scholarly journals Membrane insertion of chromogranin B for granule maturation in regulated secretion

2019 ◽  
Author(s):  
Gaya P. Yadav ◽  
Haiyuan Wang ◽  
Joke Ouwendijk ◽  
Mani Annamalai ◽  
Stephen Cross ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Secretory Granules ◽  
Neuroendocrine Cells ◽  
Membrane Insertion ◽  
List Type ◽  
Chromogranin B ◽  
Regulated Secretion ◽  
Anion Conductance ◽  
Altered Glucose ◽  
Granule Maturation

ABSTRACTRegulated secretion serves responses to specific stimuli in eukaryotes. An anion conductance was found essential for maturation and acidification of secretory granules four decades ago, but its genetic identity was unknown. We now demonstrate that chromogranin B (CHGB), an obligate granule protein, constitutes the long-sought anion channel. High-pressure freezing immuno-electron microscopy and biochemical assays showed native CHGB in close proximity to secretory granule membranes, and its membrane-bound and soluble forms both reconstituted Cl- channels. Release of secretory granules delivered CHGB clusters to plasma membranes, which dominate whole-cell anion conductance. Intragranular pH measurements and cargo maturation assays found that CHGB channels supported proinsulin - insulin conversion and dopamine-loading in neuroendocrine cells. β-cells from Chgb-/- mice exhibited significant granule deacidification, accounting for hyperproinsulinemia, altered glucose-tolerance response and lower dopamine concentration in chromaffin granules in these animals. Membrane insertion of well-conserved CHGB is thus indispensable for granule maturation in exocrine, endocrine and neuronal cells.HighlightsNative CHGB is amphipathic and distributes in the lumen and membranes of secretory granules with contrastingly different destinies and functions.Native CHGB, once delivered to cell surface via granule exocytosis, dominates anion conductance in plasma membranes.CHGB channels facilitate granule acidification and cargo maturation in cultured and primary neuroendocrine cells.CHGB channels from bovine, rat and mouse cells all serve the long-missing, intra-organellar anion shunt pathway in the secretory granules for regulated secretion.

scholarly journals Novel organelle anion channels formed by chromogranin B drive normal granule maturation in endocrine cells

2018 ◽  
Author(s):  
Gaya Yadav ◽  
Hui Zheng ◽  
Qing Yang ◽  
Lauren Douma ◽  
Mani Annamalai ◽  
...  
Keyword(s):  
Pancreatic Beta Cells ◽  
Chloride Channels ◽  
Endocrine Cells ◽  
Secretory Granules ◽  
Neuroendocrine Cells ◽  
Anion Channels ◽  
Chromogranin B ◽  
Fast Kinetics ◽  
Anion Conductance ◽  
Granule Maturation

All endocrine cells need an anion conductance for maturation of secretory granules. Identity of this family of anion channels has been elusive for forty years. We now show that a family of granule proteins, CHGB, serves the long-sought conductance. CHGB interacts with membranes through two amphipathic helices, and forms a chloride channel with a large conductance and high anion selectivity. Fast kinetics and high cooperativity suggest that CHGB tetramerizes to form a functional channel. Nonconducting mutants separate CHGB channel function in granule maturation from its role in granule biogenesis. In neuroendocrine cells, CHGB channel and a H+-ATPase drive normal insulin maturation inside or catecholamine loading into secretory granules. Tight membrane-association of CHGB after exocytotic release of secretory granules separates its intracellular functions from the extracellular functions accomplished by its proteolytic peptides. CHGB-null mice show impairment of granule acidification in pancreatic beta-cells due to lack of anion conductance. These findings together support that the phylogenetically conserved CHGB proteins constitute a fifth family of chloride channels that function in various endocrine cells.

scholarly journals A Novel Site of Action for α-SNAP in the SNARE Conformational Cycle Controlling Membrane Fusion

2008 ◽  
Vol 19 (3) ◽  
pp. 776-784 ◽  
Author(s):  
Marcin Barszczewski ◽  
John J. Chua ◽  
Alexander Stein ◽  
Ulrike Winter ◽  
Rainer Heintzmann ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Plasma Membranes ◽  
Secretory Granules ◽  
Neuroendocrine Cells ◽  
Snare Complex ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Site Of Action ◽  
Snare Motif ◽  
Liposome Fusion

Regulated exocytosis in neurons and neuroendocrine cells requires the formation of a stable soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex consisting of synaptobrevin-2/vesicle-associated membrane protein 2, synaptosome-associated protein of 25 kDa (SNAP-25), and syntaxin 1. This complex is subsequently disassembled by the concerted action of α-SNAP and the ATPases associated with different cellular activities-ATPase N-ethylmaleimide-sensitive factor (NSF). We report that NSF inhibition causes accumulation of α-SNAP in clusters on plasma membranes. Clustering is mediated by the binding of α-SNAP to uncomplexed syntaxin, because cleavage of syntaxin with botulinum neurotoxin C1 or competition by using antibodies against syntaxin SNARE motif abolishes clustering. Binding of α-SNAP potently inhibits Ca2+-dependent exocytosis of secretory granules and SNARE-mediated liposome fusion. Membrane clustering and inhibition of both exocytosis and liposome fusion are counteracted by NSF but not when an α-SNAP mutant defective in NSF activation is used. We conclude that α-SNAP inhibits exocytosis by binding to the syntaxin SNARE motif and in turn prevents SNARE assembly, revealing an unexpected site of action for α-SNAP in the SNARE cycle that drives exocytotic membrane fusion.

scholarly journals Milieu-induced, selective aggregation of regulated secretory proteins in the trans-Golgi network.

