scholarly journals Analysis of ZAPs, Zymogen Granule Membrane Associated Proteins, in the Regulated Exocytosis of the Pancreas

1994 ◽  
Vol 58 (7) ◽  
pp. 1282-1285 ◽  
Author(s):  
Shin-Ichi Fukuoka
Keyword(s):  
Zymogen Granule ◽  
Regulated Exocytosis ◽  
Granule Membrane ◽  
Associated Proteins

Involvement of a phosphoprotein on the zymogen granule membrane in the control of regulated exocytosis in the exocrine pancreas

1993 ◽  
Vol 106 (2) ◽  
pp. 663-670
Author(s):  
C.M. MacLean ◽  
S.J. Marciniak ◽  
D.V. Hall ◽  
J.M. Edwardson
Keyword(s):  
Acinar Cell ◽  
Time Course ◽  
Kinase Inhibitor ◽  
Pancreatic Acinar Cell ◽  
Zymogen Granule ◽  
Pancreatic Acini ◽  
Regulated Exocytosis ◽  
Permeabilized Cells ◽  
Granule Membrane ◽  
Gamma S

The pancreatic acinar cell is one of a number of cell types in which phosphoproteins are believed to be involved in the control of regulated exocytosis. We have examined the effects of three agents that affect secretion in the acinar cell on the phosphorylation states of proteins on the zymogen granule membrane. We show that Ca2+ and GTP gamma S, which stimulate secretion, also stimulate the phosphorylation of a protein of M(r) 45,000 (p45) on isolated zymogen granules. On the other hand, the protein kinase inhibitor genistein inhibits both secretion and phosphorylation of p45. For all three agents, p45 phosphorylation is affected over concentration ranges identical to those that affect secretion. The stimulatory effect of GTP gamma S and the inhibitory effect of genistein are also seen when the phosphorylation state of p45 on granules within permeabilized cells is examined. Ca2+, however, over the same concentration range, now causes dephosphorylation of p45. Furthermore, the time-course of this effect is similar to that of Ca(2+)-triggered secretion. Phosphorylation of p45 is exclusively on serine, with no detectable phosphorylation on either threonine or tyrosine. We propose that exocytosis in pancreatic acini is controlled at least in part through the phosphorylation/dephosphorylation of p45, with dephosphorylation acting as a trigger for exocytosis.

Rab3D localizes to zymogen granules in rat pancreatic acini and other exocrine glands

1996 ◽  
Vol 271 (3) ◽  
pp. G531-G538 ◽  
Author(s):  
H. Ohnishi ◽  
S. A. Ernst ◽  
N. Wys ◽  
M. McNiven ◽  
J. A. Williams
Keyword(s):  
Polyclonal Antiserum ◽  
Secretory Cells ◽  
Apical Region ◽  
Zymogen Granule ◽  
Zymogen Granules ◽  
Base Pairs ◽  
Pancreatic Acini ◽  
Pcr Product ◽  
The Family ◽  
Granule Membrane

Rab3 proteins are members of the family of Ras-like monomeric GTP-binding proteins that have been implicated in secretion in neuronal cells. Although an isoform of Rab3 has been assumed to exist in pancreatic acini, its identity has not yet been established. We now report that Rab3D is present in rat pancreatic acini and is localized to the zymogen granule membrane. Reverse transcription-polymerase chain reaction (PCR) was used with primers based on mouse Rab3D to amplify Rab3D from rat pancreas. The PCR product without primer sites consisted of 580 base pairs and was 94% identical to the mouse Rab3D cDNA sequence previously cloned from adipocytes. Western blotting with a polyclonal antiserum raised against Rab3D-specific carboxyterminal amino acids identified Rab3D in rat pancreatic acini and revealed its concentration on zymogen granule membranes. Immunocytochemistry of pancreatic lobules showed that Rab3D localized to the apical region in a pattern similar to amylase. Confocal fluorescence microscopy of lobules double immunolabeled with antibodies to Rab3D and the granule membrane marker protein glycoprotein-2 (GP-2) revealed a similar localization of these proteins to zymogen granules. Immunocytochemistry also revealed the presence of Rab3D in chief and enterochromaffin-like cells in the stomach, acinar cells in lacrimal and parotid gland, and Paneth cells in the intestine. These results show that Rab3D is expressed in rat pancreatic acini and other exocrine secretory cells. Its location implies it may be involved in regulated exocytosis.

