Immunocytochemical localization of beta-COP to the ER-Golgi boundary and the TGN

1995 ◽  
Vol 108 (8) ◽  
pp. 2839-2856 ◽  
Author(s):  
G. Griffiths ◽  
R. Pepperkok ◽  
J.K. Locker ◽  
T.E. Kreis
Keyword(s):  
Golgi Complex ◽  
Retrograde Transport ◽  
Convincing Evidence ◽  
Permissive Temperature ◽  
Viral Glycoprotein ◽  
Anterograde Transport ◽  
Trans Golgi Network ◽  
Culture Cells ◽  
Transport Vesicles ◽  
Golgi Network

Recent data strongly suggest that the coatomer (COP) complex is involved in membrane transport between the ER and Golgi complex. This vesicular coat has been implicated in ER to Golgi, in intra Golgi as well as in Golgi to ER traffic. In this study we present a detailed immunocytochemical analysis of the distribution of beta-COP in different tissue culture cells. Our results extend previous studies by showing, using electron microscopy, that beta-COP accumulates on vesicular profiles and buds in the intermediate compartment (IC) under conditions that block ER to Golgi transport (15 degrees C). Importantly, under these conditions beta-COP co-localizes on these structures with a passenger protein, the membrane glycoprotein of vesicular stomatis virus (ts-O45-G). Furthermore, quantitative immunofluorescence microscopy of cells with ts-045-G accumulated in the ER, IC and trans-Golgi network, shifted briefly to the permissive temperature, showed that beta-COP was associated with many of the putative transport intermediates containing the viral glycoprotein which is in transit between the ER/IC and the cis-Golgi. The simplest interpretation of these data is that COP-coated vesicles are involved in anterograde transport of ts-045-G from the IC to the Golgi complex. Since many putative COP vesicle lacked the G protein following release of the 15 degrees C block this pool could be involved in retrograde transport. We also show that beta-COP is present on the membranes of the trans-Golgi network. However, in contrast to the ER-Golgi boundary, we could find no convincing evidence that this pool of beta-COP is associated with buds or trans-Golgi network-derived transport vesicles.

scholarly journals FIP1/RCP Binding to Golgin-97 Regulates Retrograde Transport from Recycling Endosomes to the trans-Golgi Network

2010 ◽  
Vol 21 (17) ◽  
pp. 3041-3053 ◽  
Author(s):  
Jian Jing ◽  
Jagath R. Junutula ◽  
Christine Wu ◽  
Jemima Burden ◽  
Hugo Matern ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Binding Protein ◽  
Retrograde Transport ◽  
Recycling Endosome ◽  
Transport Pathways ◽  
Recycling Endosomes ◽  
Trans Golgi Network ◽  
C Terminus ◽  
Transport Vesicles ◽  
Golgi Network

Many proteins are retrieved to the trans-Golgi Network (TGN) from the endosomal system through several retrograde transport pathways to maintain the composition and function of the TGN. However, the molecular mechanisms involved in these distinct retrograde pathways remain to be fully understood. Here we have used fluorescence and electron microscopy as well as various functional transport assays to show that Rab11a/b and its binding protein FIP1/RCP are both required for the retrograde delivery of TGN38 and Shiga toxin from early/recycling endosomes to the TGN, but not for the retrieval of mannose-6-phosphate receptor from late endosomes. Furthermore, by proteomic analysis we identified Golgin-97 as a FIP1/RCP-binding protein. The FIP1/RCP-binding domain maps to the C-terminus of Golgin-97, adjacent to its GRIP domain. Binding of FIP1/RCP to Golgin-97 does not affect Golgin-97 recruitment to the TGN, but appears to regulate the targeting of retrograde transport vesicles to the TGN. Thus, we propose that FIP1/RCP binding to Golgin-97 is required for tethering and fusion of recycling endosome-derived retrograde transport vesicles to the TGN.

scholarly journals Identification of a novel, N-ethylmaleimide-sensitive cytosolic factor required for vesicular transport from endosomes to the trans-Golgi network in vitro.

