scholarly journals The disulfide-bonded loop of chromogranin B mediates membrane binding and directs sorting from the trans-Golgi network to secretory granules

1999 ◽  
Vol 18 (4) ◽  
pp. 1059-1070 ◽  
Author(s):  
Michael M. Glombik ◽  
Andreas Krömer ◽  
Thorsten Salm ◽  
Wieland B. Huttner ◽  
Hans-Hermann Gerdes
Keyword(s):  
Secretory Granules ◽  
Membrane Binding ◽  
Chromogranin B ◽  
Trans Golgi Network ◽  
Golgi Network

scholarly journals Expression of Regulated Secretory Proteins Is Sufficient to Generate Granule-like Structures in Constitutively Secreting Cells

2004 ◽  
Vol 279 (19) ◽  
pp. 20242-20249 ◽  
Author(s):  
Nicole Beuret ◽  
Hansruedi Stettler ◽  
Anja Renold ◽  
Jonas Rutishauser ◽  
Martin Spiess
Keyword(s):  
Protease Inhibitor ◽  
Secretory Granules ◽  
Secretory Proteins ◽  
Ionophore A23187 ◽  
Cell Periphery ◽  
Chromogranin B ◽  
Granule Formation ◽  
Trans Golgi Network ◽  
Secretogranin Ii ◽  
Golgi Network

The formation of secretory granules and regulated secretion are generally assumed to occur only in specialized endocrine, neuronal, or exocrine cells. We discovered that regulated secretory proteins such as the hormone precursors pro-vasopressin, pro-oxytocin, and pro-opiomelanocortin, as well as the granins secretogranin II and chromogranin B but not the constitutive secretory protein α1-protease inhibitor, accumulate in granular structures at the Golgi and in the cell periphery in transfected COS-1 fibroblast cells. The accumulations were observed in 30–70% of the transfected cells expressing the pro-hormones and for virtually all of the cells expressing the granins. Similar structures were also generated in other cell lines believed to be lacking a regulated secretory pathway. The accumulations resembled secretory granules morphologically in immunofluorescence and electron microscopy. They were devoid of markers of the endoplasmic reticulum, endosomes, and lysosomes but in part stained positive for the trans-Golgi network marker TGN46, consistent with their formation at the trans-Golgi network. When different regulated proteins were coexpressed, they were frequently found in the same granules, whereas α1-protease inhibitor could not be detected in accumulations formed by secretogranin II, demonstrating segregation of regulated from constitutive secretory proteins. In pulse-chase experiments, significant intracellular storage of secretogranin II and chromogranin B was observed and secretion of retained secretogranin II was stimulated with the calcium ionophore A23187. The results suggest that expression of regulated cargo proteins is sufficient to generate structures that resemble secretory granules in the background of constitutively secreting cells, supporting earlier proposals on the mechanism of granule formation.

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 Differential sorting behavior for soluble and transmembrane cargoes at the trans-Golgi network in endocrine cells

2020 ◽  
Vol 31 (3) ◽  
pp. 157-166 ◽  
Author(s):  
Blake H. Hummer ◽  
Drew Maslar ◽  
Margarita Soltero-Gutierrez ◽  
Noah F. de Leeuw ◽  
Cedric S. Asensio
Keyword(s):  
Endocrine Cells ◽  
Secretory Granules ◽  
Complex Model ◽  
Trans Golgi Network ◽  
Golgi Network

Formation of secretory granules (SGs) occurs at the trans-Golgi network (TGN). Here we show that transmembrane SG cargoes (phogrin and VMAT2) do not sort directly onto SGs during budding, but rather exit the TGN into nonregulated vesicles to get incorporated to SGs at a later step, suggesting a more complex model of SG biogenesis than anticipated.

scholarly journals Dynamics of Immature Secretory Granules: Role of Cytoskeletal Elements during Transport, Cortical Restriction, and F-Actin-dependent Tethering

2001 ◽  
Vol 12 (5) ◽  
pp. 1353-1365 ◽  
Author(s):  
Rüdiger Rudolf ◽  
Thorsten Salm ◽  
Amin Rustom ◽  
Hans-Hermann Gerdes
Keyword(s):  
Secretory Granules ◽  
Late Phase ◽  
Cell Cortex ◽  
Cell Periphery ◽  
Dependent Manner ◽  
Chromogranin B ◽  
Cooperative Action ◽  
Cytoskeletal Elements ◽  
Golgi Network