1991 ◽  
Vol 115 (6) ◽  
pp. 1505-1519 ◽  
Author(s):  
E Chanat ◽  
W B Huttner
Keyword(s):  
Secretory Granules ◽  
Hormonal Treatment ◽  
Neuroendocrine Cells ◽  
Secretory Proteins ◽  
Chromogranin B ◽  
Granule Formation ◽  
Trans Golgi Network ◽  
High Calcium ◽  
Golgi Network ◽  
Selective Aggregation

Regulated secretory proteins are thought to be sorted in the trans-Golgi network (TGN) via selective aggregation. The factors responsible for this aggregation are unknown. We show here that two widespread regulated secretory proteins, chromogranin B and secretogranin II (granins), remain in an aggregated state when TGN vesicles from neuroendocrine cells (PC12) are permeabilized at pH 6.4 in 1-10 mM calcium, conditions believed to exist in this compartment. Permeabilization of immature secretory granules under these conditions allowed the recovery of electron dense cores. The granin aggregates in the TGN largely excluded glycosaminoglycan chains which served as constitutively secreted bulk flow markers. The low pH, high calcium milieu was sufficient to induce granin aggregation in the RER. In the TGN of pituitary GH4C1 cells, the proportion of granins conserved as aggregates was higher upon hormonal treatment known to increase secretory granule formation. Our data suggest that a decrease in pH and an increase in calcium are sufficient to trigger the selective aggregation of the granins in the TGN, segregating them from constitutive secretory proteins.

scholarly journals Chromogranin B Serves the Long-Sought-After Anion Conductance in Regulated Secretion

2017 ◽  
Vol 112 (3) ◽  
pp. 184a
Author(s):  
Gaya P. Yadav ◽  
Qing Yang ◽  
Qiu-Xing Jiang
Keyword(s):  
Chromogranin B ◽  
Regulated Secretion ◽  
Anion Conductance

LIGHT AND ELECTRON MICROSCOPICAL STUDY OF THE EFFECT OF OESTROGEN ON THE CHICKEN OVIDUCT

1967 ◽  
Vol 56 (3) ◽  
pp. 391-402 ◽  
Author(s):  
H. I. Ljungkvist
Keyword(s):  
Secretory Granules ◽  
Apical Cell ◽  
Cell Types ◽  
Microscopical Study ◽  
List Type ◽  
New Production ◽  
General Increase ◽  
Chicken Oviduct ◽  
Electron Microscopical ◽  
Aortic Perfusion

ABSTRACT Oviducts from 20 one-day old chickens were used. Ten chickens were injected subcutaneously with 0.2 mg oestradiol for 5 days, the remaining ones serving as controls. The chickens were fixed by an aortic perfusion with 2.5% glutaraldehyde in phosphate buffer, pH 7.2. The treatment with oestrogen resulted in the following changes: general increase in oviduct length and thickness, differentiation of the epithelial membrane into three cell types: basal, apical and gland cells, increase in the number of cilia in the apical cell, probably due to a new production of cilia, formation of secretory granules in the vaginal epithelium as seen by light microscopy, formation of proteinlike secretory granules in the apical cell as seen by electron microscopy, increase in protein synthesis, observed as an augmentation of the endoplasmic reticulum.

Synaptic Transmission in the Immune System

e-Neuroforum ◽  
2017 ◽  
Vol 23 (4) ◽  
Author(s):  
Jens Rettig ◽  
David R. Stevens
Keyword(s):  
Nervous System ◽  
Immune System ◽  
Synaptic Transmission ◽  
Molecular Mechanisms ◽  
Secretory Granules ◽  
Neuroendocrine Cells ◽  
Regulated Exocytosis ◽  
Protein Receptor ◽  
Immunological Synapses ◽  
Immunological Diseases

AbstractThe release of neurotransmitters at synapses belongs to the most important processes in the central nervous system. In the last decades much has been learned about the molecular mechanisms which form the basis for this fundamental process. Highly regulated exocytosis, based on the SNARE (soluble N-ethylmaleimide-sensitive attachment protein receptor) complex and its regulatory molecules is the signature specialization of the nervous system and is shared by neurons and neuroendocrine cells. Cells of the immune system use a similar mechanism to release cytotoxic materials from secretory granules at contacts with virally or bacterially infected cells or cancer cells, in order to remove these threats. These contact zones have been termed immunological synapses in reference to the highly specific targeted exocytosis of effector molecules. Recent findings indicate that mutations in SNARE or SNARE-interacting proteins are the basis of a number of devastating immunological diseases. While SNARE complexes are ubiquitous and mediate a wide variety of membrane fusion events it is surprising that in many cases the SNARE proteins involved in immunological synapses are the same molecules which mediate regulated exocytosis of transmitters and hormones in neurons and neuroendocrine cells. These similarities raise the possibility that results obtained at immunological synapses may be applicable, in particular in the area of presynaptic function, to neuronal synapses. Since immunological synapses (IS) are assembled and disassembled in about a half an hour, the use of immune cells isolated from human blood allows not only the study of the molecular mechanisms of synaptic transmission in human cells, but is particularly suited to the examination of the assembly and disassembly of these “synapses” via live imaging. In this overview we discuss areas of similarity between synapses of the nervous and immune systems and in the process will refer to results of our experiments of the last few years.

