scholarly journals Immunocytochemical and cytochemical demonstration of a novel selective lysosomal pathway (SLP) of secretion in the exocrine pancreas.

1996 ◽  
Vol 44 (4) ◽  
pp. 357-368 ◽  
Author(s):  
G Grondin ◽  
A R Beaudoin
Keyword(s):  
Golgi Apparatus ◽  
Acinar Cell ◽  
Major Protein ◽  
Zymogen Granule ◽  
Golgi Stack ◽  
Strong Signal ◽  
Cytochemical Demonstration ◽  
Acinar Lumen ◽  
Golgi Network ◽  
Kinetics Of

The intracellular distributions of lysosomal and zymogen granule (ZG) membrane proteins were analyzed in the pancreas exocrine acinar cell by cytochemical and immunocytochemical approaches. A strong signal was observed with acid phosphatase (AcPase) in the trans-Golgi network and condensing vacuoles, whereas mature ZG and acinar lumina were devoid of any detectable reaction. The enzyme appears to exit from the regulated pathway by a shedding process during conversion of condensing vacuoles to mature granules. Trimetaphosphatase (TMPase) shows no reaction in the Golgi apparatus and condensing vacuole but is present in immature granules. The exit from the regulated pathway appears to occur at a later stage of the ZG maturation process. A third lysosomal enzyme, nicotinamide adenine dinucleotide phosphohydrolase (NADPase), was found in the median cisterna of the Golgi stack, was undetectable in condensing vacuoles and ZG, but produced a strong signal in the acinar lumen. Our observations show that only one type of intermediate organelle can explain the transport of that enzyme from the Golgi apparatus to the acinar lumen, and it is represented by a subpopulation of lysosomal bodies (LBs) highly reactive for this enzyme. In parallel, immunocytochemical observations with specific antibodies to GP2, a major protein component of the ZG membrane, have confirmed that most of the GP2 molecules in the acinar lumen do not derive from the ZG compartment but rather are from a subpopulation of highly reactive lysosomal structures. Because both NADPase and GP2 co-localize in specific lysosomal structures, and because these LBs are extruded in the acinar lumen, we conclude that this subpopulation of LBs is involved in selective transport of some lysosomal enzymes from the Golgi apparatus to the acinar lumen. This selective lysosomal pathway of secretion can explain the kinetics of GP2 transport and release from the acinar cell that cannot be explained either by the constitutive or the regulated pathway of secretion.

scholarly journals Depletion of the human N-terminal acetyltransferase hNaa30 disrupts Golgi integrity and ARFRP1 localization

2017 ◽  
Vol 37 (2) ◽  
Author(s):  
Kristian K. Starheim ◽  
Thomas V. Kalvik ◽  
Geir Bjørkøy ◽  
Thomas Arnesen
Keyword(s):  
Lipid Metabolism ◽  
Golgi Apparatus ◽  
Membrane Association ◽  
Cellular Lipid ◽  
Related Protein ◽  
Terminal Sequence ◽  
Golgi Stack ◽  
Cellular Processes ◽  
Golgi Network ◽  
Adp Ribosylation Factor

The organization of the Golgi apparatus (GA) is tightly regulated. Golgi stack scattering is observed in cellular processes such as apoptosis and mitosis, and has also been associated with disruption of cellular lipid metabolism and neurodegenerative diseases. Our studies show that depletion of the human N-α-acetyltransferase 30 (hNaa30) induces fragmentation of the Golgi stack in HeLa and CAL-62 cell lines. The GA associated GTPase ADP ribosylation factor related protein 1 (ARFRP1) was previously shown to require N-terminal acetylation for membrane association and based on its N-terminal sequence, it is likely to be a substrate of hNaa30. ARFRP1 is involved in endosome-to-trans-Golgi network (TGN) traffic. We observed that ARFRP1 shifted from a predominantly cis-Golgi and TGN localization to localizing both Golgi and non-Golgi vesicular structures in hNaa30-depleted cells. However, we did not observe loss of membrane association of ARFRP1. We conclude that hNaa30 depletion induces Golgi scattering and induces aberrant ARFRP1 Golgi localization.

scholarly journals Detection and characterization of microvesicles in the acinar lumen and in juice of unstimulated rat pancreas.

