Phosphoinositides and membrane traffic at the trans-Golgi network.

2005 ◽  
Vol 72 ◽  
pp. 31-38 ◽  
Author(s):  
Rawshan R. Choudhury ◽  
Noora Hyvola ◽  
Martin Lowe
Keyword(s):  
Plasma Membrane ◽  
Secretory Pathway ◽  
Binding Proteins ◽  
Molecular Mechanisms ◽  
Membrane Traffic ◽  
Trans Golgi Network ◽  
Cargo Proteins ◽  
Golgi Network

Cargo proteins moving along the secretory pathway are sorted at the TGN (trans-Golgi network) into distinct carriers for delivery to the plasma membrane or endosomes. Recent studies in yeast and mammals have shown that formation of these carriers is regulated by PtdIns(4)P. This phosphoinositide is abundant at the TGN and acts to recruit components required for carrier formation to the membrane. Other phosphoinositides are also present on the TGN, but the extent to which they regulate trafficking is less clear. Further characterization of phosphoinositide kinases and phosphatases together with identification of new TGN-associated phosphoinositide-binding proteins will reveal the extent to which different phosphoinositides regulate TGN trafficking, and help define the molecular mechanisms involved.

Dual-color visualization of trans-Golgi network to plasma membrane traffic along microtubules in living cells

1999 ◽  
Vol 112 (1) ◽  
pp. 21-33 ◽  
Author(s):  
D. Toomre ◽  
P. Keller ◽  
J. White ◽  
J.C. Olivo ◽  
K. Simons
Keyword(s):  
Plasma Membrane ◽  
Protein Trafficking ◽  
Membrane Traffic ◽  
Living Cells ◽  
Tubular Structures ◽  
Trans Golgi Network ◽  
Vesicular Stomatitis ◽  
Golgi Network ◽  
Color Visualization

The mechanisms and carriers responsible for exocytic protein trafficking between the trans-Golgi network (TGN) and the plasma membrane remain unclear. To investigate the dynamics of TGN-to-plasma membrane traffic and role of the cytoskeleton in these processes we transfected cells with a GFP-fusion protein, vesicular stomatitis virus G protein tagged with GFP (VSVG3-GFP). After using temperature shifts to block VSVG3-GFP in the endoplasmic reticulum and subsequently accumulate it in the TGN, dynamics of TGN-to-plasma membrane transport were visualized in real time by confocal and video microscopy. Both small vesicles (<250 nm) and larger vesicular-tubular structures (>1.5 microm long) are used as transport containers (TCs). These TCs rapidly moved out of the Golgi along curvilinear paths with average speeds of approximately 0.7 micrometer/second. Automatic computer tracking objectively determined the dynamics of different carriers. Fission and fusion of TCs were observed, suggesting that these late exocytic processes are highly interactive. To directly determine the role of microtubules in post-Golgi traffic, rhodamine-tubulin was microinjected and both labeled cargo and microtubules were simultaneously visualized in living cells. These studies demonstrated that exocytic cargo moves along microtubule tracks and reveals that carriers are capable of switching between tracks.

scholarly journals Proteomic Characterization of Isolated Arabidopsis Clathrin-Coated Vesicles Reveals Evolutionarily Conserved and Plant Specific Components

2021 ◽  
Author(s):  
Dana A. Dahhan ◽  
Gregory D. Reynolds ◽  
Jessica J. Cárdenas ◽  
Dominique Eeckhout ◽  
Alexander Johnson ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Adaptor Protein ◽  
Coated Vesicles ◽  
Mass Spectrometry Analysis ◽  
Chemical Labeling ◽  
Spectrometry Analysis ◽  
Trans Golgi Network ◽  
Evolutionarily Conserved ◽  
Golgi Network

