Golgi enzymes do not cycle through the endoplasmic reticulum during protein secretion or mitosis

Golgi-specific sialyltransferase (ST) expressed as a chimera with the rapamycin-binding domain of mTOR, FRB, relocates to the endoplasmic reticulum (ER) in cells exposed to rapamycin that also express invariant chain (Ii)-FKBP in the ER. This result has been taken to indicate that Golgi-resident enzymes cycle to the ER constitutively. We show that ST-FRB is trapped in the ER even without Ii-FKBP upon rapamycin addition. This is because ER-Golgi–cycling FKBP proteins contain a C-terminal KDEL-like sequence, bind ST-FRB in the Golgi, and are transported together back to the ER by KDEL receptor–mediated retrograde transport. Moreover, depletion of KDEL receptor prevents trapping of ST-FRB in the ER by rapamycin. Thus ST-FRB cycles artificially by binding to FKBP domain–containing proteins. In addition, Golgi-specific O-linked glycosylation of a resident ER protein occurs only upon artificial fusion of Golgi membranes with ER. Together these findings support the consensus view that there is no appreciable mixing of Golgi-resident enzymes with ER under normal conditions.

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Prohibitin: A Novel Molecular Player in KDEL Receptor Signalling

BioMed Research International ◽

10.1155/2015/319454 ◽

2015 ◽

Vol 2015 ◽

pp. 1-13 ◽

Cited By ~ 8

Author(s):

Monica Giannotta ◽

Giorgia Fragassi ◽

Antonio Tamburro ◽

Capone Vanessa ◽

Alberto Luini ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Complex ◽

Membrane Trafficking ◽

Transmembrane Domain ◽

Cell Cycle Control ◽

Retrograde Transport ◽

Multifunctional Protein ◽

G Protein Coupled ◽

Kdel Receptor ◽

Prohibitin 1

The KDEL receptor (KDELR) is a seven-transmembrane-domain protein involved in retrograde transport of protein chaperones from the Golgi complex to the endoplasmic reticulum. Our recent findings have shown that the Golgi-localised KDELR acts as a functional G-protein-coupled receptor by binding to and activating Gs and Gq. These G proteins induce activation of PKA and Src and regulate retrograde and anterograde Golgi trafficking. Here we used an integrated coimmunoprecipitation and mass spectrometry approach to identify prohibitin-1 (PHB) as a KDELR interactor. PHB is a multifunctional protein that is involved in signal transduction, cell-cycle control, and stabilisation of mitochondrial proteins. We provide evidence that depletion of PHB induces intense membrane-trafficking activity at the ER–Golgi interface, as revealed by formation of GM130-positive Golgi tubules, and recruitment of p115,β-COP, and GBF1 to the Golgi complex. There is also massive recruitment of SEC31 to endoplasmic-reticulum exit sites. Furthermore, absence of PHB decreases the levels of the Golgi-localised KDELR, thus preventing KDELR-dependent activation of Golgi-Src and inhibiting Golgi-to-plasma-membrane transport of VSVG. We propose a model whereby in analogy to previous findings (e.g., the RAS-RAF signalling pathway), PHB can act as a signalling scaffold protein to assist in KDELR-dependent Src activation.

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SLY1 and Syntaxin 18 specify a distinct pathway for procollagen VII export from the endoplasmic reticulum

eLife ◽

10.7554/elife.02784 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 47

Author(s):

Cristina Nogueira ◽

Patrik Erlmann ◽

Julien Villeneuve ◽

António JM Santos ◽

Emma Martínez-Alonso ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Fusion ◽

Protein Secretion ◽

Family Members ◽

Retrograde Transport ◽

Cytoplasmic Domain ◽

Snare Complex ◽

Snare Proteins ◽

General Protein

TANGO1 binds and exports Procollagen VII from the endoplasmic reticulum (ER). In this study, we report a connection between the cytoplasmic domain of TANGO1 and SLY1, a protein that is required for membrane fusion. Knockdown of SLY1 by siRNA arrested Procollagen VII in the ER without affecting the recruitment of COPII components, general protein secretion, and retrograde transport of the KDEL-containing protein BIP, and ERGIC53. SLY1 is known to interact with the ER-specific SNARE proteins Syntaxin 17 and 18, however only Syntaxin 18 was required for Procollagen VII export. Neither SLY1 nor Syntaxin 18 was required for the export of the equally bulky Procollagen I from the ER. Altogether, these findings reveal the sorting of bulky collagen family members by TANGO1 at the ER and highlight the existence of different export pathways for secretory cargoes one of which is mediated by the specific SNARE complex containing SLY1 and Syntaxin 18.

