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.

scholarly journals In vitro mutagenesis of trypsinogen: role of the amino terminus in intracellular protein targeting to secretory granules.

1987 ◽  
Vol 105 (2) ◽  
pp. 659-668 ◽  
Author(s):  
T L Burgess ◽  
C S Craik ◽  
L Matsuuchi ◽  
R B Kelly
Keyword(s):  
Signal Peptide ◽  
Secretory Pathway ◽  
Protein Targeting ◽  
Secretory Granules ◽  
Tumor Cell Line ◽  
Signal Peptidase ◽  
Secretory Proteins ◽  
In Vitro Mutagenesis ◽  
Regulated Secretory Pathway

The mouse anterior pituitary tumor cell line, AtT-20, targets secretory proteins into two distinct intracellular pathways. When the DNA that encodes trypsinogen is introduced into AtT-20 cells, the protein is sorted into the regulated secretory pathway as efficiently as the endogenous peptide hormone ACTH. In this study we have used double-label immunoelectron microscopy to demonstrate that trypsinogen colocalizes in the same secretory granules as ACTH. In vitro mutagenesis was used to test whether the information for targeting trypsinogen to the secretory granules resides at the amino (NH2) terminus of the protein. Mutations were made in the DNA that encodes trypsinogen, and the mutant proteins were expressed in AtT-20 cells to determine whether intracellular targeting could be altered. Replacing the trypsinogen signal peptide with that of the kappa-immunoglobulin light chain, a constitutively secreted protein, does not alter targeting to the regulated secretory pathway. In addition, deletion of the NH2-terminal "pro" sequence of trypsinogen has virtually no effect on protein targeting. However, this deletion does affect the signal peptidase cleavage site, and as a result the enzymatic activity of the truncated trypsin protein is abolished. We conclude that neither the signal peptide nor the 12 NH2-terminal amino acids of trypsinogen are essential for sorting to the regulated secretory pathway of AtT-20 cells.

Determinants for chromogranin A sorting into the regulated secretory pathway are also sufficient to generate granule-like structures in non-endocrine cells

2009 ◽  
Vol 418 (1) ◽  
pp. 81-91 ◽  
Author(s):  
Hansruedi Stettler ◽  
Nicole Beuret ◽  
Cristina Prescianotto-Baschong ◽  
Bérengère Fayard ◽  
Laurent Taupenot ◽  
...  
Keyword(s):  
Chromogranin A ◽  
Endocrine Cells ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Secretory Granules ◽  
Peptide Hormone ◽  
Secretory Proteins ◽  
Cell Periphery ◽  
Granule Formation ◽  
Regulated Secretory Pathway

In endocrine cells, prohormones and granins are segregated in the TGN (trans-Golgi network) from constitutively secreted proteins, stored in concentrated form in dense-core secretory granules, and released in a regulated manner on specific stimulation. The mechanism of granule formation is only partially understood. Expression of regulated secretory proteins, both peptide hormone precursors and granins, had been found to be sufficient to generate structures that resemble secretory granules in the background of constitutively secreting, non-endocrine cells. To identify which segment of CgA (chromogranin A) is important to induce the formation of such granule-like structures, a series of deletion constructs fused to either GFP (green fluorescent protein) or a short epitope tag was expressed in COS-1 fibroblast cells and analysed by fluorescence and electron microscopy and pulse-chase labelling. Full-length CgA as well as deletion constructs containing the N-terminal 77 residues generated granule-like structures in the cell periphery that co-localized with co-expressed SgII (secretogranin II). These are essentially the same segments of the protein that were previously shown to be required for granule sorting in wild-type PC12 (pheochromocytoma cells) cells and for rescuing a regulated secretory pathway in A35C cells, a variant PC12 line deficient in granule formation. The results support the notion that self-aggregation is at the core of granule formation and sorting into the regulated pathway.

scholarly journals Reconstitution in vitro of the pH-dependent aggregation of pancreatic zymogens en route to the secretory granule: implication of GP-2

1993 ◽  
Vol 291 (1) ◽  
pp. 289-296 ◽  
Author(s):  
F A Leblond ◽  
G Viau ◽  
J Lainé ◽  
D Lebel
Keyword(s):  
Secretory Granule ◽  
Secretory Pathway ◽  
Relative Proportion ◽  
Secretory Protein ◽  
Secretory Proteins ◽  
Zymogen Granule ◽  
Granule Membrane ◽  
Regulated Secretory Pathway ◽  
Golgi Network

Regulated secretory proteins are thought to be sorted in the trans-Golgi network (TGN) via selective aggregation. To elucidate the biogenesis of the secretory granule in the exocrine pancreas, we reconstituted in vitro the conditions of pH and ions believed to exist in the TGN using the end product of this sorting process, the zymogen granule contents. Protein aggregation was dependent on pH (acidic) and on the presence of cations (10 mM Ca2+, 150 mM K+) to reproduce the pattern of proteins found in the granule. The constitutive secretory protein IgG was excluded from these aggregates. Zymogen aggregation correlated with the relative proportion of the major granule membrane protein GP-2 in the assay. These results show that the glycosylphosphatidylinositol-anchored protein GP-2 co-aggregates with zymogens in the acidic environment believed to exist in the pancreatic TGN, and thus suggest that GP-2 would function as a membrane anchor for zymogen aggregates, facilitating their entrapment in budding vesicles directed towards the regulated secretory pathway.

