Lumenal protein multimerization in the distal secretory pathway/secretory granules

2002 ◽  
Vol 14 (4) ◽  
pp. 448-453 ◽  
Author(s):  
Peter Arvan ◽  
Bao-yan Zhang ◽  
Lijun Feng ◽  
Ming Liu ◽  
Regina Kuliawat
Keyword(s):  
Secretory Pathway ◽  
Secretory Granules ◽  
Protein Multimerization

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.

scholarly journals PACS-1 and Adaptor Protein-1 Mediate ACTH Trafficking to the Regulated Secretory Pathway

2018 ◽  
Author(s):  
Brennan S. Dirk ◽  
Christopher End ◽  
Emily N. Pawlak ◽  
Logan R. Van Nynatten ◽  
Rajesh Abraham Jacob ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Adaptor Protein ◽  
Cell Types ◽  
Cellular Membrane ◽  
Extracellular Milieu ◽  
Specialized Form ◽  
Regulated Secretory Pathway ◽  
Clathrin Adaptor

ABSTRACTThe regulated secretory pathway is a specialized form of protein secretion found in endocrine and neuroendocrine cell types. Pro-opiomelanocortin (POMC) is a pro-hormone that utilizes this pathway to be trafficked to dense core secretory granules (DCSGs). Within this organelle, POMC is processed to multiple bioactive hormones that play key roles in cellular physiology. However, the complete set of cellular membrane trafficking proteins that mediate the correct sorting of POMC to DCSGs remain unknown. Here, we report the roles of the phosphofurin acidic cluster sorting protein – 1 (PACS-1) and the clathrin adaptor protein 1 (AP-1) in the targeting of POMC to DCSGs. Upon knockdown of PACS-1 and AP-1, POMC is readily secreted into the extracellular milieu and fails to be targeted to DCSGs.

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.

PKA inhibitor, H-89, affects the intracellular transit of regulated secretory proteins in rat lacrimal glands

1998 ◽  
Vol 274 (1) ◽  
pp. C262-C271 ◽  
Author(s):  
P. Robin ◽  
B. Rossignol ◽  
M. N. Raymond
Keyword(s):  
Protein Kinase ◽  
Secretory Pathway ◽  
Kinase Inhibitors ◽  
Secretory Granules ◽  
Protein Kinase Inhibitors ◽  
Secretory Proteins ◽  
Lacrimal Glands ◽  
Calphostin C ◽  
A 23187 ◽  
Golgi Network

We tested the effect of H-89, a protein kinase A (PKA) inhibitor, on the intracellular transit of the regulated secretory proteins in rat lacrimal glands. We show that H-89, by itself, induces the secretion of newly synthesized proteins trafficking in its presence but not of proteins already stored in the mature secretory granules. This secretion does not depend on the presence of extracellular Ca2+. The proteins released are identical to those secreted after cholinergic stimulation or under the action of the ionophore A-23187, but the secretion level is ∼40% lower. The effect of H-89 seems to be due to PKA inhibition because other protein kinase inhibitors (calphostin C, chelerythrine, H-85) do not induce secretion. We further show that H-89 does not modify the rate of glycoprotein galactosylation but induces the secretion of newly galactosylated glycoproteins. Finally, we used a “20°C block” procedure to show that H-89 affects a trans-Golgi network (TGN) or post-TGN step of the secretory pathway. Our results demonstrate that, in lacrimal cells, H-89 affects the intracellular trafficking of secretory proteins, suggesting a role for PKA in this process.

