scholarly journals Genetic Ablation of Phosphatidylinositol Transfer Protein Function in Murine Embryonic Stem Cells

2002 ◽  
Vol 13 (3) ◽  
pp. 739-754 ◽  
Author(s):  
James G. Alb ◽  
Scott E. Phillips ◽  
Kathleen Rostand ◽  
Xiaoxia Cui ◽  
Jef Pinxteren ◽  
...  
Keyword(s):  
Mast Cell ◽  
Membrane Trafficking ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Embryonic Stem ◽  
Endocytic Pathway ◽  
Dense Core ◽  
Homologous Proteins ◽  
Genetic Ablation ◽  
Phosphatidylinositol Transfer

Phosphatidylinositol transfer proteins (PITPs) regulate the interface between signal transduction, membrane-trafficking, and lipid metabolic pathways in eukaryotic cells. The best characterized mammalian PITPs are PITPα and PITPβ, two highly homologous proteins that are encoded by distinct genes. Insights into PITPα and PITPβ function in mammalian systems have been gleaned exclusively from cell-free or permeabilized cell reconstitution and resolution studies. Herein, we report for the first time the use of genetic approaches to directly address the physiological functions of PITPα and PITPβ in murine cells. Contrary to expectations, we find that ablation of PITPα function in murine cells fails to compromise growth and has no significant consequence for bulk phospholipid metabolism. Moreover, the data show that PITPα does not play an obvious role in any of the cellular activities where it has been reconstituted as an essential stimulatory factor. These activities include protein trafficking through the constitutive secretory pathway, endocytic pathway function, biogenesis of mast cell dense core secretory granules, and the agonist-induced fusion of dense core secretory granules to the mast cell plasma membrane. Finally, the data demonstrate that PITPα-deficient cells not only retain their responsiveness to bulk growth factor stimulation but also retain their pluripotency. In contrast, we were unable to evict both PITPβ alleles from murine cells and show that PITPβ deficiency results in catastrophic failure early in murine embryonic development. We suggest that PITPβ is an essential housekeeping PITP in murine cells, whereas PITPα plays a far more specialized function in mammals than that indicated by in vitro systems that show PITP dependence.

scholarly journals PACS-1 and Adaptor Protein-1 Mediate ACTH Trafficking to the Regulated Secretory Pathway

2018 ◽  
Author(s):  
Brennan S. Dirk ◽  
Christopher End ◽  
Emily N. Pawlak ◽  
Logan R. Van Nynatten ◽  
Rajesh Abraham Jacob ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Adaptor Protein ◽  
Cell Types ◽  
Cellular Membrane ◽  
Extracellular Milieu ◽  
Specialized Form ◽  
Regulated Secretory Pathway ◽  
Clathrin Adaptor

ABSTRACTThe regulated secretory pathway is a specialized form of protein secretion found in endocrine and neuroendocrine cell types. Pro-opiomelanocortin (POMC) is a pro-hormone that utilizes this pathway to be trafficked to dense core secretory granules (DCSGs). Within this organelle, POMC is processed to multiple bioactive hormones that play key roles in cellular physiology. However, the complete set of cellular membrane trafficking proteins that mediate the correct sorting of POMC to DCSGs remain unknown. Here, we report the roles of the phosphofurin acidic cluster sorting protein – 1 (PACS-1) and the clathrin adaptor protein 1 (AP-1) in the targeting of POMC to DCSGs. Upon knockdown of PACS-1 and AP-1, POMC is readily secreted into the extracellular milieu and fails to be targeted to DCSGs.

scholarly journals Specific and Nonspecific Membrane-binding Determinants Cooperate in Targeting Phosphatidylinositol Transfer Protein β-Isoform to the MammalianTrans-Golgi Network

