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.

scholarly journals Visualization of the Dynamics of Synaptic Vesicle and Plasma Membrane Proteins in Living Axons

1998 ◽  
Vol 140 (3) ◽  
pp. 659-674 ◽  
Author(s):  
Takao Nakata ◽  
Sumio Terada ◽  
Nobutaka Hirokawa
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Fluorescent Protein ◽  
Plasma Membrane Proteins ◽  
Trans Golgi Network ◽  
Direction Of Movement ◽  
Green Fluorescent ◽  
Golgi Network

Newly synthesized membrane proteins are transported by fast axonal flow to their targets such as the plasma membrane and synaptic vesicles. However, their transporting vesicles have not yet been identified. We have successfully visualized the transporting vesicles of plasma membrane proteins, synaptic vesicle proteins, and the trans-Golgi network residual proteins in living axons at high resolution using laser scan microscopy of green fluorescent protein-tagged proteins after photobleaching. We found that all of these proteins are transported by tubulovesicular organelles of various sizes and shapes that circulate within axons from branch to branch and switch the direction of movement. These organelles are distinct from the endosomal compartments and constitute a new entity of membrane organelles that mediate the transport of newly synthesized proteins from the trans-Golgi network to the plasma membrane.

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 Visualization of Rab9-mediated vesicle transport from endosomes to the trans-Golgi in living cells

2002 ◽  
Vol 156 (3) ◽  
pp. 511-518 ◽  
Author(s):  
Pierre Barbero ◽  
Lenka Bittova ◽  
Suzanne R. Pfeffer
Keyword(s):  
Fluorescent Protein ◽  
Cultured Cells ◽  
Late Endosome ◽  
Live Cells ◽  
Transport Vesicles ◽  
Late Endosomes ◽  
Mannose 6 Phosphate ◽  
Green Fluorescent ◽  
Golgi Network ◽  
Protein Variants

Mannose 6-phosphate receptors (MPRs) are transported from endosomes to the trans-Golgi via a transport process that requires the Rab9 GTPase and the cargo adaptor TIP47. We have generated green fluorescent protein variants of Rab9 and determined their localization in cultured cells. Rab9 is localized primarily in late endosomes and is readily distinguished from the trans-Golgi marker galactosyltransferase. Coexpression of fluorescent Rab9 and Rab7 revealed that these two late endosome Rabs occupy distinct domains within late endosome membranes. Cation-independent mannose 6-phosphate receptors are enriched in the Rab9 domain relative to the Rab7 domain. TIP47 is likely to be present in this domain because it colocalizes with the receptors in fixed cells, and a TIP47 mutant disrupted endosome morphology and sequestered MPRs intracellularly. Rab9 is present on endosomes that display bidirectional microtubule-dependent motility. Rab9-positive transport vesicles fuse with the trans-Golgi network as followed by video microscopy of live cells. These data provide the first indication that Rab9-mediated endosome to trans-Golgi transport can use a vesicle (rather than a tubular) intermediate. Our data suggest that Rab9 remains vesicle associated until docking with the Golgi complex and is rapidly removed concomitant with or just after membrane fusion.

scholarly journals LIM Kinase 1 and Cofilin Regulate Actin Filament Population Required for Dynamin-dependent Apical Carrier Fission from the Trans-Golgi Network

2009 ◽  
Vol 20 (1) ◽  
pp. 438-451 ◽  
Author(s):  
Susana B. Salvarezza ◽  
Sylvie Deborde ◽  
Ryan Schreiner ◽  
Fabien Campagne ◽  
Michael M. Kessels ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Fluorescent Protein ◽  
Live Imaging ◽  
Actin Dynamics ◽  
Lim Kinase ◽  
Trans Golgi Network ◽  
Lim Kinase 1 ◽  
Photoactivatable Gfp ◽  
Green Fluorescent ◽  
Golgi Network