Regulation of renin processing and secretion: chemiosmotic control and novel secretory pathway

1993 ◽  
Vol 265 (2) ◽  
pp. C305-C320 ◽  
Author(s):  
J. A. King ◽  
D. J. Lush ◽  
J. C. Fray
Keyword(s):  
Secretory Pathway ◽  
Secretory Granules ◽  
Surface Membrane ◽  
Water Flux ◽  
Cellular Level ◽  
Macula Densa ◽  
Secretory Proteins ◽  
Juxtaglomerular Cells ◽  
Cl Channels ◽  
Health And Disease

The renin-angiotensin-aldosterone system (RAAS) plays an important role in cardiovascular and electrolyte regulation in health and disease. Juxtaglomerular cells in the kidney regulate endocrine RAAS by physiologically controlling conversion of prorenin and secretion of renin. The classical baroceptor, neurogenic, and macula densa mechanisms regulate renin expression at the cellular level by Ca2+, adenosine 3',5'-cyclic monophosphate (cAMP), and chemiosmotic forces (K+, Cl-, and water flux coupled to H+ movement). The baroceptor mechanism (through Ca2+) activates K+ and Cl- channels in the surface membrane and deactivates a KCl-H+ exchange chemiosmotic transporter in the secretory granular membrane. The neurogenic mechanism (through cAMP) promotes prorenin processing to renin. The macula densa mechanism (through K+ and Cl-) involves the processing of prorenin to renin. Ca2+, by inhibiting the KCl-H+ exchange transporter, prevents secretory granules from engaging in chemiosmotically mediated exocytosis. cAMP, on the other hand, by stimulating H+ influx, provides the acidic granular environment for prorenin processing to renin. It is concluded that, in the presence of a favorable chemiosmotic environment, prorenin is processed to renin, which may then be secreted by regulative degranulation or divergence translocation, a novel secretory pathway used by several secretory proteins, including renin.

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.

scholarly journals Oligomerization of green fluorescent protein in the secretory pathway of endocrine cells

2001 ◽  
Vol 360 (3) ◽  
pp. 645-649 ◽  
Author(s):  
Renu K. JAIN ◽  
Paul B. M. JOYCE ◽  
Miguel MOLINETE ◽  
Philippe A. HALBAN ◽  
Sven-Ulrik GORR
Keyword(s):  
Green Fluorescent Protein ◽  
Endocrine Cells ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Secretory Granules ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Reporter Protein ◽  
Regulated Secretory Pathway ◽  
Green Fluorescent

Green fluorescent protein (GFP) is used extensively as a reporter protein to monitor cellular processes, including intracellular protein trafficking and secretion. In general, this approach depends on GFP acting as a passive reporter protein. However, it was recently noted that GFP oligomerizes in the secretory pathway of endocrine cells. To characterize this oligomerization and its potential role in GFP transport, cytosolic and secretory forms of enhanced GFP (EGFP) were expressed in GH4C1 and AtT-20 endocrine cells. Biochemical analysis showed that cytosolic EGFP existed as a 27kDa monomer, whereas secretory forms of EGFP formed disulphide-linked oligomers. EGFP contains two cysteine residues (Cys49 and Cys71), which could play a role in this oligomerization. Site-directed mutagenesis of Cys49 and Cys71 showed that both cysteine residues were involved in disulphide interactions. Substitution of either cysteine residue resulted in a reduction or loss of oligomers, although dimers of the secretory form of EGFP remained. Mutation of these residues did not adversely affect the fluorescence of EGFP. EGFP oligomers were stored in secretory granules and secreted by the regulated secretory pathway in endocrine AtT-20 cells. Similarly, the dimeric mutant forms of EGFP were still secreted via the regulated secretory pathway, indicating that the higher-order oligomers were not necessary for sorting in AtT-20 cells. These results suggest that the oligomerization of EGFP must be considered when the protein is used as a reporter molecule in the secretory pathway.

scholarly journals Genetic Ablation of Phosphatidylinositol Transfer Protein Function in Murine Embryonic Stem Cells

2002 ◽  
Vol 13 (3) ◽  
pp. 739-754 ◽  
Author(s):  
James G. Alb ◽  
Scott E. Phillips ◽  
Kathleen Rostand ◽  
Xiaoxia Cui ◽  
Jef Pinxteren ◽  
...  
Keyword(s):  
Mast Cell ◽  
Membrane Trafficking ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Embryonic Stem ◽  
Endocytic Pathway ◽  
Dense Core ◽  
Homologous Proteins ◽  
Genetic Ablation ◽  
Phosphatidylinositol Transfer