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.

scholarly journals Passive Sorting in Maturing Granules of AtT-20 Cells: The Entry and Exit of Salivary Amylase and Proline-rich Protein

1997 ◽  
Vol 138 (1) ◽  
pp. 45-54 ◽  
Author(s):  
Anna M. Castle ◽  
Amy Y. Huang ◽  
J. David Castle
Keyword(s):  
Secretory Pathway ◽  
Critical Role ◽  
Endogenous Hormones ◽  
Salivary Amylase ◽  
Regulated Secretion ◽  
J 13 ◽  
Regulated Secretory Pathway ◽  
Granule Maturation ◽  
Proline Rich Protein

Previous studies have suggested that salivary amylase and proline-rich protein are sorted differently when expressed in AtT-20 cells (Castle, A.M., L.E. Stahl, and J.D. Castle. 1992. J. Biol. Chem. 267:13093– 13100; Colomer, V., K. Lal, T.C. Hoops, and M.J. Rindler. 1994.EMBO (Eur. Mol. Biol. Organ.) J. 13:3711– 3719). We now show that both exocrine proteins behave similarly and enter the regulated secretory pathway as judged by immunolocalization and secretagogue- dependent stimulation of secretion. Analysis of stimulated secretion of newly synthesized proline-rich protein, amylase, and endogenous hormones indicates that the exogenous proteins enter the granule pool with about the same efficiency as the endogenous hormones. However, in contrast to the endogenous hormones, proline-rich protein and amylase are progressively removed from the granule pool during the process of granule maturation such that only small portions remain in mature granules where they colocalize with the stored hormones. The exogenous proteins that are not stored are recovered from the incubation medium and are presumed to have undergone constitutive-like secretion. These results point to a level of sorting for regulated secretion after entry of proteins into forming granules and indicate that retention is essential for efficient storage. Consequently, the critical role of putative sorting receptors for regulated secretion may be in retention rather than in granule entry.

scholarly journals Pancreatic plasma membranes: promiscuous partners in membrane fusion

1994 ◽  
Vol 298 (3) ◽  
pp. 599-604 ◽  
Author(s):  
E G Lee ◽  
S J Marciniak ◽  
C M MacLean ◽  
J M Edwardson
Keyword(s):  
Membrane Fusion ◽  
Plasma Membranes ◽  
Secretory Granules ◽  
The Other ◽  
Zymogen Granules ◽  
Other Hand

We have developed a system in which the fusion of pancreatic plasma membranes with zymogen granules can be studied in vitro. We show here that pancreatic plasma membranes fuse not only with pancreatic zymogen granules but also with parotid secretory granules. In contrast, parotid membranes fuse only with parotid granules and not with pancreatic granules. The extent of fusion is insensitive to Ca2+ for all combinations of plasma membranes and granules. Guanosine 5′-[gamma-thio]triphosphate (GTP[S]), on the other hand, stimulates fusion of pancreatic membranes with both pancreatic granules and parotid granules, but inhibits fusion between parotid membranes and parotid granules.

scholarly journals Localization of protein disulfide isomerase on plasma membranes of rat exocrine pancreatic cells.

1988 ◽  
Vol 36 (8) ◽  
pp. 1069-1074 ◽  
Author(s):  
S Akagi ◽  
A Yamamoto ◽  
T Yoshimori ◽  
R Masaki ◽  
R Ogawa ◽  
...  
Keyword(s):  
Protein Disulfide Isomerase ◽  
Plasma Membranes ◽  
Secretory Granules ◽  
Amorphous Material ◽  
Protein A ◽  
Disulfide Isomerase ◽  
Gold Particles ◽  
Pancreatic Cells ◽  
Acinar Lumen ◽  
Protein Disulfide

We investigated immunocytochemically the ultrastructural localization of protein disulfide isomerase (PDI) in rat pancreatic exocrine cells by use of the post-embedding protein A-gold technique. We found that not only the endoplasmic reticulum (ER) and nuclear envelope but also the trans-Golgi cisternae, secretory granules, and plasma membranes were heavily labeled with gold particles. Labeling density of the gold particles in the rough ER and plasma membranes of the exocrine pancreatic cells was twofold and twentyfold greater, respectively, than that of hepatocytes. In the acinar lumen, amorphous material presumably corresponding to the secreted zymogens was also labeled with gold particles. These results suggest that in rat exocrine pancreatic cells a significant amount of PDI is transported to the plasma membrane and secreted to the acinar lumen.

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