2006 ◽  
Vol 17 (6) ◽  
pp. 2498-2512 ◽  
Author(s):  
Scott E. Phillips ◽  
Kristina E. Ile ◽  
Malika Boukhelifa ◽  
Richard P.H. Huijbregts ◽  
Vytas A. Bankaitis
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Membrane Binding ◽  
Bound State ◽  
Missense Mutations ◽  
Cis Acting ◽  
Domain Mapping ◽  
Phosphatidylinositol Transfer ◽  
Golgi Network

Phosphatidylinositol transfer proteins (PITPs) regulate the interface between lipid metabolism and specific steps in membrane trafficking through the secretory pathway in eukaryotes. Herein, we describe the cis-acting information that controls PITPβ localization in mammalian cells. We demonstrate PITPβ localizes predominantly to the trans-Golgi network (TGN) and that this localization is independent of the phospholipid-bound state of PITPβ. Domain mapping analyses show the targeting information within PITPβ consists of three short C-terminal specificity elements and a nonspecific membrane-binding element defined by a small motif consisting of adjacent tryptophan residues (the W202W203motif). Combination of the specificity elements with the W202W203motif is necessary and sufficient to generate an efficient TGN-targeting module. Finally, we demonstrate that PITPβ association with the TGN is tolerant to a range of missense mutations at residue serine 262, we describe the TGN localization of a novel PITPβ isoform with a naturally occurring S262Q polymorphism, and we find no other genetic or pharmacological evidence to support the concept that PITPβ localization to the TGN is obligately regulated by conventional protein kinase C (PKC) or the Golgi-localized PKC isoforms δ or ε. These latter findings are at odds with a previous report that conventional PKC-mediated phosphorylation of residue Ser262is required for PITPβ targeting to Golgi membranes.

scholarly journals Membrane Traffic in Aspergillus oryzae and Related Filamentous Fungi

2021 ◽  
Vol 7 (7) ◽  
pp. 534
Author(s):  
Yujiro Higuchi
Keyword(s):  
Filamentous Fungi ◽  
Aspergillus Oryzae ◽  
Protein Secretion ◽  
Membrane Trafficking ◽  
Secretory Pathway ◽  
Molecular Mechanisms ◽  
Membrane Traffic ◽  
Industrial Applications ◽  
Endocytic Pathway ◽  
Great Ability

The industrially important filamentous fungus Aspergillus oryzae, known as the yellow Koji mold and also designated the Japanese National fungus, has been investigated for understanding the intracellular membrane trafficking machinery due to the great ability of valuable enzyme production. The underlying molecular mechanisms of the secretory pathway delineate the main secretion route from the hyphal tip via the vesicle cluster Spitzenkörper, but also there is a growing body of evidence that septum-directed and unconventional secretion occurs in A. oryzae hyphal cells. Moreover, not only the secretory pathway but also the endocytic pathway is crucial for protein secretion, especially having a role in apical endocytic recycling. As a hallmark of multicellular filamentous fungal cells, endocytic organelles early endosome and vacuole are quite dynamic: the former exhibits constant long-range motility through the hyphal cells and the latter displays pleiomorphic structures in each hyphal region. These characteristics are thought to have physiological roles, such as supporting protein secretion and transporting nutrients. This review summarizes molecular and physiological mechanisms of membrane traffic, i.e., secretory and endocytic pathways, in A. oryzae and related filamentous fungi and describes the further potential for industrial applications.

scholarly journals 60 YEARS OF POMC: Biosynthesis, trafficking, and secretion of pro-opiomelanocortin-derived peptides

2016 ◽  
Vol 56 (4) ◽  
pp. T77-T97 ◽  
Author(s):  
Niamh X Cawley ◽  
Zhaojin Li ◽  
Y Peng Loh
Keyword(s):  
Endocrine Cells ◽  
Secretory Pathway ◽  
Current Knowledge ◽  
Secretory Granules ◽  
Peptide Hormones ◽  
The Body ◽  
Dense Core ◽  
Biologically Active ◽  
Dependent Manner ◽  
Basic Residue