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Trimeric G proteins regulate the cytosol-induced redistribution of Golgi enzymes into the endoplasmic reticulum

Journal of Cell Science ◽

10.1242/jcs.108.4.1805 ◽

1995 ◽

Vol 108 (4) ◽

pp. 1805-1815 ◽

Cited By ~ 2

Author(s):

J. Hidalgo ◽

M. Muniz ◽

A. Velasco

Keyword(s):

Endoplasmic Reticulum ◽

G Proteins ◽

Retrograde Transport ◽

Microtubule Depolymerization ◽

High Concentration ◽

Intact Cells ◽

Cytosolic Proteins ◽

Permeabilized Cells ◽

Integral Proteins ◽

In Cells

Streptolysin O-permeabilized cells incubated with a high concentration (5-10 mg/ml) of cytosolic proteins and ATP-generating system exhibit redistribution into the endoplasmic reticulum (ER) of Golgi integral proteins (mannosidase II, galactosyltransferase, TGN 38), detected by immunofluorescence. In addition, mannosidase II is detected in the ER of cells exposed to a high concentration of cytosolic proteins and processed for immunolectron microscopy by immunoperoxidase. The redistribution process requires ATP and is not affected by previous microtubule depolymerization. Ultrastructural observations indicate that Golgi disassembly occurs by budding of coated vesicles. This stage of the process is inhibited by GTP-gamma S, AIF(3–5), transducin beta gamma subunits, and mastoparan, indicating the involvement of trimeric G proteins. At a later stage, vesicles lose their coats and fuse with the ER. This part of the process does not occur in cells incubated at either 15 degrees C or 20 degrees C, or exposed to N-ethylmaleimide. In cells treated with either cholera or pertussis toxin Golgi redistribution into the ER shows a 50-fold lower requirement for cytosolic factors than in untreated cells. These data suggest a regulatory role for both alpha s and alpha i trimeric G proteins in the normal Golgi-ER retrograde transport taking place in intact cells.

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Endoplasmic reticulum stress activates SRC, relocating chaperones to the cell surface where GRP78/CD109 blocks TGF-β signaling

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1714866115 ◽

2018 ◽

Vol 115 (18) ◽

pp. E4245-E4254 ◽

Cited By ~ 38

Author(s):

Yuan-Li Tsai ◽

Dat P. Ha ◽

He Zhao ◽

Anthony J. Carlos ◽

Shan Wei ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Cell Surface ◽

Er Stress ◽

Paradigm Shift ◽

Retrograde Transport ◽

Suppressor Effect ◽

Cell Surface Grp78 ◽

Β Receptor ◽

Kdel Receptor ◽

Tumor Suppressor Effect

The discovery that endoplasmic reticulum (ER) luminal chaperones such as GRP78/BiP can escape to the cell surface upon ER stress where they regulate cell signaling, proliferation, apoptosis, and immunity represents a paradigm shift. Toward deciphering the mechanisms, we report here that, upon ER stress, IRE1α binds to and triggers tyrosine kinase SRC activation, leading to ASAP1 phosphorylation and Golgi accumulation of ASAP1 and Arf1-GTP, resulting in KDEL receptor dispersion from the Golgi and suppression of retrograde transport. At the cell surface, GRP78 binds to and acts in concert with a glycosylphosphatidylinositol-anchored protein, CD109, in blocking TGF-β signaling by promoting the routing of the TGF-β receptor to the caveolae, thereby disrupting its binding to and activation of Smad2. Collectively, we uncover a SRC-mediated signaling cascade that leads to the relocalization of ER chaperones to the cell surface and a mechanism whereby GRP78 counteracts the tumor-suppressor effect of TGF-β.