1991 ◽  
Vol 112 (5) ◽  
pp. 823-831 ◽  
Author(s):  
Y Goda ◽  
S R Pfeffer
Keyword(s):  
Early Stage ◽  
Gradient Centrifugation ◽  
Vesicular Transport ◽  
Free System ◽  
Transport Reaction ◽  
Trans Golgi Network ◽  
Transport Vesicles ◽  
Golgi Network ◽  
Gamma S

We have recently described a cell-free system that reconstitutes the vesicular transport of 300-kD mannose 6-phosphate receptors from late endosomes to the trans-Golgi network (TGN). We report here that the endosome----TGN transport reaction was significantly inhibited by low concentrations of the alkylating agent, N-ethylmaleimide (NEM). Addition of fresh cytosol to NEM-inactivated reaction mixtures restored transport to at least 80% of control levels. Restorative activity was only present in cytosol fractions, and was sensitive to trypsin treatment or incubation at 100 degrees C. A variety of criteria demonstrated that the restorative activity was distinct from NSF, an NEM-sensitive protein that facilitates the transport of proteins from the ER to the Golgi complex and between Golgi cisternae. Cytosol fractions immunodepleted of greater than or equal to 90% of NSF protein, or heated to 37 degrees C to inactivate greater than or equal to 93% of NSF activity, were fully able to restore transport to NEM-treated reaction mixtures. The majority of restorative activity sedimented as a uniform species of 50-100 kD upon glycerol gradient centrifugation. We have termed this activity ETF-1, for endosome----TGN transport factor-1. Kinetic experiments showed that ETF-1 acts at a very early stage in vesicular transport, which may reflect a role for this factor in the formation of nascent transport vesicles. GTP hydrolysis appears to be required throughout the transport reaction. The ability of GTP gamma S to inhibit endosome----TGN transport required the presence of donor, endosome membranes, and cytosol, which may reflect a role for guanine nucleotides in vesicle budding. Finally, ETF-1 appears to act before a step that is blocked by GTP gamma S, during the process by which proteins are transported from endosomes to the TGN in vitro.

Retrograde trafficking of both Golgi complex and TGN markers to the ER induced by nordihydroguaiaretic acid and cyclofenil diphenol

1998 ◽  
Vol 111 (7) ◽  
pp. 951-965 ◽  
Author(s):  
D. Drecktrah ◽  
P. de Figueiredo ◽  
R.M. Mason ◽  
W.J. Brown
Keyword(s):  
Golgi Complex ◽  
Membrane Trafficking ◽  
Molecular Mechanisms ◽  
Nordihydroguaiaretic Acid ◽  
Retrograde Transport ◽  
Lipoxygenase Inhibitor ◽  
Golgi Stack ◽  
Golgi Network ◽  
In Vivo Effects

Previous studies have shown that the Golgi stack and the trans-Golgi network (TGN) may play a role in capturing escaped resident endoplasmic reticulum (ER) proteins, and directing their retrograde transport back to that organelle. Whether this retrograde movement represents a highly specific or more generalized membrane trafficking pathway is unclear. To better understand both the retrograde and anterograde trafficking pathways of the secretory apparatus, we examined more closely the in vivo effects of two structurally unrelated compounds, the potent lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA), and the non-steroidal estrogen cyclofenil diphenol (CFD), both of which are known to inhibit secretion. In the presence of these compounds, transport of vesicular stomatitis virus G membrane glycoprotein from the ER to the Golgi complex, and from the TGN to the cell surface, was inhibited potently and rapidly. Surprisingly, we found that NDGA and CFD stimulated the rapid, but not concomitant, retrograde movement of both Golgi stack and TGN membrane proteins back to the ER until both organelles were morphologically absent from cells. Both NDGA- and CFD-stimulated TGN and Golgi retrograde membrane trafficking were inhibited by microtubule depolymerizing agents and energy poisons. Removal of NDGA and CFD resulted in the complete, but not concomitant, reformation of both Golgi stacks and their closely associated TGN compartments. These studies suggest that NDGA and CFD unmask a generalized bulk recycling pathway to the ER for both Golgi and TGN membranes and, further, that NDGA and CFD are useful for investigating the molecular mechanisms that control the formation and maintenance of both the Golgi stack proper and the TGN.