Phosphatidylinositol transfer proteins (PITPs) regulate the interface between signal transduction, membrane-trafficking, and lipid metabolic pathways in eukaryotic cells. The best characterized mammalian PITPs are PITPα and PITPβ, two highly homologous proteins that are encoded by distinct genes. Insights into PITPα and PITPβ function in mammalian systems have been gleaned exclusively from cell-free or permeabilized cell reconstitution and resolution studies. Herein, we report for the first time the use of genetic approaches to directly address the physiological functions of PITPα and PITPβ in murine cells. Contrary to expectations, we find that ablation of PITPα function in murine cells fails to compromise growth and has no significant consequence for bulk phospholipid metabolism. Moreover, the data show that PITPα does not play an obvious role in any of the cellular activities where it has been reconstituted as an essential stimulatory factor. These activities include protein trafficking through the constitutive secretory pathway, endocytic pathway function, biogenesis of mast cell dense core secretory granules, and the agonist-induced fusion of dense core secretory granules to the mast cell plasma membrane. Finally, the data demonstrate that PITPα-deficient cells not only retain their responsiveness to bulk growth factor stimulation but also retain their pluripotency. In contrast, we were unable to evict both PITPβ alleles from murine cells and show that PITPβ deficiency results in catastrophic failure early in murine embryonic development. We suggest that PITPβ is an essential housekeeping PITP in murine cells, whereas PITPα plays a far more specialized function in mammals than that indicated by in vitro systems that show PITP dependence.

scholarly journals The sorting of proglucagon to secretory granules is mediated by carboxypeptidase E and intrinsic sorting signals

2013 ◽  
Vol 217 (2) ◽  
pp. 229-240 ◽  
Author(s):  
Rebecca McGirr ◽  
Leonardo Guizzetti ◽  
Savita Dhanvantari
Keyword(s):  
Secretory Pathway ◽  
Secretory Granules ◽  
Alpha Helix ◽  
Intestinal Cell ◽  
Alpha Cells ◽  
Pancreatic Alpha Cells ◽  
Carboxypeptidase E ◽  
L Cells ◽  
Sorting Signals ◽  
Regulated Secretory Pathway

Proglucagon is expressed in pancreatic alpha cells, intestinal L cells and brainstem neurons. Tissue-specific processing of proglucagon yields the peptide hormones glucagon in the alpha cell and glucagon-like peptide (GLP)-1 and GLP-2 in L cells. Both glucagon and GLP-1 are secreted in response to nutritional status and are critical for regulating glycaemia. The sorting of proglucagon to the dense-core secretory granules of the regulated secretory pathway is essential for the appropriate secretion of glucagon and GLP-1. We examined the roles of carboxypeptidase E (CPE), a prohormone sorting receptor, the processing enzymes PC1/3 and PC2 and putative intrinsic sorting signals in proglucagon sorting. In Neuro 2a cells that lacked CPE, PC1/3 and PC2, proglucagon co-localised with the Golgi marker p115 as determined by quantitative immunofluorescence microscopy. Expression of CPE, but not of PC1/3 or PC2, enhanced proglucagon sorting to granules. siRNA-mediated knockdown of CPE disrupted regulated secretion of glucagon from pancreatic-derived alphaTC1–6 cells, but not of GLP-1 from intestinal cell-derived GLUTag cells. Mutation of the PC cleavage site K70R71, the dibasic R17R18 site within glucagon or the alpha-helix of glucagon, all significantly affected the sub-cellular localisation of proglucagon. Protein modelling revealed that alpha helices corresponding to glucagon, GLP-1 and GLP-2, are arranged within a disordered structure, suggesting some flexibility in the sorting mechanism. We conclude that there are multiple mechanisms for sorting proglucagon to the regulated secretory pathway, including a role for CPE in pancreatic alpha cells, initial cleavage at K70R71 and multiple sorting signals.

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