In eukaryotes, clathrin-coated vesicles (CCVs) facilitate the internalization of material from the cell surface as well as the movement of cargo in post-Golgi trafficking pathways. This diversity of functions is partially provided by multiple monomeric and multimeric clathrin adaptor complexes that provide compartment and cargo selectivity. The adaptor-protein AP-1 complex operates as part of the secretory pathway at the trans-Golgi network, while the AP-2 complex and the TPLATE complex (TPC) jointly operate at the plasma membrane to execute clathrin-mediated endocytosis. Key to our further understanding of clathrin-mediated trafficking in plants will be the comprehensive identification and characterization of the network of evolutionarily conserved and plant-specific core and accessory machinery involved in the formation and targeting of CCVs. To facilitate these studies, we have analyzed the proteome of enriched trans-Golgi network/early endosome-derived and endocytic CCVs isolated from dividing and expanding suspension-cultured Arabidopsis cells. Tandem mass spectrometry analysis results were validated by differential chemical labeling experiments to identify proteins co-enriching with CCVs. Proteins enriched in CCVs included previously characterized CCV components and cargos such as the vacuolar sorting receptors in addition to conserved and plant-specific components whose function in clathrin-mediated trafficking has not been previously defined. Notably, in addition to AP-1 and AP-2, all subunits of the AP-4 complex, but not AP-3 or AP-5, were found to be in high abundance in the CCV proteome. The association of AP-4 with suspension-cultured Arabidopsis CCVs is further supported via additional biochemical data.

scholarly journals Dual pulse-chase microscopy reveals early divergence in the biosynthetic trafficking of the Na,K-ATPase and E-cadherin

2015 ◽  
Vol 26 (24) ◽  
pp. 4401-4411 ◽  
Author(s):  
Glen A. Farr ◽  
Michael Hull ◽  
Emily H. Stoops ◽  
Rosalie Bateson ◽  
Michael J. Caplan
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Golgi Complex ◽  
Plasma Membranes ◽  
Secretory Pathway ◽  
Trans Golgi Network ◽  
Polarized Epithelial Cells ◽  
Golgi Network ◽  
E Cadherin

Recent evidence indicates that newly synthesized membrane proteins that share the same distributions in the plasma membranes of polarized epithelial cells can pursue a variety of distinct trafficking routes as they travel from the Golgi complex to their common destination at the cell surface. In most polarized epithelial cells, both the Na,K-ATPase and E-cadherin are localized to the basolateral domains of the plasma membrane. To examine the itineraries pursued by newly synthesized Na,K-ATPase and E-cadherin in polarized MDCK epithelial cells, we used the SNAP and CLIP labeling systems to fluorescently tag temporally defined cohorts of these proteins and observe their behaviors simultaneously as they traverse the secretory pathway. These experiments reveal that E-cadherin is delivered to the cell surface substantially faster than is the Na,K-ATPase. Furthermore, the surface delivery of newly synthesized E-cadherin to the plasma membrane was not prevented by the 19°C temperature block that inhibits the trafficking of most proteins, including the Na,K-ATPase, out of the trans-Golgi network. Consistent with these distinct behaviors, populations of newly synthesized E-cadherin and Na,K-ATPase become separated from one another within the trans-Golgi network, suggesting that they are sorted into different carrier vesicles that mediate their post-Golgi trafficking.

scholarly journals Conserved role for Gga proteins in phosphatidylinositol 4-kinase localization to the trans-Golgi network

2017 ◽  
Vol 114 (13) ◽  
pp. 3433-3438 ◽  
Author(s):  
Lydia Daboussi ◽  
Giancarlo Costaguta ◽  
Razmik Ghukasyan ◽  
Gregory S. Payne
Keyword(s):  
Binding Proteins ◽  
Molecular Mechanisms ◽  
Coated Vesicles ◽  
Coat Proteins ◽  
Trans Golgi Network ◽  
Phosphatidylinositol Kinases ◽  
Vhs Domain ◽  
Phosphatidylinositol 4 ◽  
Golgi Network