1986 ◽  
Vol 34 (8) ◽  
pp. 1079-1084 ◽  
Author(s):  
A R Beaudoin ◽  
G Grondin ◽  
A Vachereau ◽  
P St-Jean ◽  
C Cabana
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Freeze Fracture ◽  
Freeze Substitution ◽  
Major Protein ◽  
Zymogen Granule ◽  
Rat Pancreas ◽  
Granule Membrane ◽  
Acinar Lumen

Small vesicles were visualized in the lumen of rat pancreas acini by freeze-substitution and conventional electron microscopy. Microvesicles were subsequently isolated from pancreatic juice. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that these vesicles contain only one major protein. The major protein was identified by an immunoblot technique as GP-2, an 80 kD glycoprotein also found in the zymogen granule membrane. The immunocytochemical localization of rabbit anti-GP-2 and anti-amylase by the protein A-gold technique confirmed that GP-2 was associated with clusters of microvesicles, whereas amylase was virtually excluded. Freeze-fracture of the microvesicles revealed that their membrane was devoid of intramembrane particles. Biochemical analysis indicated also that the membrane did not contain any detectable cholesterol. These results demonstrate that GP-2 is released from the acinar cell in the gland lumen within microvesicles by a hitherto undescribed mode of secretion.

Specific interactions of pancreatic amylase at acidic pH. Amylase and the major protein of the zymogen granule membrane (GP-2) bind to immobilized or polymerized amylase

1992 ◽  
Vol 70 (10-11) ◽  
pp. 1105-1114 ◽  
Author(s):  
Michèle Jacob ◽  
Jean Lainé ◽  
Denis LeBel
Keyword(s):  
Binding Sites ◽  
Major Protein ◽  
Zymogen Granule ◽  
Dependent Manner ◽  
Acidic Ph ◽  
Trans Golgi Network ◽  
Neutral Ph ◽  
Granule Membrane ◽  
Ph Dependent ◽  
Golgi Network

Regulated secretory proteins are thought to be sorted in the trans-Golgi network towards the secretory granule via acidic aggregation. In the exocrine pancreas, amylase is one of the major zymogens. It is a basic protein of pI 8.6 and does not precipitate in acidic conditions. To identify the mechanism by which amylase aggregates in the acidic cisternæ of the pancreatic trans-Golgi network, we have developed an in vitro model in which amylase was fixed to plastic microtiter plates. The fixed amylase was probed with two ligands: amylase itself and GP-2, the major protein of the zymogen granule membrane. Biotinylated amylase bound to fixed amylase in a strict pH-dependent manner with optimal binding between pH 5.0 and 5.7. The affinity of binding was in the nanogram range (Kd ≈ 20.0 ng/mL) at pH 5.5. Acid binding of amylase was not reversible by incubation at neutral pH, nor could it be displaced by native amylase. GP-2 binding to fixed amylase was also pH dependent with optimal binding between pH 5.0 and 5.7. As for amylase, it was not reversible by incubation at neutral pH. GP-2 binding sites on fixed amylase appeared to be different from those of biotinylated amylase. While native and biotinylated amylase did not bind to GP-2, polymerized amylase precipitated GP-2 at acidic pH. Taken together these data suggest that slight modifications are sufficient to reveal on the amylase molecule binding sites for GP-2 and for amylase itself. These new binding capacities acquired at acidic pH could be involved in the cascade of reactions that lead to the in vivo formation of the immature secretory granule.Key words: regulated secretion, sorting, granules, trans-Golgi network.

scholarly journals Mutations in GDAP1 Influence Structure and Function of the Trans-Golgi Network

2021 ◽  
Vol 22 (2) ◽  
pp. 914
Author(s):  
Katarzyna Binięda ◽  
Weronika Rzepnikowska ◽  
Damian Kolakowski ◽  
Joanna Kaminska ◽  
Andrzej Antoni Szczepankiewicz ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Experimental Therapy ◽  
Negative Effects ◽  
Gene Encoding ◽  
Charcot Marie Tooth Disease ◽  
Terra Incognita ◽  
And Function ◽  
Golgi Network ◽  
Cell Phenotypes ◽  
Tooth Disease

