scholarly journals Copper stimulates trafficking of a distinct pool of the Menkes copper ATPase (ATP7A) to the plasma membrane and diverts it into a rapid recycling pool

2004 ◽  
Vol 378 (3) ◽  
pp. 1031-1037 ◽  
Author(s):  
Luke PASE ◽  
Ilia VOSKOBOINIK ◽  
Mark GREENOUGH ◽  
James CAMAKARIS
Keyword(s):  
Plasma Membrane ◽  
Cell Biology ◽  
Trans Golgi Network ◽  
Excess Copper ◽  
Cell Surface Biotinylation ◽  
Golgi Compartment ◽  
High Level ◽  
P Type ◽  
Golgi Network ◽  
Regulated Trafficking

MNK (Menkes copper-translocating P-type ATPase, or the Menkes protein; ATP7A) plays a key role in regulating copper homoeostasis in humans. MNK has been shown to have a dual role in the cell: it delivers copper to cuproenzymes in the Golgi compartment and effluxes excess copper from the cell. These roles can be achieved through copper-regulated trafficking of MNK. It has previously been shown to undergo trafficking from the trans-Golgi network to the plasma membrane in response to elevated copper concentrations, and to be endocytosed from the plasma membrane to the trans-Golgi network upon the removal of elevated copper. However, the fundamental question as to whether copper influences trafficking of MNK to or from the plasma membrane remained unanswered. In this study we utilized various methods of cell-surface biotinylation to attempt to resolve this issue. These studies suggest that copper induces trafficking of MNK to the plasma membrane but does not affect its rate of internalization from the plasma membrane. We also found that only a specific pool of MNK can traffic to the plasma membrane in response to elevated copper. Significantly, copper appeared to divert MNK into a fast-recycling pool and prevented it from recycling to the Golgi compartment, thus maintaining a high level of MNK in the proximity of the plasma membrane. These findings shed new light on the cell biology of MNK and the mechanism of copper homoeostasis in general.

scholarly journals Phospholipase C β3 is a key component in the Gβγ/PKCη/PKD-mediated regulation of trans-Golgi network to plasma membrane transport

2007 ◽  
Vol 406 (1) ◽  
pp. 157-165 ◽  
Author(s):  
Alberto M. Díaz Añel
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Membrane Transport ◽  
Second Messengers ◽  
Activation Loop ◽  
Trans Golgi Network ◽  
Transport Vesicles ◽  
Golgi Compartment ◽  
Golgi Network ◽  
Interfering Rna

The requirement of DAG (diacylglycerol) to recruit PKD (protein kinase D) to the TGN (trans-Golgi network) for the targeting of transport carriers to the cell surface, has led us to a search for new components involved in this regulatory pathway. Previous findings reveal that the heterotrimeric Gβγ (GTP-binding protein βγ subunits) act as PKD activators, leading to fission of transport vesicles at the TGN. We have recently shown that PKCη (protein kinase Cη) functions as an intermediate member in the vesicle generating pathway. DAG is capable of activating this kinase at the TGN, and at the same time is able to recruit PKD to this organelle in order to interact with PKCη, allowing phosphorylation of PKD's activation loop. The most qualified candidates for the production of DAG at the TGN are PI-PLCs (phosphatidylinositol-specific phospholipases C), since some members of this family can be directly activated by Gβγ, utilizing PtdIns(4,5)P2 as a substrate, to produce the second messengers DAG and InsP3. In the present study we show that βγ-dependent Golgi fragmentation, PKD1 activation and TGN to plasma membrane transport were affected by a specific PI-PLC inhibitor, U73122 [1-(6-{[17-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione]. In addition, a recently described PI-PLC activator, m-3M3FBS [2,4,6-trimethyl-N-(m-3-trifluoromethylphenyl)benzenesulfonamide], induced vesiculation of the Golgi apparatus as well as PKD1 phosphorylation at its activation loop. Finally, using siRNA (small interfering RNA) to block several PI-PLCs, we were able to identify PLCβ3 as the sole member of this family involved in the regulation of the formation of transport carriers at the TGN. In conclusion, we demonstrate that fission of transport carriers at the TGN is dependent on PI-PLCs, specifically PLCβ3, which is necessary to activate PKCη and PKD in that Golgi compartment, via DAG production.

scholarly journals Loss of P4 ATPases Drs2p and Dnf3p Disrupts Aminophospholipid Transport and Asymmetry in Yeast Post-Golgi Secretory Vesicles

2006 ◽  
Vol 17 (4) ◽  
pp. 1632-1642 ◽  
Author(s):  
Nele Alder-Baerens ◽  
Quirine Lisman ◽  
Lambert Luong ◽  
Thomas Pomorski ◽  
Joost C.M. Holthuis
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Plasma Membranes ◽  
Atp Hydrolysis ◽  
Budding Yeast ◽  
Secretory Vesicles ◽  
Trans Golgi Network ◽  
P Type ◽  
Golgi Network