Phosphoinositides serve as key membrane determinants for assembly of clathrin coat proteins that drive formation of clathrin-coated vesicles. At the trans-Golgi network (TGN), phosphatidylinositol 4-phosphate (PtdIns4P) plays important roles in recruitment of two major clathrin adaptors, Gga (Golgi-localized, gamma-adaptin ear homology, Arf-binding) proteins and the AP-1 (assembly protein-1) complex. The molecular mechanisms that mediate localization of phosphatidylinositol kinases responsible for synthesis of PtdIns4P at the TGN are not well characterized. We identify two motifs in the yeast phosphatidylinositol 4-kinase, Pik1, which are required for binding to the VHS domain of Gga2. Mutations in these motifs that inhibit Gga2–VHS binding resulted in reduced Pik1 localization and delayed accumulation of PtdIns4P and recruitment of AP-1 to the TGN. The Pik1 homolog in mammals, PI4KIIIβ, interacted preferentially with the VHS domain of GGA2 compared with VHS domains of GGA1 and GGA3. Depletion of GGA2, but not GGA1 or GGA3, specifically affected PI4KIIIβ localization. These results reveal a conserved role for Gga proteins in regulating phosphatidylinositol 4-kinase function at the TGN.

scholarly journals Transport of influenza HA from the trans-Golgi network to the apical surface of MDCK cells permeabilized in their basolateral plasma membranes: energy dependence and involvement of GTP-binding proteins.

1990 ◽  
Vol 111 (6) ◽  
pp. 2893-2908 ◽  
Author(s):  
D Gravotta ◽  
M Adesnik ◽  
D D Sabatini
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Binding Proteins ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Apical Surface ◽  
Gtp Binding Proteins ◽  
Trans Golgi Network ◽  
Gtp Binding ◽  
Golgi Network

A procedure employing streptolysin O to effect the selective permeabilization of either the apical or basolateral plasma membrane domains of MDCK cell monolayers grown on a filter support was developed which permeabilizes the entire monolayer, leaves the opposite cell surface domain intact, and does not abolish the integrity of the tight junctions. This procedure renders the cell interior accessible to exogenous macromolecules and impermeant reagents, permitting the examination of their effects on membrane protein transport to the intact surface. The last stages of the transport of the influenza virus hemagglutinin (HA) to the apical surface were studied in pulse-labeled, virus-infected MDCK cells that were incubated at 19.5 degrees C for 90 min to accumulate newly synthesized HA in the trans-Golgi network (TGN), before raising the temperature to 35 degrees C to allow synchronized transport to the plasma membrane. In cells permeabilized immediately after the cold block, 50% of the intracellular HA molecules were subsequently delivered to the apical surface. This transport was dependent on the presence of an exogenous ATP supply and was markedly inhibited by the addition of GTP-gamma-S at the time of permeabilization. On the other hand, the GTP analogue had no effect when it was added to cells that, after the cold block, were incubated for 15 min at 35 degrees C before permeabilization, even though at this time most HA molecules were still intracellular and their appearance at the cell surface was largely dependent on exogenous ATP. These findings indicate that GTP-binding proteins are involved in the constitutive process that effects vesicular transport from the TGN to the plasma membrane and that they are charged early in this process. Transport of HA to the cell surface could be made dependent on the addition of exogenous cytosol when, after permeabilization, cells were washed to remove endogenous cytosolic components. This opens the way towards the identification of cell components that mediate the sorting of apical and basolateral membrane components in the TGN and their polarized delivery to the cell surface.

Membrane traffic related to endosome dynamics and protein secretion in filamentous fungi

2021 ◽  
Author(s):  
Yujiro Higuchi
Keyword(s):  
Filamentous Fungi ◽  
Protein Secretion ◽  
Secretory Pathway ◽  
Molecular Mechanisms ◽  
Membrane Traffic ◽  
Early Endosome ◽  
Protein Glycosylation ◽  
Hyphal Cell ◽  
Cell Factories ◽  
Membrane Contacts

ABSTRACT In eukaryotic cells, membrane-surrounded organelles are orchestrally organized spatiotemporally under environmental situations. Among such organelles, vesicular transports and membrane contacts occur to communicate each other, so-called membrane traffic. Filamentous fungal cells are highly polarized and thus membrane traffic is developed to have versatile functions. Early endosome (EE) is an endocytic organelle that dynamically exhibits constant long-range motility through the hyphal cell, which is proven to have physiological roles, such as other organelle distribution and signal transduction. Since filamentous fungal cells are also considered as cell factories, to produce valuable proteins extracellularly, molecular mechanisms of secretory pathway including protein glycosylation have been well investigated. In this review, molecular and physiological aspects of membrane traffic especially related to EE dynamics and protein secretion in filamentous fungi are summarized, and perspectives for application are also described.

scholarly journals Membrane traffic between secretory compartments is differentially affected during mitosis.

