scholarly journals Formation of secretory vesicles in permeabilized cells: a salt extract from yeast membranes promotes budding of nascent secretory vesicles from the trans-Golgi network of endocrine cells

1996 ◽  
Vol 314 (3) ◽  
pp. 723-726 ◽  
Author(s):  
Wai Lam W. LING ◽  
Dennis SHIELDS
Keyword(s):  
Mammalian Cells ◽  
Endocrine Cells ◽  
Secretory Vesicle ◽  
Gh3 Cells ◽  
Vesicle Formation ◽  
Secretory Vesicles ◽  
Vesicle Budding ◽  
Trans Golgi Network ◽  
Golgi Network ◽  
Salt Extract

The mechanism of secretory-vesicle formation from the trans-Golgi network (TGN) of endocrine cells is poorly understood. To identify cytosolic activities that facilitate the formation and fission of nascent secretory vesicles, we treated permeabilized pituitary GH3 cells with high salt to remove endogenous budding factors. Using this cell preparation, secretory-vesicle budding from the TGN required addition of exogenous cytosol and energy. Mammalian cytosols (GH3 cells and bovine brain) promoted post-TGN vesicle formation. Most significantly, a salt extract of membranes from the yeast Saccharomyces cerevisiae, a cell lacking a regulated secretory pathway, stimulated secretory vesicle budding in the absence of mammalian cytosolic factors. These results demonstrate that the factors which promote secretory-vesicle release from the TGN are conserved between yeast and mammalian cells.

scholarly journals Formation of nascent secretory vesicles from the trans-Golgi network of endocrine cells is inhibited by tyrosine kinase and phosphatase inhibitors.

1996 ◽  
Vol 135 (6) ◽  
pp. 1471-1483 ◽  
Author(s):  
C D Austin ◽  
D Shields
Keyword(s):  
Endocrine Cells ◽  
Kinase Inhibitors ◽  
Tyrosine Phosphatase ◽  
Secretory Vesicle ◽  
Vesicle Formation ◽  
Secretory Vesicles ◽  
Vesicle Release ◽  
Vesicle Budding ◽  
Phosphatase Inhibitors ◽  
Gtp Binding

Recent evidence suggests that secretory vesicle formation from the TGN is regulated by cytosolic signaling pathways involving small GTP-binding proteins, heterotrimeric G proteins, inositol phospholipid metabolism, and protein serine/threonine phosphorylation. At the cell surface, protein phosphorylation and dephosphorylation on tyrosine residues can rapidly modulate cytosolic signaling pathways in response to extracellular stimuli and have been implicated in the internalization and sorting of signaling receptors. to determine if phosphotyrosine metabolism might also regulate secretory vesicle budding from the TGN, we treated permeabilized rat pituitary GH3 cells with inhibitors of either tyrosine phosphatases or tyrosine kinases. We demonstrate that the tyrosine phosphatase inhibitors pervanadate and zinc potently inhibited budding of nascent secretory vesicles. Tyrphostin A25 (TA25) and other tyrosine kinase inhibitors also prevented secretory vesicle release, suggesting that vesicle formation requires both phosphatase and kinase activities. A stimulatory peptide derived from the NH2 terminus of the small GTP-binding protein ADP ribosylation factor 1 (ARF1) antagonized the inhibitory effect of TA25, indicating that both agents influence the same pathway leading to secretory vesicle formation. Antiphosphotyrosine immunoblotting revealed that protein tyrosine phosphorylation was enhanced after treatment with tyrosine phosphatase or kinase inhibitors. Subcellular fractionation identified several tyrosine phosphorylated polypeptides of approximately 175, approximately 130, and 90-110 kD that were enriched in TGN-containing Golgi fractions and tightly membrane associated. The phosphorylation of these polypeptides correlated with inhibition of vesicle budding. Our results suggest that in endocrine cells, protein tyrosine phosphrylation and dephosphorylation are required for secretory vesicle release from the TGN.

scholarly journals Ride the wave: Retrograde trafficking becomes Ca2+ dependent with BAIAP3