Secretory granules store neuropeptides and hormones and exhibit regulated exocytosis upon appropriate cellular stimulation. They are generated in the trans-Golgi network as immature secretory granules, short-lived vesicular intermediates, which undergo a complex and poorly understood maturation process. Due to their short half-life and low abundance, real-time studies of immature secretory granules have not been previously possible. We describe here a pulse/chase-like system based on the expression of a human chromogranin B-GFP fusion protein in neuroendocrine PC12 cells, which permits direct visualization of the budding of immature secretory granules and their dynamics during maturation. Live cell imaging revealed that newly formed immature secretory granules are transported in a direct and microtubule-dependent manner within a few seconds to the cell periphery. Our data suggest that the cooperative action of microtubules and actin filaments restricts immature secretory granules to the F-actin-rich cell cortex, where they move randomly and mature completely within a few hours. During this maturation period, secretory granules segregate into pools of different motility. In a late phase of maturation, 60% of secretory granules were found to be immobile and about half of these underwent F-actin-dependent tethering.

scholarly journals 100-kD proteins of Golgi- and trans-Golgi network-associated coated vesicles have related but distinct membrane binding properties

1992 ◽  
Vol 117 (6) ◽  
pp. 1171-1179 ◽  
Author(s):  
DH Wong ◽  
FM Brodsky
Keyword(s):  
Membrane Binding ◽  
Coated Vesicles ◽  
Coat Proteins ◽  
Coated Pits ◽  
Binding Properties ◽  
Vitro Assay ◽  
Normal Cycle ◽  
Trans Golgi Network ◽  
Golgi Region ◽  
Golgi Network

The 100-110-kD proteins (alpha-, beta-, beta'-, and gamma-adaptins) of clathrin-coated vesicles and the 110-kD protein (beta-COP) of the nonclathrin-coated vesicles that mediate constitutive transport through the Golgi have homologous protein sequences. To determine whether homologous processes are involved in assembly of the two types of coated vesicles, the membrane binding properties of their coat proteins were compared. After treatment of MDBK cells with the fungal metabolite Brefeldin A (BFA), beta-COP was redistributed to the cytoplasm within 15 s, gamma-adaptin and clathrin in the trans-Golgi network (TGN) dispersed within 30 s, but the alpha-adaptin and clathrin present on coated pits and vesicles derived from the plasma membrane remained membrane associated even after a 15-min exposure to BFA. In PtK1 cells and MDCK cells, BFA did not affect beta-COP binding or Golgi morphology but still induced redistribution of gamma-adaptin and clathrin from TGN membranes to the cytoplasm. Thus BFA affects the binding of coat proteins to membranes in the Golgi region (Golgi apparatus and TGN) but not plasma membranes. However, the Golgi binding interactions of beta-COP and gamma-adaptin are distinct and differentially sensitive to BFA. BFA treatment did not release gamma-adaptin or clathrin from purified clathrin-coated vesicles, suggesting that their distribution to the cytoplasm after BFA treatment of cells was due to interference with their rebinding to TGN membranes after a normal cycle of disassembly. This was confirmed using an in vitro assay in which gamma-adaptin binding to TGN membranes was blocked by BFA and enhanced by GTP gamma S, similar to the binding of beta-COP to Golgi membranes. These results suggest the involvement of GTP-dependent proteins in the association of the 100-kD coat proteins with membranes in the Golgi region of the cell.

Molecular chaperones in pancreatic tissue: the presence of cpn10, cpn60 and hsp70 in distinct compartments along the secretory pathway of the acinar cells

1994 ◽  
Vol 107 (3) ◽  
pp. 539-549 ◽  
Author(s):  
C.S. Velez-Granell ◽  
A.E. Arias ◽  
J.A. Torres-Ruiz ◽  
M. Bendayan
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Acinar Cells ◽  
Pancreatic Tissue ◽  
Secretory Proteins ◽  
Trans Golgi Network ◽  
Hsp70 Protein ◽  
Golgi Network