Pro-opiomelanocortin (POMC) is a prohormone that encodes multiple smaller peptide hormones within its structure. These peptide hormones can be generated by cleavage of POMC at basic residue cleavage sites by prohormone-converting enzymes in the regulated secretory pathway (RSP) of POMC-synthesizing endocrine cells and neurons. The peptides are stored inside the cells in dense-core secretory granules until released in a stimulus-dependent manner. The complexity of the regulation of the biosynthesis, trafficking, and secretion of POMC and its peptides reflects an impressive level of control over many factors involved in the ultimate role of POMC-expressing cells, that is, to produce a range of different biologically active peptide hormones ready for action when signaled by the body. From the discovery of POMC as the precursor to adrenocorticotropic hormone (ACTH) and β-lipotropin in the late 1970s to our current knowledge, the understanding of POMC physiology remains a monumental body of work that has provided insight into many aspects of molecular endocrinology. In this article, we describe the intracellular trafficking of POMC in endocrine cells, its sorting into dense-core secretory granules and transport of these granules to the RSP. Additionally, we review the enzymes involved in the maturation of POMC to its various peptides and the mechanisms involved in the differential processing of POMC in different cell types. Finally, we highlight studies pertaining to the regulation of ACTH secretion in the anterior and intermediate pituitary and POMC neurons of the hypothalamus.

scholarly journals Tumor protein D52 controls trafficking of an apical endolysosomal secretory pathway in pancreatic acinar cells

2013 ◽  
Vol 305 (6) ◽  
pp. G439-G452 ◽  
Author(s):  
Scott W. Messenger ◽  
Diana D. H. Thomas ◽  
Michelle A. Falkowski ◽  
Jennifer A. Byrne ◽  
Fred S. Gorelick ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Phosphorylation Site ◽  
Brefeldin A ◽  
Acinar Cells ◽  
Pancreatic Acinar Cell ◽  
Pancreatic Acinar Cells ◽  
Zymogen Granule ◽  
Adenoviral Delivery

Zymogen granule (ZG) formation in acinar cells involves zymogen cargo sorting from trans-Golgi into immature secretory granules (ISGs). ISG maturation progresses by removal of lysosomal membrane and select content proteins, which enter endosomal intermediates prior to their apical exocytosis. Constitutive and stimulated secretion through this mechanism is termed the constitutive-like and minor-regulated pathways, respectively. However, the molecular components that control membrane trafficking within these endosomal compartments are largely unknown. We show that tumor protein D52 is highly expressed in endosomal compartments following pancreatic acinar cell stimulation and regulates apical exocytosis of an apically directed endolysosomal compartment. Secretion from the endolysosomal compartment was detected by cell-surface antigen labeling of lysosome-associated membrane protein LAMP1, which is absent from ZGs, and had incomplete overlap with surface labeling of synaptotagmin 1, a marker of ZG exocytosis. Although culturing (16–18 h) of isolated acinar cells is accompanied by a loss of secretory responsiveness, the levels of SNARE proteins necessary for ZG exocytosis were preserved. However, levels of endolysosomal proteins D52, EEA1, Rab5, and LAMP1 markedly decreased with culture. When D52 levels were restored by adenoviral delivery, the levels of these regulatory proteins and secretion of both LAMP1 (endolysosomal) and amylase was strongly enhanced. These secretory effects were absent in alanine and aspartate substitutions of serine 136, the major D52 phosphorylation site, and were inhibited by brefeldin A, which does not directly affect the ZG compartment. Our results indicate that D52 directly regulates apical endolysosomal secretion and are consistent with previous studies, suggesting that this pathway indirectly regulates ZG secretion of digestive enzymes.

scholarly journals RNAi screen identifies a role for adaptor protein AP-3 in sorting to the regulated secretory pathway