GGA proteins regulate retrograde transport of BACE1 from endosomes to the trans-Golgi network

2005 ◽  
Vol 29 (3) ◽  
pp. 453-461 ◽  
Author(s):  
Tina Wahle ◽  
Kai Prager ◽  
Nikolai Raffler ◽  
Christian Haass ◽  
Michael Famulok ◽  
...  
Keyword(s):  
Retrograde Transport ◽  
Trans Golgi Network ◽  
Golgi Network

scholarly journals Retrograde Transport of Golgi-localized Proteins to the ER

1998 ◽  
Vol 140 (1) ◽  
pp. 1-15 ◽  
Author(s):  
Nelson B. Cole ◽  
Jan Ellenberg ◽  
Jia Song ◽  
Diane DiEuliis ◽  
Jennifer Lippincott-Schwartz
Keyword(s):  
Plasma Membrane ◽  
Golgi Complex ◽  
Fusion Proteins ◽  
Secretory Pathway ◽  
Molecular Mechanisms ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Viral Glycoprotein ◽  
Lumenal Domain

The ER is uniquely enriched in chaperones and folding enzymes that facilitate folding and unfolding reactions and ensure that only correctly folded and assembled proteins leave this compartment. Here we address the extent to which proteins that leave the ER and localize to distal sites in the secretory pathway are able to return to the ER folding environment during their lifetime. Retrieval of proteins back to the ER was studied using an assay based on the capacity of the ER to retain misfolded proteins. The lumenal domain of the temperature-sensitive viral glycoprotein VSVGtsO45 was fused to Golgi or plasma membrane targeting domains. At the nonpermissive temperature, newly synthesized fusion proteins misfolded and were retained in the ER, indicating the VSVGtsO45 ectodomain was sufficient for their retention within the ER. At the permissive temperature, the fusion proteins were correctly delivered to the Golgi complex or plasma membrane, indicating the lumenal epitope of VSVGtsO45 also did not interfere with proper targeting of these molecules. Strikingly, Golgi-localized fusion proteins, but not VSVGtsO45 itself, were found to redistribute back to the ER upon a shift to the nonpermissive temperature, where they misfolded and were retained. This occurred over a time period of 15 min–2 h depending on the chimera, and did not require new protein synthesis. Significantly, recycling did not appear to be induced by misfolding of the chimeras within the Golgi complex. This suggested these proteins normally cycle between the Golgi and ER, and while passing through the ER at 40°C become misfolded and retained. The attachment of the thermosensitive VSVGtsO45 lumenal domain to proteins promises to be a useful tool for studying the molecular mechanisms and specificity of retrograde traffic to the ER.

scholarly journals Oligomerization of atrans-Golgi/trans-Golgi Network Retained Protein Occurs in the Golgi Complex and May Be Part of Its Retention

1995 ◽  
Vol 270 (15) ◽  
pp. 8815-8821 ◽  
Author(s):  
Jacomine Krijnse Locker ◽  
Dirk-Jan E. Opstelten ◽  
Maria Ericsson ◽  
Marian C. Horzinek ◽  
Peter J. M. Rottier
Keyword(s):  
Golgi Complex ◽  
Trans Golgi Network ◽  
Golgi Network

Nucleotide depletion increases trafficking of gentamicin to the Golgi complex in LLC-PK1 cells

2002 ◽  
Vol 283 (6) ◽  
pp. F1422-F1429 ◽  
Author(s):  
Ruben M. Sandoval ◽  
Robert L. Bacallao ◽  
Kenneth W. Dunn ◽  
Jeffrey D. Leiser ◽  
Bruce A. Molitoris
Keyword(s):  
Cell Culture ◽  
Golgi Complex ◽  
Lens Culinaris ◽  
Ricinus Communis ◽  
Physiological State ◽  
Complex Mechanism ◽  
Trans Golgi Network ◽  
Golgi Network ◽  
Texas Red ◽  
Number Of Cells

Having shown rapid trafficking of aminoglycosides to the Golgi complex in cell culture, we focused on the injurious interaction that occurs when gentamicin administration is preceded by renal ischemia. Using Texas red-labeled gentamicin as a tracer, we determined that 15 min of cellular nucleotide depletion did not significantly increase subsequent uptake. However, cells previously depleted of nucleotides accumulated significantly more Texas red-labeled gentamicin within a dispersed Golgi complex. Using Ricinus communis and Lens culinaris lectins, which label specific compartments of the Golgi complex ( trans-Golgi network/ trans and medial/ cis compartments, respectively), we determined that the medial/ cis compartment dispersed after 15 min of nucleotide depletion but the trans-Golgi network/ trans compartment remained unaffected. An increase in the number of cells exhibiting disrupted medial/ cis-Golgi morphology after repletion in physiological media containing gentamicin was also seen. In summary, the increase in nephrotoxicity seen when ischemia precedes aminoglycoside uptake may be part of a complex mechanism initially involving increased Golgi accumulation and prolonged Golgi dispersion. The Golgi complex must then endure the effects of gentamicin accumulated in larger quantities in an aberrant physiological state.