Eukaryotic plasma membranes generally display asymmetric lipid distributions with the aminophospholipids concentrated in the cytosolic leaflet. This arrangement is maintained by aminophospholipid translocases (APLTs) that use ATP hydrolysis to flip phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the external to the cytosolic leaflet. The identity of APLTs has not been established, but prime candidates are members of the P4 subfamily of P-type ATPases. Removal of P4 ATPases Dnf1p and Dnf2p from budding yeast abolishes inward translocation of 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminocaproyl] (NBD)-labeled PS, PE, and phosphatidylcholine (PC) across the plasma membrane and causes cell surface exposure of endogenous PE. Here, we show that yeast post-Golgi secretory vesicles (SVs) contain a translocase activity that flips NBD-PS, NBD-PE, and NBD-PC to the cytosolic leaflet. This activity is independent of Dnf1p and Dnf2p but requires two other P4 ATPases, Drs2p and Dnf3p, that reside primarily in the trans-Golgi network. Moreover, SVs have an asymmetric PE arrangement that is lost upon removal of Drs2p and Dnf3p. Our results indicate that aminophospholipid asymmetry is created when membrane flows through the Golgi and that P4-ATPases are essential for this process.

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.

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.

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.

Immunoisolation of Kex2p-containing organelles from yeast demonstrates colocalisation of three processing proteinases to a single Golgi compartment

1993 ◽  
Vol 106 (3) ◽  
pp. 815-822
Author(s):  
N.J. Bryant ◽  
A. Boyd
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Secretory Pathway ◽  
High Yield ◽  
Trans Golgi Network ◽  
Processing Enzymes ◽  
Terminal Domain ◽  
Golgi Compartment ◽  
Functional Equivalent ◽  
Golgi Network

One of the Golgi compartments of Saccharomyces cerevisiae is defined by the presence of a specific endoproteinase, Kex2p, which cleaves precursor polypeptides at pairs of basic residues. We have used antibodies directed against the cytoplasmically disposed C-terminal domain of Kex2p to develop an immuno-affinity procedure for the isolation of Kex2p-containing organelles. The method gives a high yield of sealed organelles that are essentially free of contamination from other secretory pathway organelles while being significantly enriched for two other late Golgi enzymes, dipeptidylaminopeptidase A and the Kex1 carboxypeptidase. Our findings provide clear evidence for a single yeast Golgi compartment containing all three late-processing enzymes, which is likely to be the functional equivalent in yeast of the mammalian trans-Golgi network.

scholarly journals AGS3-dependent trans-Golgi network membrane trafficking is essential for compaction in mouse embryos

2020 ◽  
Vol 133 (23) ◽  
pp. jcs243238
Author(s):  
Zheng-Wen Nie ◽  
Ying-Jie Niu ◽  
Wenjun Zhou ◽  
Dong-Jie Zhou ◽  
Ju-Yeon Kim ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Fluorescent Protein ◽  
Mouse Embryos ◽  
Cell Contact ◽  
Protein Signaling ◽  
Cell Stage ◽  
Trans Golgi Network ◽  
Early Mouse ◽  
Golgi Network

ABSTRACTActivator of G-protein signaling 3 (AGS3, also known as GPSM1) regulates the trans-Golgi network. The AGS3 GoLoco motif binds to Gαi and thereby regulates the transport of proteins to the plasma membrane. Compaction of early embryos is based on the accumulation of E-cadherin (Cdh1) at cell-contacted membranes. However, how AGS3 regulates the transport of Cdh1 to the plasma membrane remains undetermined. To investigate this, AGS3 was knocked out using the Cas9-sgRNA system. Both trans-Golgi network protein 46 (TGN46, also known as TGOLN2) and transmembrane p24-trafficking protein 7 (TMED7) were tracked in early mouse embryos by tagging these proteins with a fluorescent protein label. We observed that the majority of the AGS3-edited embryos were developmentally arrested and were fragmented after the four-cell stage, exhibiting decreased accumulation of Cdh1 at the membrane. The trans-Golgi network and TMED7-positive vesicles were also dispersed and were not polarized near the membrane. Additionally, increased Gαi1 (encoded by GNAI1) expression could rescue AGS3-overexpressed embryos. In conclusion, AGS3 reinforces the dynamics of the trans-Golgi network and the transport of TMED7-positive cargo containing Cdh1 to the cell-contact surface during early mouse embryo development.

scholarly journals Yeast synaptobrevin, Snc1, engages distinct routes of postendocytic recycling mediated by a sorting nexin, Rcy1-COPI, and retromer

2020 ◽  
Vol 31 (9) ◽  
pp. 944-962
Author(s):  
Jordan T. Best ◽  
Peng Xu ◽  
Jack G. McGuire ◽  
Shannon N. Leahy ◽  
Todd R. Graham
Keyword(s):  
Plasma Membrane ◽  
Multiple Modes ◽  
Sorting Nexin ◽  
Trans Golgi Network ◽  
Golgi Network

The yeast synaptobrevin, Snc1, uses multiple modes of postendocytic recycling to facilitate its return to either the trans-Golgi network or the plasma membrane. Snc1 primarily recycles via pathways dependent on either Rcy1/COPI or Snx4, while a smaller portion of the SNARE appears to be capable of being retrieved by retromer.

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