Three chaperones, the chaperonins cpn10 and cpn60, and the hsp70 protein, were revealed by immunochemistry and cytochemistry in pancreatic rat acinar cells. Western immunoblotting analysis of rat pancreas homogenates has shown that antibodies against cpn10, cpn60 and hsp70 protein recognize single protein bands of 25 kDa, 60 kDa and 70 kDa, respectively. Single bands for the cpn10 and cpn60 were also detected in pancreatic juice. Immunofluorescence studies on rat pancreatic tissue revealed a strong positive signal in the apical region of the acinar cells for cpn10 and cpn60, while an immunoreaction was detected at the juxtanuclear Golgi region with the anti-hsp70 antibody. Immunocytochemical gold labeling confirmed the presence of these three chaperones in distinct cell compartments of pancreatic acinar cells. Chaperonin 10 and cpn60 were located in the endoplasmic reticulum, Golgi apparatus, condensing vacuoles and secretory granules. Interestingly, the labeling for both cpn10 and cpn60 followed the increasing concentration gradient of secretory proteins along the RER-Golgi-granule secretory pathway. On the contrary, the labeling for hsp70 was mainly concentrated in the endoplasmic reticulum and the Golgi apparatus. In the latter, the hsp70 was found to be primary located in the trans-most cisternae and to colocalize with acid phosphatase in the trans-Golgi network. The three chaperones were also present in mitochondria. In view of the role played by the chaperones in the proper folding, sorting and aggregation of proteins, we postulate that hsp70 assists the adequate sorting and packaging of proteins from the ER to the trans-Golgi network while cpn10 and cpn60 play key roles in the proper packaging and aggregation of secretory proteins as well as, most probably, in the prevention of early enzyme activation in secretory granules.

Structural and histochemical studies of Golgi complex differentiation in salivary gland cells during Drosophila development

1992 ◽  
Vol 102 (1) ◽  
pp. 169-184 ◽  
Author(s):  
G.N. Thomopoulos ◽  
E.P. Neophytou ◽  
M. Alexiou ◽  
A. Vadolas ◽  
S. Limberi-Thomopoulos ◽  
...  
Keyword(s):  
Salivary Gland ◽  
Golgi Complex ◽  
Secretory Granules ◽  
Differential Staining ◽  
Reaction Products ◽  
Trans Golgi Network ◽  
Gland Cells ◽  
Salivary Gland Cells ◽  
Complex Carbohydrates ◽  
Golgi Network

Morphological alterations in the Golgi complex (GC) and changes in the distribution of acid phosphatase (AcPase), thiamine pyrophosphatase (TPPase), complex carbohydrates and reduced osmium tetroxide compounds in this organelle were studied in the salivary gland cells of Drosophila during larval and prepupal development. The morphology and the AcPase, TPPase and complex carbohydrates cytochemical patterns of the Golgi complex varied characteristically during cell differentiation. At the early 3rd instar period the Golgi complex consisted mainly of vesiculated cisternae, and AcPase activity was observed in all cisternae but not in the secretory granules. As development proceeded to the late 3rd instar the Golgi complex displayed its typical appearance, consisting of four to six cisternae, and only the two to three cisternae towards the trans-face as well as the trans-Golgi network and some of the immature secretory granules exhibited AcPase reactivity. In the course of a ‘wave’ of production of the ‘glue’ secretory granules proceeding proximally through the gland, the number of AcPase positive cisternae changed correspondingly. After secretion of the ‘glue’ secretory granules, the size of the Golgi complex decreased and almost all cisternae displayed AcPase reactivity. The detection of TPPase activity presented some specificity problems, since staining was observed not only in the GC cisternae but in the endoplasmic reticulum (ER) and microvilli. The reaction products were seen in a few GC vesicles during the early 3rd instar and in the trans side of the organelle at the end of the 3rd instar. During production of the secretory granules, every GC cisterna was intensely stained. These results agree with previous findings suggesting that AcPase and TPPase in secretory cells may be primarily involved in the processing of exportable proteins. The vicinal (vic)-glycol groups of the complex carbohydrates were detected using the periodic acid/thiocarbohydrazide/silver proteinate (PA-TCH-SP) technique. During synthesis of the ‘glue’ secretory granules, the reaction products were observed over the GC cisternae and the trans-Golgi network, with increasing intensity from the cis to the trans side of the organelle. No PA-TCH-SP staining was observed over the GC cisternae during the early 3rd instar. Following discharge of the ‘glue’ secretory granules, all GC cisternae displayed uniform PA-TCH-SP staining. After OsO4 impregnation, the reaction products were observed mainly in ER and mitochondria and rarely in the GC. In numerous cells, only the mitochondria were stained, while in many cases the ER of neighboring cells exhibited differential staining.(ABSTRACT TRUNCATED AT 400 WORDS)

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