Charcot-Marie-Tooth disease (CMT) is a heritable neurodegenerative disease that displays great genetic heterogeneity. The genes and mutations that underlie this heterogeneity have been extensively characterized by molecular genetics. However, the molecular pathogenesis of the vast majority of CMT subtypes remains terra incognita. Any attempts to perform experimental therapy for CMT disease are limited by a lack of understanding of the pathogenesis at a molecular level. In this study, we aim to identify the molecular pathways that are disturbed by mutations in the gene encoding GDAP1 using both yeast and human cell, based models of CMT-GDAP1 disease. We found that some mutations in GDAP1 led to a reduced expression of the GDAP1 protein and resulted in a selective disruption of the Golgi apparatus. These structural alterations are accompanied by functional disturbances within the Golgi. We screened over 1500 drugs that are available on the market using our yeast-based CMT-GDAP1 model. Drugs were identified that had both positive and negative effects on cell phenotypes. To the best of our knowledge, this study is the first report of the Golgi apparatus playing a role in the pathology of CMT disorders. The drugs we identified, using our yeast-based CMT-GDAP1 model, may be further used in translational research.

The role of the membrane-spanning domain and stalk region of N-acetylglucosaminyltransferase I in retention, kin recognition and structural maintenance of the Golgi apparatus in HeLa cells

1996 ◽  
Vol 109 (7) ◽  
pp. 1975-1989 ◽  
Author(s):  
T. Nilsson ◽  
C. Rabouille ◽  
N. Hui ◽  
R. Watson ◽  
G. Warren
Keyword(s):  
Golgi Apparatus ◽  
Cell Lines ◽  
Hela Cells ◽  
Kin Recognition ◽  
Dramatic Effect ◽  
Golgi Stack ◽  
Stable Cell Lines ◽  
Membrane Spanning ◽  
Necessary And Sufficient

Using a series of chimeric and truncated N-acetylglucosaminyltransferase I (NAGT I) molecules we have shown that part of the lumenal stalk region is both necessary and sufficient for kin recognition of mannosidase II and retention in the Golgi stack. The membrane-spanning domain was not required for retention, but replacing part or all of this domain with leucine residues did have a dramatic effect on Golgi morphology. In stable cell lines, stacked cisternae were replaced by tubulo-vesicular clusters containing the mutated NAGT I. The loss of stacked cisternae was proportional to the number of leucines used to replace the membrane-spanning domain.

Identification of a di-leucine motif within the C terminus domain of the Menkes disease protein that mediates endocytosis from the plasma membrane

1999 ◽  
Vol 112 (11) ◽  
pp. 1721-1732 ◽  
Author(s):  
M.J. Francis ◽  
E.E. Jones ◽  
E.R. Levy ◽  
R.L. Martin ◽  
S. Ponnambalam ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Transmembrane Domain ◽  
Menkes Disease ◽  
Cytoplasmic Domain ◽  
Endocytic Pathway ◽  
Site Directed Mutagenesis ◽  
Trans Golgi Network ◽  
C Terminus ◽  
Golgi Network

The protein encoded by the Menkes disease gene (MNK) is localised to the Golgi apparatus and cycles between the trans-Golgi network and the plasma membrane in cultured cells on addition and removal of copper to the growth medium. This suggests that MNK protein contains active signals that are involved in the retention of the protein to the trans-Golgi network and retrieval of the protein from the plasma membrane. Previous studies have identified a signal involved in Golgi retention within transmembrane domain 3 of MNK. To identify a motif sufficient for retrieval of MNK from the plasma membrane, we analysed the cytoplasmic domain, downstream of transmembrane domain 7 and 8. Chimeric constructs containing this cytoplasmic domain fused to the reporter molecule CD8 localised the retrieval signal(s) to 62 amino acids at the C terminus. Further studies were performed on putative internalisation motifs, using site-directed mutagenesis, protein expression, chemical treatment and immunofluorescence. We observed that a di-leucine motif (L1487L1488) was essential for rapid internalisation of chimeric CD8 proteins and the full-length Menkes cDNA from the plasma membrane. We suggest that this motif mediates the retrieval of MNK from the plasma membrane into the endocytic pathway, via the recycling endosomes, but is not sufficient on its own to return the protein to the Golgi apparatus. These studies provide a basis with which to identify other motifs important in the sorting and delivery of MNK from the plasma membrane to the Golgi apparatus.