scholarly journals A new role for RINT-1 in SNARE complex assembly at the trans-Golgi network in coordination with the COG complex

2013 ◽  
Vol 24 (18) ◽  
pp. 2907-2917 ◽  
Author(s):  
Kohei Arasaki ◽  
Daichi Takagi ◽  
Akiko Furuno ◽  
Miwa Sohda ◽  
Yoshio Misumi ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Trafficking ◽  
Retrograde Transport ◽  
Snare Complex ◽  
Initial Contact ◽  
Complex Assembly ◽  
Trans Golgi Network ◽  
Cog Complex ◽  
Protein Receptor ◽  
Golgi Network

Docking and fusion of transport vesicles/carriers with the target membrane involve a tethering factor–mediated initial contact followed by soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE)–catalyzed membrane fusion. The multisubunit tethering CATCHR family complexes (Dsl1, COG, exocyst, and GARP complexes) share very low sequence homology among subunits despite likely evolving from a common ancestor and participate in fundamentally different membrane trafficking pathways. Yeast Tip20, as a subunit of the Dsl1 complex, has been implicated in retrograde transport from the Golgi apparatus to the endoplasmic reticulum. Our previous study showed that RINT-1, the mammalian counterpart of yeast Tip20, mediates the association of ZW10 (mammalian Dsl1) with endoplasmic reticulum–localized SNARE proteins. In the present study, we show that RINT-1 is also required for endosome-to–trans-Golgi network trafficking. RINT-1 uncomplexed with ZW10 interacts with the COG complex, another member of the CATCHR family complex, and regulates SNARE complex assembly at the trans-Golgi network. This additional role for RINT-1 may in part reflect adaptation to the demand for more diverse transport routes from endosomes to the trans-Golgi network in mammals compared with those in a unicellular organism, yeast. The present findings highlight a new role of RINT-1 in coordination with the COG complex.

scholarly journals A cytosolic complex of p62 and rab6 associates with TGN38/41 and is involved in budding of exocytic vesicles from the trans-Golgi network

1993 ◽  
Vol 122 (4) ◽  
pp. 775-788 ◽  
Author(s):  
SM Jones ◽  
JR Crosby ◽  
J Salamero ◽  
KE Howell
Keyword(s):  
Binding Proteins ◽  
Integral Membrane Protein ◽  
Preliminary Evidence ◽  
Velocity Sedimentation ◽  
Free System ◽  
Gtp Binding Proteins ◽  
Trans Golgi Network ◽  
Gtp Binding ◽  
Transport Vesicles ◽  
Golgi Network

TGN38/41, an integral membrane protein predominantly localized to the trans-Golgi network, has been shown to cycle to the plasma membrane and return to the TGN within 30 min. (Ladinsky, M. S., and K. E. Howell. 1992. Eur. J. Cell Biol. 59:92-105). In characterizing the proteins which associate with TGN38/41, a peripheral 62-kD protein, two forms of rab6 and two other small GTP-binding proteins were identified by coimmunoprecipitation. However, approximately 90% of the 62-kD protein is cytosolic and is associated with the same subset of small GTP-binding proteins. Both the membrane and cytoplasmic complexes were characterized by sizing column fractionation and velocity sedimentation. The membrane complex was approximately 250 kD (11.6 S) consisting of the cytosolic complex and a heterodimer of TGN38/41 (160 kD). The cytosolic complex was approximately 86 kD (6.1 S) consisting of p62 and one small GTP-binding protein. Preliminary evidence indicates that phosphorylation of the p62 molecule regulates the dissociation of the cytosolic complex from TGN38/41. Functionally the cytosolic p62 complex must bind to TGN38/41 for the budding of exocytic transport vesicles from the TGN as assayed in a cell-free system (Salamero, J., E. S. Sztul, and K. E. Howell. 1990. Proc. Natl. Acad. Sci. USA. 87:7717-7721). Interference with p62, rab6 or TGN38, and TGN41 cytoplasmic domains by immunodepletion or competing peptides completely inhibited the budding of exocytic transport vesicles. These results support an essential role for interaction of the cytosolic p62/rab6 complex with TGN38/41 in budding of exocytic vesicles from the TGN.

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