The functions of the actin cytoskeleton in post-Golgi trafficking are still poorly understood. Here, we report the role of LIM Kinase 1 (LIMK1) and its substrate cofilin in the trafficking of apical and basolateral proteins in Madin-Darby canine kidney cells. Our data indicate that LIMK1 and cofilin organize a specialized population of actin filaments at the Golgi complex that is selectively required for the emergence of an apical cargo route to the plasma membrane (PM). Quantitative pulse-chase live imaging experiments showed that overexpression of kinase-dead LIMK1 (LIMK1-KD), or of LIMK1 small interfering RNA, or of an activated cofilin mutant (cofilin S3A), selectively slowed down the exit from the trans-Golgi network (TGN) of the apical PM marker p75-green fluorescent protein (GFP) but did not interfere with the apical PM marker glycosyl phosphatidylinositol-YFP or the basolateral PM marker neural cell adhesion molecule-GFP. High-resolution live imaging experiments of carrier formation and release by the TGN and analysis of peri-Golgi actin dynamics using photoactivatable GFP suggest a scenario in which TGN-localized LIMK1-cofilin regulate a population of actin filaments required for dynamin-syndapin-cortactin–dependent generation and/or fission of precursors to p75 transporters.

scholarly journals Retrograde transport of mannose-6-phosphate receptor depends on several sorting machineries as analyzed by sulfatable nanobodies

2019 ◽  
Author(s):  
Dominik P. Buser ◽  
Martin Spiess
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Protein Transport ◽  
Retrograde Transport ◽  
Lysosomal Hydrolases ◽  
Late Endosomes ◽  
Mannose 6 Phosphate ◽  
Clathrin Adaptor ◽  
Golgi Network

AbstractRetrograde protein transport from the cell surface and endosomes to the trans-Golgi network (TGN) is essential for membrane homeostasis in general and for the recycling of mannose-6-phosphate receptors (MPRs) for sorting of lysosomal hydrolases in particular. Several different sorting machineries have been implicated in retrieval from early or late endosomes to the TGN, mostly for the cation-independent MPR (CIMPR), mainly by analysis of steady-state localization and by interaction studies. We employed a nanobody-based sulfation tool to more directly determine transport kinetics from the plasma membrane to the TGN – the site of sulfation – for the cation-dependent MPR (CDMPR) with and without silencing of candidate machinery proteins. The clathrin adaptor AP-1 that operates bidirectionally at the TGN-to-endosome interface, which had been shown to cause reduced sulfation when rapidly depleted, produced hypersulfation of nanobodies internalized by CDMPR upon long-term silencing, reflecting accumulation in the TGN. In contrast, knockdown of retromer (Vps26), epsinR, or Rab9 reduced CDMPR arrival to the TGN. No effect was observed upon silencing of TIP47. Most surprisingly, depletion of the GGA (Golgi-localized, γ-adaptin ear-containing, Arf-binding) proteins inhibited retrograde transport rather than TGN exit. This study illustrates the usefulness of derivatized, sulfation-competent nanobodies to analyze retrograde protein transport to identify the contributions of different machineries.

Human Na+/H+ exchanger isoform 6 is found in recycling endosomes of cells, not in mitochondria

2002 ◽  
Vol 282 (5) ◽  
pp. C1031-C1041 ◽  
Author(s):  
Christopher L. Brett ◽  
Ying Wei ◽  
Mark Donowitz ◽  
Rajini Rao
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Phylogenetic Analyses ◽  
Human Homolog ◽  
Recycling Endosomes ◽  
Mitochondrial Markers ◽  
Vacuolar Compartment ◽  
Green Fluorescent ◽  
Golgi Network

Since the discovery of the first intracellular Na+/H+exchanger in yeast, Nhx1, multiple homologs have been cloned and characterized in plants. Together, studies in these organisms demonstrate that Nhx1 is located in the prevacuolar/vacuolar compartment of cells where it sequesters Na+ into the vacuole, regulates intravesicular pH, and contributes to vacuolar biogenesis. In contrast, the human homolog of Nhx1, Na+/H+ exchanger isoform 6 (NHE6), has been reported to localize to mitochondria when transiently expressed as a fusion with green fluorescent protein. This result warrants reevaluation because it conflicts with predictions from phylogenetic analyses. Here we demonstrate that when epitope-tagged NHE6 is transiently expressed in cultured mammalian cells, it does not colocalize with mitochondrial markers. It also does not colocalize with markers of the lysosome, late endosome, trans-Golgi network, or Golgi cisternae. Rather, NHE6 is distributed in recycling compartments and transiently appears on the plasma membrane. These results suggest that, like its homologs in yeast and plants, NHE6 is an endosomal Na+/H+ exchanger that may regulate intravesicular pH and volume and contribute to lysosomal biogenesis.