2010 ◽  
Vol 191 (6) ◽  
pp. 1173-1187 ◽  
Author(s):  
Cédric S. Asensio ◽  
Daniel W. Sirkis ◽  
Robert H. Edwards
Keyword(s):  
Secretory Pathway ◽  
Adaptor Protein ◽  
Neuroendocrine Cells ◽  
Endocytic Pathway ◽  
Dense Core ◽  
Primary Defect ◽  
Regulated Exocytosis ◽  
Drosophila Melanogaster S2 Cells ◽  
Large Dense Core Vesicles ◽  
Regulated Secretory Pathway

The regulated release of proteins depends on their inclusion within large dense-core vesicles (LDCVs) capable of regulated exocytosis. LDCVs form at the trans-Golgi network (TGN), but the mechanism for protein sorting to this regulated secretory pathway (RSP) and the cytosolic machinery involved in this process have remained poorly understood. Using an RNA interference screen in Drosophila melanogaster S2 cells, we now identify a small number of genes, including several subunits of the heterotetrameric adaptor protein AP-3, which are required for sorting to the RSP. In mammalian neuroendocrine cells, loss of AP-3 dysregulates exocytosis due to a primary defect in LDCV formation. Previous work implicated AP-3 in the endocytic pathway, but we find that AP-3 promotes sorting to the RSP within the biosynthetic pathway at the level of the TGN. Although vesicles with a dense core still form in the absence of AP-3, they contain substantially less synaptotagmin 1, indicating that AP-3 concentrates the proteins required for regulated exocytosis.

scholarly journals A Yeast t-SNARE Involved in Endocytosis

1998 ◽  
Vol 9 (10) ◽  
pp. 2873-2889 ◽  
Author(s):  
Karin Séron ◽  
Ville Tieaho ◽  
Cristina Prescianotto-Baschong ◽  
Thomas Aust ◽  
Marie-Odile Blondel ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Secretory Pathway ◽  
Essential Gene ◽  
Specific Interaction ◽  
Endocytic Pathway ◽  
Intracellular Membrane ◽  
Atpase Subunit ◽  
Early Endosomes ◽  
Endocytic Vesicles ◽  
Positively Charged

The ORF YOL018c (TLG2) of Saccharomyces cerevisiae encodes a protein that belongs to the syntaxin protein family. The proteins of this family, t-SNAREs, are present on target organelles and are thought to participate in the specific interaction between vesicles and acceptor membranes in intracellular membrane trafficking. TLG2 is not an essential gene, and its deletion does not cause defects in the secretory pathway. However, its deletion in cells lacking the vacuolar ATPase subunit Vma2p leads to loss of viability, suggesting that Tlg2p is involved in endocytosis. In tlg2Δ cells, internalization was normal for two endocytic markers, the pheromone α-factor and the plasma membrane uracil permease. In contrast, degradation of α-factor and uracil permease was delayed intlg2Δ cells. Internalization of positively charged Nanogold shows that the endocytic pathway is perturbed in the mutant, which accumulates Nanogold in primary endocytic vesicles and shows a greatly reduced complement of early endosomes. These results strongly suggest that Tlg2p is a t-SNARE involved in early endosome biogenesis.

scholarly journals Constitutive and basal secretion from the endocrine cell line, AtT-20.

1991 ◽  
Vol 112 (5) ◽  
pp. 843-852 ◽  
Author(s):  
L Matsuuchi ◽  
R B Kelly
Keyword(s):  
Cell Line ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Secretory Protein ◽  
Dense Core ◽  
Wild Type ◽  
Dna Encoding ◽  
Basal Secretion ◽  
Constitutive Secretion ◽  
Regulated Secretory Pathway