Pancreatic Zymogen Granule Membrane Proteins: Molecular Details Begin to Emerge

Digestion ◽  
1994 ◽  
Vol 55 (4) ◽  
pp. 191-199 ◽  
Author(s):  
Andreas C.C. Wagner ◽  
John A. Williams
Keyword(s):  
Membrane Proteins ◽  
Zymogen Granule ◽  
Granule Membrane ◽  
Granule Membrane Proteins

Antibodies to GP2, the major zymogen granule membrane glycoprotein, in inflammatory bowel diseases

Gut ◽  
2010 ◽  
Vol 61 (1) ◽  
pp. 162.3-164 ◽  
Author(s):  
Katrijn Op De Beéck ◽  
Séverine Vermeire ◽  
Paul Rutgeerts ◽  
Xavier Bossuyt
Keyword(s):  
Inflammatory Bowel Diseases ◽  
Membrane Glycoprotein ◽  
Zymogen Granule ◽  
Bowel Diseases ◽  
Granule Membrane ◽  
Inflammatory Bowel

Identification of the catalytic subunit of the ATP diphosphohydrolase by photoaffinity labeling of high-affinity ATP-binding sites of pancreatic zymogen granule membranes with 8-azido-[α-32P]ATP

1986 ◽  
Vol 64 (1) ◽  
pp. 13-20 ◽  
Author(s):  
Denis LeBel ◽  
Marlyne Beattie
Keyword(s):  
Binding Sites ◽  
Divalent Cations ◽  
Photoaffinity Labeling ◽  
Competitive Inhibitor ◽  
Atp Binding ◽  
Zymogen Granule ◽  
Atp Diphosphohydrolase ◽  
High Affinity ◽  
Granule Membrane ◽  
Triton X

Photoaffinity labeling has been performed on pancreatic zymogen granule membranes using 8-azido-[α-32P]ATP (8-N3-ATP). Proteins of 92, 67, 53, and 35 kdaltons (kDa) were specifically labeled. ATP (100 μM) inhibited very strongly the labeling with 8-N3-ATP, while ADP was much less potent, AMP and cAMP being inefficient. The apparent constants for 8-N3-ATP binding were in the micromolar concentration range for the four labeled proteins. Without irradiation, 8-N3-ATP was a competitive inhibitor (Ki = 2.66 μM) for the hydrolysis of ATP by the ATP diphosphohydrolase. The optimal conditions for the photolabeling of the 92- and 53-kDa proteins were pH 6.0 in presence of divalent cations. On the other hand the 67- and 35-kDa proteins required an alkaline pH and the addition of EDTA in the photolabeling medium. No proteins could be labeled on intact zymogen granules, showing that all the high-affinity ATP-binding sites of the membrane were located at the interior of the granule. Both the 92- and 53-kDa glycoproteins could bind to concanavalin A–Sepharose and be extracted in the detergent phase in the Triton X-114 phase separation system. These latter properties are typical of integral membrane proteins. In addition, the 53-kDa labeled protein was sensitive to endo-β-N-acetylglucosaminidase digestion. Photolabeling with 8-N3-ATP of two different preparations of purified ATP diphosphohydrolase also led to the labeling of a 53-kDa protein. Thus among the four proteins labeled with 8-N3-ATP on the pancreatic zymogen granule membrane, the 53-kDa integral membrane glycoprotein was shown to bear the catalytic site of the ATP diphosphohydrolase.