2017 ◽  
Vol 216 (7) ◽  
pp. 1887-1889 ◽  
Author(s):  
Jakob B. Sørensen
Keyword(s):  
Angiogenesis Inhibitor ◽  
Secretory Vesicle ◽  
Vesicle Formation ◽  
Retrograde Trafficking ◽  
Trans Golgi Network ◽  
Cell Biol ◽  
Golgi Network

The functions of four of the five proteins in the mammalian uncoordinated-13 (Munc13) family have been identified as priming factors in SNARE-dependent exocytosis. In this issue, Zhang et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201702099) show that the fifth member, BAIAP3 (brain-specific angiogenesis inhibitor I–associated protein 3), acts in retrograde trafficking by returning secretory vesicle material to the trans-Golgi network. In its absence, secretory vesicle formation is impaired, leading to accumulation of immature vesicles, or lysosomal vesicle degradation.

GGA proteins: new players in the sorting game

2001 ◽  
Vol 114 (19) ◽  
pp. 3413-3418 ◽  
Author(s):  
Annette L. Boman
Keyword(s):  
Membrane Trafficking ◽  
Sequence Homology ◽  
Mammalian Cells ◽  
Gene Knockout ◽  
Protein Domains ◽  
Vesicle Formation ◽  
Trans Golgi Network ◽  
And Function ◽  
Golgi Network ◽  
Cargo Sorting

The GGA proteins are a novel family of proteins that were discovered nearly simultaneously by several labs studying very different aspects of membrane trafficking. Since then, several studies have described the GGA proteins and their functions in yeast and mammalian cells. Four protein domains are present in all GGA proteins, as defined by sequence homology and function. These different domains interact directly with ARF proteins, cargo and clathrin. Alteration of the levels of GGA proteins by gene knockout or overexpression affects specific trafficking events between the trans-Golgi network and endosomes. These data suggest that GGAs function as ARF-dependent, monomeric clathrin adaptors to facilitate cargo sorting and vesicle formation at the trans-Golgi network.

scholarly journals Phospholipase D Stimulates Release of Nascent Secretory Vesicles from the trans-Golgi Network

1997 ◽  
Vol 138 (3) ◽  
pp. 495-504 ◽  
Author(s):  
Ye-Guang Chen ◽  
Anirban Siddhanta ◽  
Cary D. Austin ◽  
Scott M. Hammond ◽  
Tsung-Chang Sung ◽  
...  
Keyword(s):  
Phospholipase D ◽  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Gh3 Cells ◽  
Mutant Form ◽  
Secretory Vesicles ◽  
Permeabilized Cell ◽  
Vesicle Budding ◽  
Golgi Membranes

Phospholipase D (PLD) is a phospholipid hydrolyzing enzyme whose activation has been implicated in mediating signal transduction pathways, cell growth, and membrane trafficking in mammalian cells. Several laboratories have demonstrated that small GTP-binding proteins including ADP-ribosylation factor (ARF) can stimulate PLD activity in vitro and an ARF-activated PLD activity has been found in Golgi membranes. Since ARF-1 has also been shown to enhance release of nascent secretory vesicles from the TGN of endocrine cells, we hypothesized that this reaction occurred via PLD activation. Using a permeabilized cell system derived from growth hormone and prolactin-secreting pituitary GH3 cells, we demonstrate that immunoaffinity-purified human PLD1 stimulated nascent secretory vesicle budding from the TGN approximately twofold. In contrast, a similarly purified but enzymatically inactive mutant form of PLD1, designated Lys898Arg, had no effect on vesicle budding when added to the permeabilized cells. The release of nascent secretory vesicles from the TGN was sensitive to 1% 1-butanol, a concentration that inhibited PLD-catalyzed formation of phosphatidic acid. Furthermore, ARF-1 stimulated endogenous PLD activity in Golgi membranes approximately threefold and this activation correlated with its enhancement of vesicle budding. Our results suggest that ARF regulation of PLD activity plays an important role in the release of nascent secretory vesicles from the TGN.

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