scholarly journals Role for Dynamin in Late Endosome Dynamics and Trafficking of the Cation-independent Mannose 6-Phosphate Receptor

2000 ◽  
Vol 11 (2) ◽  
pp. 481-495 ◽  
Author(s):  
Paolo Nicoziani ◽  
Frederik Vilhardt ◽  
Alicia Llorente ◽  
Leila Hilout ◽  
Pierre J. Courtoy ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Dominant Negative ◽  
Confocal Imaging ◽  
Trans Golgi Network ◽  
Late Endosomes ◽  
Molecular Machinery ◽  
Mannose 6 Phosphate ◽  
Golgi Network ◽  
Dynamin 2

It is well established that dynamin is involved in clathrin-dependent endocytosis, but relatively little is known about possible intracellular functions of this GTPase. Using confocal imaging, we found that endogenous dynamin was associated with the plasma membrane, the trans-Golgi network, and a perinuclear cluster of cation-independent mannose 6-phosphate receptor (CI-MPR)–containing structures. By electron microscopy (EM), it was shown that these structures were late endosomes and that the endogenous dynamin was preferentially localized to tubulo-vesicular appendices on these late endosomes. Upon induction of the dominant-negative dynK44A mutant, confocal microscopy demonstrated a redistribution of the CI-MPR in mutant-expressing cells. Quantitative EM analysis of the ratio of CI-MPR to lysosome-associated membrane protein-1 in endosome profiles revealed a higher colocalization of the two markers in dynK44A-expressing cells than in control cells. Western blot analysis showed that dynK44A-expressing cells had an increased cellular procathepsin D content. Finally, EM revealed that in dynK44A-expressing cells, endosomal tubules containing CI-MPR were formed. These results are in contrast to recent reports that dynamin-2 is exclusively associated with endocytic structures at the plasma membrane. They suggest instead that endogenous dynamin also plays an important role in the molecular machinery behind the recycling of the CI-MPR from endosomes to the trans-Golgi network, and we propose that dynamin is required for the final scission of vesicles budding from endosome tubules.

The Golgi-targeting sequence of the peripheral membrane protein p230

1999 ◽  
Vol 112 (11) ◽  
pp. 1645-1654 ◽  
Author(s):  
L. Kjer-Nielsen ◽  
C. van Vliet ◽  
R. Erlich ◽  
B.H. Toh ◽  
P.A. Gleeson
Keyword(s):  
Fusion Protein ◽  
Fluorescent Protein ◽  
Brefeldin A ◽  
Deletion Mutants ◽  
Alanine Scanning Mutagenesis ◽  
Binding Characteristics ◽  
Cos Cells ◽  
Trans Golgi Network ◽  
Green Fluorescent ◽  
Golgi Network

Vesicle transport requires the recruitment of cytosolic proteins to specific membrane compartments. We have previously characterised a brefeldin A-sensitive trans-Golgi network-localised protein (p230) that is associated with a population of non-clathrin-coated vesicles. p230 recycles between the cytosol and the cytoplasmic face of buds/vesicles of trans-Golgi network membranes in a G protein-regulated manner. Identifying the mechanism responsible for Golgi targeting of p230 is important for the elucidation of its function. By transfection of COS cells with deletion mutants of p230 we here demonstrate that the C-terminal domain is necessary for targeting to the Golgi. Furthermore, the C-terminal 98 amino acid domain of p230 attached to the green fluorescent protein (GFP-p230-C98aa) was efficiently Golgi-localised in transfected COS cells. Deletion mutants of GFP-p230-C98aa together with alanine scanning mutagenesis identified a minimum stretch of 42 amino acids that is essential for Golgi targeting, suggesting that the conformation of the domain is critical for efficient targeting. In COS cells expressing high levels of GFP-p230-C98aa fusion protein, endogenous p230 was no longer associated with Golgi membranes, suggesting that the GFP fusion protein and endogenous p230 may compete for the same membrane target structures. The Golgi binding of GFP-p230-C98aa is brefeldin A-sensitive and is regulated by G proteins. These studies have identified a minimal sequence responsible for specific targeting of p230 to the Golgi apparatus, which displays similar membrane binding characteristics to wild-type p230.