scholarly journals Association of nucleoside diphosphate kinase with pancreatic zymogen granules: effects of local GTP generation on granule membrane characteristics

1996 ◽  
Vol 316 (1) ◽  
pp. 99-106 ◽  
Author(s):  
Stefan J. MARCINIAK ◽  
J. Michael EDWARDSON
Keyword(s):  
Plasma Membranes ◽  
Direct Action ◽  
Nucleoside Diphosphate Kinase ◽  
Pancreatic Acinar Cell ◽  
Zymogen Granule ◽  
Zymogen Granules ◽  
Cytoplasmic Face ◽  
Granule Membrane ◽  
Stimulation Of

It is well established that both GTP-binding proteins and phosphoproteins are involved in the control of exocytosis in the exocrine pancreas. Exocytotic membrane fusion is stimulated by guanosine 5′-[γ-thio]triphosphate, and the phosphorylation states of several proteins, including at least one on the zymogen granule membrane, are known to change during exocytosis. We show here that a nucleoside diphosphate kinase is associated with the cytoplasmic face of pancreatic zymogen granules. This enzyme behaves as a phosphoprotein of apparent molecular mass 21 kDa on SDS/polyacrylamide gels, and is able to produce GTP by using ATP to phosphorylate endogenous GDP. GTP production by nucleoside diphosphate kinase is stimulated by the wasp venom peptide mastoparan, both through a direct action on the enzyme and through its ability to increase the availability of endogenous GDP. Two effects of the GTP produced by nucleoside diphosphate kinase are demonstrated: phosphorylation of a 37 kDa zymogen granule protein on histidine residues, and stimulation of the fusion of zymogen granules with pancreatic plasma membranes in vitro. These results suggest that granule-associated nucleoside diphosphate kinase is able to maintain local GTP concentrations, and raise the possibility that it might be involved in the control of exocytosis in the pancreatic acinar cell.

scholarly journals Mistargeting of secretory cargo in retromer-deficient cells

2020 ◽  
pp. dmm.046417
Author(s):  
Sarah D. Neuman ◽  
Erica L. Terry ◽  
Jane E. Selegue ◽  
Amy T. Cavanagh ◽  
Arash Bashirullah
Keyword(s):  
Membrane Proteins ◽  
Salivary Glands ◽  
Secretory Granule ◽  
Complex Function ◽  
Amyloid Β ◽  
Secretory Cells ◽  
Regulated Exocytosis ◽  
Retromer Complex ◽  
Granule Membrane ◽  
Granule Membrane Proteins

Intracellular trafficking is a basic and essential cellular function required for delivery of proteins to the appropriate subcellular destination; this process is especially demanding in professional secretory cells, which synthesize and secrete massive quantities of cargo proteins via regulated exocytosis. The Drosophila larval salivary glands are professional secretory cells that synthesize and secrete mucin proteins at the onset of metamorphosis. Using the larval salivary glands as a model system, we have identified a role for the highly conserved retromer complex in trafficking of secretory granule membrane proteins. We demonstrate that retromer-dependent trafficking via endosomal tubules is induced at the onset of secretory granule biogenesis, and that recycling via endosomal tubules is required for delivery of essential secretory granule membrane proteins to nascent granules. Without retromer function, nascent granules do not contain the proper membrane proteins; as a result, cargo from these defective granules is mistargeted to Rab7-positive endosomes, where it progressively accumulates to generate dramatically enlarged endosomes. Retromer complex dysfunction is strongly associated with neurodegenerative diseases, including Alzheimer's disease, characterized by accumulation of amyloid β (Aβ). We show that ectopically expressed amyloid precursor protein (APP) undergoes regulated exocytosis in salivary glands and accumulates within enlarged endosomes in retromer-deficient cells. These results highlight recycling of secretory granule membrane proteins as a critical step during secretory granule maturation and provide new insights into our understanding of retromer complex function in secretory cells. These findings also suggest that missorting of secretory cargo, including APP, may contribute to the progressive nature of neurodegenerative disease.

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