1990 ◽  
Vol 1 (5) ◽  
pp. 415-424 ◽  
Author(s):  
T Kreiner ◽  
H P Moore
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Secretory Pathway ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Membrane Traffic ◽  
Human Growth ◽  
Secretory Proteins ◽  
Viral Membrane ◽  
Mitotic Cells

Membrane traffic has been shown to be regulated during cell division. In particular, with the use of viral membrane proteins as markers, endoplasmic reticulum (ER)-to-Golgi transport in mitotic cells has been shown to be essentially blocked. However, the effect of mitosis on other steps in the secretory pathway is less clear, because an early block makes examination of following steps difficult. Here, we report studies on the functional characteristics of secretory pathways in mitotic mammalian tissue culture cells by the use of a variety of markers. Chinese hamster ovary cells were transfected with cDNAs encoding secretory proteins. Consistent with earlier results following viral membrane proteins, we found that the overall secretory pathway is nonfunctional in mitotic cells, and a major block to secretion is at the step between ER and Golgi: the overall rate of secretion of human growth hormone is reduced at least 10-fold in mitotic cells, and export of truncated vesicular stomatitis virus G protein from the ER is inhibited to about the same extent, as judged by acquisition of endoglycosidase H resistance. To ascertain the integrity of transport from the trans-Golgi to plasma membrane, we followed the secretion of sulfated glycosaminoglycan (GAG) chains, which are synthesized in the Golgi and thus are not subject to the earlier ER-to-Golgi block. GAG chains are valid markers for the pathway taken by constitutive secretory proteins; both protein secretion and GAG chain secretion are sensitive to treatment with n-ethyl-maleimide and monensin and are blocked at 19 degrees C. We found that the extent of GAG-chain secretion is not altered during mitosis, although the initial rate of secretion is reduced about twofold in mitotic compared with interphase cells. Thus, during mitosis, transport from the trans-Golgi to plasma membrane is much less hindered than ER-to-Golgi traffic. We conclude that transport steps are not affected to the same extent during mitosis.

scholarly journals Lysosomal Hydrolase Mannose 6-Phosphate Uncovering Enzyme Resides in the trans-Golgi Network

2001 ◽  
Vol 12 (6) ◽  
pp. 1623-1631 ◽  
Author(s):  
Jack Rohrer ◽  
Rosalind Kornfeld
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Lysosomal Hydrolases ◽  
Intracellular Location ◽  
Trans Golgi Network ◽  
Cytosolic Domain ◽  
Cytosolic Domains ◽  
Mannose 6 Phosphate ◽  
Green Fluorescent ◽  
Golgi Network

A crucial step in lysosomal biogenesis is catalyzed by “uncovering” enzyme (UCE), which removes a coveringN-acetylglucosamine from the mannose 6-phosphate (Man-6-P) recognition marker on lysosomal hydrolases. This study shows that UCE resides in the trans-Golgi network (TGN) and cycles between the TGN and plasma membrane. The cytosolic domain of UCE contains two potential endocytosis motifs: 488YHPL and C-terminal 511NPFKD. YHPL is shown to be the more potent of the two in retrieval of UCE from the plasma membrane. A green-fluorescent protein-UCE transmembrane-cytosolic domain fusion protein colocalizes with TGN 46, as does endogenous UCE in HeLa cells, showing that the transmembrane and cytosolic domains determine intracellular location. These data imply that the Man-6-P recognition marker is formed in the TGN, the compartment where Man-6-P receptors bind cargo and are packaged into clathrin-coated vesicles.

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