A variant of the ACTH-secreting pituitary cell line, AtT-20, has been isolated that does not make ACTH, sulfated proteins characteristic of the regulated secretory pathway, or dense-core secretory granules but retains constitutive secretion. Unlike wild type AtT-20 cells, the variant cannot store or release on stimulation, free glycosaminoglycan (GAG) chains. In addition, the variant cells cannot store trypsinogen or proinsulin, proteins that are targeted to dense core secretory granules in wild type cells. The regulated pathway could not be restored by transfecting with DNA encoding trypsinogen, a soluble regulated secretory protein targeted to secretory granules. A comparison of secretion from variant and wild type cells allows a distinction to be made between constitutive secretion and basal secretion, the spontaneous release of regulated proteins that occurs in the absence of stimulation.

scholarly journals Anaphylactic Degranulation of Mast Cells: Focus on Compound Exocytosis

2019 ◽  
Vol 2019 ◽  
pp. 1-12 ◽  
Author(s):  
Ofir Klein ◽  
Ronit Sagi-Eisenberg
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Molecular Mechanisms ◽  
Secretory Granules ◽  
Cell Types ◽  
Secretory Cells ◽  
Regulated Exocytosis ◽  
Open Questions ◽  
Conflicting Evidence ◽  
Compound Exocytosis

Anaphylaxis is a notorious type 2 immune response which may result in a systemic response and lead to death. A precondition for the unfolding of the anaphylactic shock is the secretion of inflammatory mediators from mast cells in response to an allergen, mostly through activation of the cells via the IgE-dependent pathway. While mast cells are specialized secretory cells that can secrete through a variety of exocytic modes, the most predominant mode exerted by the mast cell during anaphylaxis is compound exocytosis—a specialized form of regulated exocytosis where secretory granules fuse to one another. Here, we review the modes of regulated exocytosis in the mast cell and focus on compound exocytosis. We review historical landmarks in the research of compound exocytosis in mast cells and the methods available for investigating compound exocytosis. We also review the molecular mechanisms reported to underlie compound exocytosis in mast cells and expand further with reviewing key findings from other cell types. Finally, we discuss the possible reasons for the mast cell to utilize compound exocytosis during anaphylaxis, the conflicting evidence in different mast cell models, and the open questions in the field which remain to be answered.

scholarly journals Glycosylation Modulates Plasma Membrane Trafficking of CD24 in Breast Cancer Cells

2021 ◽  
Vol 22 (15) ◽  
pp. 8165
Author(s):  
Amanda Chantziou ◽  
Kostas Theodorakis ◽  
Hara Polioudaki ◽  
Eelco de Bree ◽  
Marilena Kampa ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Plasma Membrane ◽  
Subcellular Localization ◽  
Cancer Cells ◽  
Membrane Trafficking ◽  
Breast Cancer Cells ◽  
Endocytic Pathway ◽  
Cell Periphery ◽  
Wild Type ◽  
Mcf 7

In breast cancer, expression of Cluster of Differentiation 24 (CD24), a small GPI-anchored glycoprotein at the cell periphery, is associated with metastasis and immune escape, while its absence is associated with tumor-initiating capacity. Since the mechanism of CD24 sorting is unknown, we investigated the role of glycosylation in the subcellular localization of CD24. Expression and localization of wild type N36- and/or N52-mutated CD24 were analyzed using immunofluorescence in luminal (MCF-7) and basal B (MDA-MB-231 and Hs578T) breast cancer cells lines, as well as HEK293T cells. Endogenous and exogenously expressed wild type and mutated CD24 were found localized at the plasma membrane and the cytoplasm, but not the nucleoplasm. The cell lines showed different kinetics for the sorting of CD24 through the secretory/endocytic pathway. N-glycosylation, especially at N52, and its processing in the Golgi were critical for the sorting and expression of CD24 at the plasma membrane of HEK293T and basal B type cells, but not of MCF-7 cells. In conclusion, our study highlights the contribution of N-glycosylation for the subcellular localization of CD24. Aberrant N-glycosylation at N52 of CD24 could account for the lack of CD24 expression at the cell surface of basal B breast cancer cells.

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