Neuroendocrine secretory protein 7B2: structure, expression and functions

2001 ◽  
Vol 357 (2) ◽  
pp. 329-342 ◽  
Author(s):  
Majambu MBIKAY ◽  
Nabil G. SEIDAH ◽  
Michel CHRÉTIEN
Keyword(s):  
Secretory Pathway ◽  
Secretory Granules ◽  
Secretory Protein ◽  
Neuroendocrine Cells ◽  
High Similarity ◽  
Granule Formation ◽  
Proteolytic Activation ◽  
Polyproline Motif

7B2 is an acidic protein residing in the secretory granules of neuroendocrine cells. Its sequence has been elucidated in many phyla and species. It shows high similarity among mammals. A Pro-Pro-Asn-Pro-Cys-Pro polyproline motif is its most conserved feature, being carried by both vertebrate and invertebrate sequences. It is biosynthesized as a precursor protein that is cleaved into an N-terminal fragment and a C-terminal peptide. In neuroendocrine cells, 7B2 functions as a specific chaperone for the proprotein convertase (PC) 2. Through the sequence around its Pro-Pro-Asn-Pro-Cys-Pro motif, it binds to an inactive proPC2 and facilitates its transport from the endoplasmic reticulum to later compartments of the secretory pathway where the zymogen is proteolytically matured and activated. Its C-terminal peptide can inhibit PC2 in vitro and may contribute to keep the enzyme transiently inactive in vivo. The PC2–7B2 model defines a new neuroendocrine paradigm whereby proteolytic activation of prohormones and proneuropeptides in the secretory pathway is spatially and temporally regulated by the dynamics of interactions between converting enzymes and their binding proteins. Interestingly, unlike PC2-null mice, which are viable, 7B2-null mutants die early in life from Cushing's disease due to corticotropin (‘ACTH’) hypersecretion by the neurointermediate lobe, suggesting a possible involvement of 7B2 in secretory granule formation and in secretion regulation. The mechanism of this regulation is yet to be elucidated. 7B2has been shown to be a good marker of several neuroendocrine cell dysfunctions in humans. The possibility that anomalies in its structure and expression could be aetiological causes of some of these dysfunctions warrants investigation.

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.

scholarly journals Proinsulin Endoproteolysis Confers Enhanced Targeting of Processed Insulin to the Regulated Secretory Pathway

2000 ◽  
Vol 11 (6) ◽  
pp. 1959-1972 ◽  
Author(s):  
Regina Kuliawat ◽  
Daniel Prabakaran ◽  
Peter Arvan
Keyword(s):  
Secretory Pathway ◽  
Proteolytic Enzymes ◽  
Secretory Granules ◽  
Protein Solubility ◽  
Vitro Assay ◽  
Processing Enzymes ◽  
Prohormone Processing ◽  
Cargo Proteins ◽  
Regulated Secretory Pathway

Recently, two different prohormone-processing enzymes, prohormone convertase 1 (PC1) and carboxypeptidase E, have been implicated in enhancing the storage of peptide hormones in endocrine secretory granules. It is important to know the extent to which such molecules may act as “sorting receptors” to allow the selective trafficking of cargo proteins from the trans-Golgi network into forming granules, versus acting as enzymes that may indirectly facilitate intraluminal storage of processed hormones within maturing granules. GH4C1 cells primarily store prolactin in granules; they lack PC1 and are defective for intragranular storage of transfected proinsulin. However, proinsulin readily enters the immature granules of these cells. Interestingly, GH4C1 clones that stably express modest levels of PC1 store more proinsulin-derived protein in granules. Even in the presence of PC1, a sizable portion of the proinsulin that enters granules goes unprocessed, and this portion largely escapes granule storage. Indeed, all of the increased granule storage can be accounted for by the modest portion converted to insulin. These results are not unique to GH4C1 cells; similar results are obtained upon PC1 expression in PC12 cells as well as in AtT20 cells (in which PC1 is expressed endogenously at higher levels). An in vitro assay of protein solubility indicates a difference in the biophysical behavior of proinsulin and insulin in the PC1 transfectants. We conclude that processing to insulin, facilitated by the catalytic activities of granule proteolytic enzymes, assists in the targeting (storage) of the hormone.

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 Synaptotagmin IV is necessary for the maturation of secretory granules in PC12 cells

2006 ◽  
Vol 173 (2) ◽  
pp. 241-251 ◽  
Author(s):  
Malika Ahras ◽  
Grant P. Otto ◽  
Sharon A. Tooze
Keyword(s):  
Pc12 Cells ◽  
Secretory Granules ◽  
Granule Formation ◽  
Synaptotagmin Iv ◽  
Prohormone Convertase 2 ◽  
Granule Maturation ◽  
Golgi Network ◽  
Interfering Rna

In neuroendocrine PC12 cells, immature secretory granules (ISGs) mature through homotypic fusion and membrane remodeling. We present evidence that the ISG-localized synaptotagmin IV (Syt IV) is involved in ISG maturation. Using an in vitro homotypic fusion assay, we show that the cytoplasmic domain (CD) of Syt IV, but not of Syt I, VII, or IX, inhibits ISG homotypic fusion. Moreover, Syt IV CD binds specifically to ISGs and not to mature secretory granules (MSGs), and Syt IV binds to syntaxin 6, a SNARE protein that is involved in ISG maturation. ISG homotypic fusion was inhibited in vivo by small interfering RNA–mediated depletion of Syt IV. Furthermore, the Syt IV CD, as well as Syt IV depletion, reduces secretogranin II (SgII) processing by prohormone convertase 2 (PC2). PC2 is found mostly in the proform, suggesting that activation of PC2 is also inhibited. Granule formation, and the sorting of SgII and PC2 from the trans-Golgi network into ISGs and MSGs, however, is not affected. We conclude that Syt IV is an essential component for secretory granule maturation.

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