Retrograde trafficking of both Golgi complex and TGN markers to the ER induced by nordihydroguaiaretic acid and cyclofenil diphenol

1998 ◽  
Vol 111 (7) ◽  
pp. 951-965 ◽  
Author(s):  
D. Drecktrah ◽  
P. de Figueiredo ◽  
R.M. Mason ◽  
W.J. Brown
Keyword(s):  
Golgi Complex ◽  
Membrane Trafficking ◽  
Molecular Mechanisms ◽  
Nordihydroguaiaretic Acid ◽  
Retrograde Transport ◽  
Lipoxygenase Inhibitor ◽  
Golgi Stack ◽  
Golgi Network ◽  
In Vivo Effects

Previous studies have shown that the Golgi stack and the trans-Golgi network (TGN) may play a role in capturing escaped resident endoplasmic reticulum (ER) proteins, and directing their retrograde transport back to that organelle. Whether this retrograde movement represents a highly specific or more generalized membrane trafficking pathway is unclear. To better understand both the retrograde and anterograde trafficking pathways of the secretory apparatus, we examined more closely the in vivo effects of two structurally unrelated compounds, the potent lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA), and the non-steroidal estrogen cyclofenil diphenol (CFD), both of which are known to inhibit secretion. In the presence of these compounds, transport of vesicular stomatitis virus G membrane glycoprotein from the ER to the Golgi complex, and from the TGN to the cell surface, was inhibited potently and rapidly. Surprisingly, we found that NDGA and CFD stimulated the rapid, but not concomitant, retrograde movement of both Golgi stack and TGN membrane proteins back to the ER until both organelles were morphologically absent from cells. Both NDGA- and CFD-stimulated TGN and Golgi retrograde membrane trafficking were inhibited by microtubule depolymerizing agents and energy poisons. Removal of NDGA and CFD resulted in the complete, but not concomitant, reformation of both Golgi stacks and their closely associated TGN compartments. These studies suggest that NDGA and CFD unmask a generalized bulk recycling pathway to the ER for both Golgi and TGN membranes and, further, that NDGA and CFD are useful for investigating the molecular mechanisms that control the formation and maintenance of both the Golgi stack proper and the TGN.

Role of sulfated O-linked glycoproteins in zymogen granule formation

2002 ◽  
Vol 115 (14) ◽  
pp. 2941-2952 ◽  
Author(s):  
Robert C. De Lisle
Keyword(s):  
Acinar Cell ◽  
Secretory Granules ◽  
Sodium Chlorate ◽  
Pancreatic Acinar Cell ◽  
Secretory Proteins ◽  
Zymogen Granule ◽  
Acidic Ph ◽  
Zymogen Granules ◽  
Granule Formation

Packaging of proteins into regulated secretory granules is mediated by the mildly acidic pH of the trans Golgi network and immature secretory granules. This need for an acidic pH indicates that ionic interactions are important. The mouse pancreatic acinar cell contains four major sulfated glycoproteins,including the zymogen granule structural component Muclin. I tested the hypothesis that sulfation and the O-linked glycosylation to which the sulfates are attached are required for normal formation of zymogen granules in the exocrine pancreas. Post-translational processing was perturbed with two chemicals: sodium chlorate was used to inhibit sulfation and benzyl-N-acetyl-α-galactosaminide was used to inhibit O-linked oligosaccharide elongation. Both chemicals resulted in the accumulation in the Golgi region of the cell of large vacuoles that appear to be immature secretory granules, and the effect was much more extensive with benzyl-N-acetyl-α-galactosaminide than chlorate. Both chemical treatments inhibited basal secretion at prolonged chase times, and again benzyl-N-acetyl-α-galactosaminide had a greater effect than chlorate. In addition, benzyl-N-acetyl-α-galactosaminide, but not chlorate, totally inhibited stimulated secretion of newly synthesized proteins. These data provide evidence for a role of sulfated O-linked glycoproteins in protein condensation and maturation of zymogen granules. Under maximal inhibition of O-linked oligosaccharide biosynthesis, anterograde post-Golgi traffic in the regulated pathway is almost totally shut down, demonstrating the importance of these post-translational modifications in progression of secretory proteins through the regulated pathway and normal granule formation in the pancreatic acinar cell.

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