scholarly journals Subcellular localization and functional expression of the glycerol uptake protein 1 (GUP1) of Saccharomyces cerevisiae tagged with green fluorescent protein

2005 ◽  
Vol 390 (1) ◽  
pp. 145-155 ◽  
Author(s):  
Gianluca Bleve ◽  
Giuseppe Zacheo ◽  
Maria Stella Cappello ◽  
Franco Dellaglio ◽  
Francesco Grieco
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Subcellular Localization ◽  
Fluorescent Protein ◽  
Functional Expression ◽  
Biologically Active ◽  
Intracellular Location ◽  
Green Fluorescent ◽  
Glycerol Uptake

GFP (green fluorescent protein) from Aequorea victoria was used as an in vivo reporter protein when fused to the N- and C-termini of the glycerol uptake protein 1 (Gup1p) of Saccharomyces cerevisiae. The subcellular localization and functional expression of biologically active Gup1–GFP chimaeras was monitored by confocal laser scanning and electron microscopy, thus supplying the first study of GUP1 dynamics in live yeast cells. The Gup1p tagged with GFP is a functional glycerol transporter localized at the plasma membrane and endoplasmic reticulum levels of induced cells. The factors involved in proper localization and turnover of Gup1p were revealed by expression of the Gup1p–GFP fusion protein in a set of strains bearing mutations in specific steps of the secretory and endocytic pathways. The chimaerical protein was targeted to the plasma membrane through a Sec6-dependent process; on treatment with glucose, it was endocytosed through END3 and targeted for degradation in the vacuole. Gup1p belongs to the list of yeast proteins rapidly down-regulated by changing the carbon source in the culture medium, in agreement with the concept that post-translational modifications triggered by glucose affect proteins of peripheral functions. The immunoelectron microscopy assays of cells expressing either Gup1–GFP or GFP–Gup1 fusions suggested the Gup1p membrane topology: the N-terminus lies in the periplasmic space, whereas its C-terminal tail has an intracellular location. An extra cytosolic location of the N-terminal tail is not generally predicted or determined in yeast membrane transporters.

scholarly journals Phosphatidylinositol 3-kinase is not required for recycling of mannose 6-phosphate receptors from late endosomes to the trans-Golgi network.

1997 ◽  
Vol 8 (4) ◽  
pp. 577-582 ◽  
Author(s):  
Y Nakajima ◽  
S R Pfeffer
Keyword(s):  
Transport Process ◽  
Kinase Inhibitor ◽  
Phosphatidylinositol 3 Kinase ◽  
Lysosomal Hydrolases ◽  
Trans Golgi Network ◽  
Cell Extracts ◽  
Late Endosomes ◽  
Mannose 6 Phosphate ◽  
Golgi Network ◽  
Phosphatidylinositol 3

Mannose 6-phosphate receptors carry newly synthesized lysosomal hydrolases from the trans-Golgi network to endosomes, then return to the trans-Golgi network for another round of enzyme delivery. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, interferes with the delivery of newly synthesized lysosomal enzymes to lysosomes. We used two independent assays of mannose 6-phosphate receptor trafficking to determine the precise step that is blocked by wortmannin. Using an assay that monitors resialylation of desialylated cell surface 300-kDa mannose 6-phosphate receptors, we found that receptor endocytosis and transport to the trans-Golgi network were not inhibited by 2 microM wortmannin. In addition, this concentration of drug had no effect on the transport of the mannose 6-phosphate receptor from late endosomes to the trans-Golgi network using a system that reconstitutes this transport process in cell extracts. Under the same conditions, wortmannin significantly inhibited the generation of mature cathepsin D. In addition, the structurally unrelated phosphatidylinositol 3-kinase inhibitor, LY294002, was also without effect when added to in vitro endosome-trans-Golgi network transport reactions. These experiments demonstrate that the interruption in lysosomal enzyme targeting is most likely due to a wortmannin-sensitive process required for the export of these receptors from the trans-Golgi network, consistent with the established role of phosphatidylinositol 3-kinase in the equivalent transport process in Saccharomyces cerevisiae.

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