH4Cl or 1 microM monensin inhibited transfer of newly synthesized proteins from the late GC----SG. RER----Golgi or intra-Golgi transport thus appears to require ATP and possibly guanosine 5'-triphosphate (GTP)-binding proteins. Acidic compartments appear to be essential for sorting of secretory proteins from the GC----SG.

scholarly journals Parotid Secretory Granules: Crossroads of Secretory Pathways and Protein Storage

2005 ◽  
Vol 84 (6) ◽  
pp. 500-509 ◽  
Author(s):  
S.-U. Gorr ◽  
S.G. Venkatesh ◽  
D.S. Darling
Keyword(s):  
Secretory Pathway ◽  
Secretory Granules ◽  
Acinar Cells ◽  
Model Systems ◽  
Salivary Proteins ◽  
Secretory Proteins ◽  
Protein Distribution ◽  
Secretory Pathways ◽  
Parotid Acinar Cells ◽  
Induced Aggregation

Saliva plays an important role in digestion, host defense, and lubrication. The parotid gland contributes a variety of secretory proteins—including amylase, proline-rich proteins, and parotid secretory protein (PSP)—to these functions. The regulated secretion of salivary proteins ensures the availability of the correct mix of salivary proteins when needed. In addition, the major salivary glands are targets for gene therapy protocols aimed at targeting therapeutic proteins either to the oral cavity or to circulation. To be successful, such protocols must be based on a solid understanding of protein trafficking in salivary gland cells. In this paper, model systems available to study the secretion of salivary proteins are reviewed. Parotid secretory proteins are stored in large dense-core secretory granules that undergo stimulated secretion in response to extracellular stimulation. Secretory proteins that are not stored in large secretory granules are secreted by either the minor regulated secretory pathway, constitutive secretory pathways (apical or basolateral), or the constitutive-like secretory pathway. It is proposed that the maturing secretory granules act as a distribution center for secretory proteins in salivary acinar cells. Protein distribution or sorting is thought to involve their selective retention during secretory granule maturation. Unlike regulated secretory proteins in other cell types, salivary proteins do not exhibit calcium-induced aggregation. Instead, sulfated proteoglycans play a role in the storage of secretory proteins in parotid acinar cells. This work suggests that unique sorting and retention mechanisms are responsible for the distribution of secretory proteins to different secretory pathways from the maturing secretory granules in parotid acinar cells.

scholarly journals A Link between Secretion and Pre-mRNA Processing Defects in Saccharomyces cerevisiae and the Identification of a Novel Splicing Gene, RSE1

1998 ◽  
Vol 18 (12) ◽  
pp. 7139-7146 ◽  
Author(s):  
Esther J. Chen ◽  
Alison R. Frand ◽  
Elizabeth Chitouras ◽  
Chris A. Kaiser
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Secretory Pathway ◽  
Small Gtpase ◽  
Mrna Splicing ◽  
Secretory Proteins ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Golgi Transport ◽  
Pathway Function ◽  
A Cell

ABSTRACT Secretory proteins in eukaryotic cells are transported to the cell surface via the endoplasmic reticulum (ER) and the Golgi apparatus by membrane-bounded vesicles. We screened a collection of temperature-sensitive mutants of Saccharomyces cerevisiaefor defects in ER-to-Golgi transport. Two of the genes identified in this screen were PRP2, which encodes a known pre-mRNA splicing factor, and RSE1, a novel gene that we show to be important for pre-mRNA splicing. Both prp2-13 andrse1-1 mutants accumulate the ER forms of invertase and the vacuolar protease CPY at restrictive temperature. The secretion defect in each mutant can be suppressed by increasing the amount ofSAR1, which encodes a small GTPase essential for COPII vesicle formation from the ER, or by deleting the intron from theSAR1 gene. These data indicate that a failure to spliceSAR1 pre-mRNA is the specific cause of the secretion defects in prp2-13 and rse1-1. Moreover, these data imply that Sar1p is a limiting component of the ER-to-Golgi transport machinery and suggest a way that secretory pathway function might be coordinated with the amount of gene expression in a cell.

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.

scholarly journals The unfolded protein response coordinates the production of endoplasmic reticulum protein and endoplasmic reticulum membrane.

1997 ◽  
Vol 8 (9) ◽  
pp. 1805-1814 ◽  
Author(s):  
J S Cox ◽  
R E Chapman ◽  
P Walter
Keyword(s):  
Endoplasmic Reticulum ◽  
Unfolded Protein Response ◽  
Secretory Pathway ◽  
Lipid Synthesis ◽  
Secretory Proteins ◽  
Endoplasmic Reticulum Membrane ◽  
Yeast Saccharomyces Cerevisiae ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response

The endoplasmic reticulum (ER) is a multifunctional organelle responsible for production of both lumenal and membrane components of secretory pathway compartments. Secretory proteins are folded, processed, and sorted in the ER lumen and lipid synthesis occurs on the ER membrane itself. In the yeast Saccharomyces cerevisiae, synthesis of ER components is highly regulated: the ER-resident proteins by the unfolded protein response and membrane lipid synthesis by the inositol response. We demonstrate that these two responses are intimately linked, forming different branches of the same pathway. Furthermore, we present evidence indicating that this coordinate regulation plays a role in ER biogenesis.

scholarly journals Cofilin recruits F-actin to SPCA1 and promotes Ca2+-mediated secretory cargo sorting

2014 ◽  
Vol 206 (5) ◽  
pp. 635-654 ◽  
Author(s):  
Christine Kienzle ◽  
Nirakar Basnet ◽  
Alvaro H. Crevenna ◽  
Gisela Beck ◽  
Bianca Habermann ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Nitrilotriacetic Acid ◽  
Calcium Atpase ◽  
Secretory Proteins ◽  
Dependent Manner ◽  
Agarose Beads ◽  
P Type ◽  
Golgi Network ◽  
Phosphorylation Domain ◽  
Cargo Sorting

The actin filament severing protein cofilin-1 (CFL-1) is required for actin and P-type ATPase secretory pathway calcium ATPase (SPCA)-dependent sorting of secretory proteins at the trans-Golgi network (TGN). How these proteins interact and activate the pump to facilitate cargo sorting, however, is not known. We used purified proteins to assess interaction of the cytoplasmic domains of SPCA1 with actin and CFL-1. A 132–amino acid portion of the SPCA1 phosphorylation domain (P-domain) interacted with actin in a CFL-1–dependent manner. This domain, coupled to nickel nitrilotriacetic acid (Ni-NTA) agarose beads, specifically recruited F-actin in the presence of CFL-1 and, when expressed in HeLa cells, inhibited Ca2+ entry into the TGN and secretory cargo sorting. Mutagenesis of four amino acids in SPCA1 that represent the CFL-1 binding site also affected Ca2+ import into the TGN and secretory cargo sorting. Altogether, our findings reveal the mechanism of CFL-1–dependent recruitment of actin to SPCA1 and the significance of this interaction for Ca2+ influx and secretory cargo sorting.

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