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Dissociation of Coatomer from Membranes Is Required for Brefeldin A–induced Transfer of Golgi Enzymes to the Endoplasmic Reticulum

The Journal of Cell Biology ◽

10.1083/jcb.137.2.319 ◽

1997 ◽

Vol 137 (2) ◽

pp. 319-333 ◽

Cited By ~ 60

Author(s):

Jochen Scheel ◽

Rainer Pepperkok ◽

Martin Lowe ◽

Gareth Griffiths ◽

Thomas E. Kreis

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Transport ◽

Golgi Complex ◽

Mammalian Cells ◽

Brefeldin A ◽

Retrograde Transport ◽

Marker Proteins ◽

Kdel Receptor

Addition of brefeldin A (BFA) to mammalian cells rapidly results in the removal of coatomer from membranes and subsequent delivery of Golgi enzymes to the endoplasmic reticulum (ER). Microinjected anti-EAGE (intact IgG or Fab-fragments), antibodies against the “EAGE”-peptide of β-COP, inhibit BFA-induced redistribution of β-COP in vivo and block transfer of resident proteins of the Golgi complex to the ER; tubulo-vesicular clusters accumulate and Golgi membrane proteins concentrate in cytoplasmic patches containing β-COP. These patches are devoid of marker proteins of the ER, the intermediate compartment (IC), and do not contain KDEL receptor. Interestingly, relocation of KDEL receptor to the IC, where it colocalizes with ERGIC53 and ts-O45-G, is not inhibited under these conditions. While no stacked Golgi cisternae remain in these injected cells, reassembly of stacks of Golgi cisternae following BFA wash-out is inhibited to only ∼50%. Mono- or divalent anti-EAGE stabilize binding of coatomer to membranes in vitro, at least as efficiently as GTPγS. Taken together these results suggest that enhanced binding of coatomer to membranes completely inhibits the BFA-induced retrograde transport of Golgi resident proteins to the ER, probably by inhibiting fusion of Golgi with ER membranes, but does not interfere with the disassembly of the stacked Golgi cisternae and recycling of KDEL receptor to the IC. These results confirm our previous results suggesting that COPI is involved in anterograde membrane transport from the ER/IC to the Golgi complex (Pepperkok et al., 1993), and corroborate that COPI regulates retrograde membrane transport between the Golgi complex and ER in mammalian cells.

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Faculty Opinions recommendation of Src regulates Golgi structure and KDEL receptor-dependent retrograde transport to the endoplasmic reticulum.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718877827.793499934 ◽

2014 ◽

Author(s):

Alberto Luini

Keyword(s):

Endoplasmic Reticulum ◽

Retrograde Transport ◽

Golgi Structure ◽

Kdel Receptor

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gp74 a membrane glycoprotein of the cis-Golgi network that cycles through the endoplasmic reticulum and intermediate compartment

The Journal of Cell Biology ◽

10.1083/jcb.124.5.649 ◽

1994 ◽

Vol 124 (5) ◽

pp. 649-665 ◽

Cited By ~ 23

Author(s):

J Alcalde ◽

G Egea ◽

IV Sandoval

Keyword(s):

Monoclonal Antibody ◽

Endoplasmic Reticulum ◽

Low Temperature ◽

Golgi Complex ◽

Low Temperatures ◽

Membrane Glycoprotein ◽

Golgi Membranes ◽

Intermediate Compartment ◽

Golgi Network ◽

In Cells

A monoclonal antibody CC92 (IgM), raised against a fraction of rat liver enriched in Golgi membranes, recognizes a novel Endo H-resistant 74-kD membrane glycoprotein (gp74). The bulk of gp74 is confined to the cis-Golgi network (CGN). Outside the Golgi gp74 is found in tubulovesicular structures and ER foci. In cells incubated at 37 degrees C the majority of gp74 is segregated from the intermediate compartment (IC) marker p58. However, in cells treated with organelle perturbants such as low temperature, BFA, and [AIF4]- the patterns of the two proteins become indistinguishable. Both proteins are retained in the Golgi complex at 20 degrees C and in the IC at 15 degrees C. Incubation of cells with BFA results in relocation of gp74 to p58 positive IC elements. [AIF4]- induces the redistribution of gp74 from the Golgi to p58-positive vesicles and does not retard the translocation of gp74 to IC elements in cells treated with BFA. Disruption of microtubules by nocodazol results in the rapid disappearance of the Golgi elements stained by gp74 and redistribution of the protein into vesicle-like structures. The responses of gp74 to cell perturbants are in sharp contrast with those of cis/middle and trans-Golgi resident proteins whose location is not affected by low temperatures or [AIF4]-, are translocated to the ER upon addition of BFA, and stay in slow disintegrating Golgi elements in cells treated with nocodazol. The results suggest that gp74 is an itinerant protein that resides most of the time in the CGN and cycles through the ER/IC following the pathway used by p58.

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Phospholipase A2Antagonists Inhibit Nocodazole-induced Golgi Ministack Formation: Evidence of an ER Intermediate and Constitutive Cycling

Molecular Biology of the Cell ◽

10.1091/mbc.10.12.4021 ◽

1999 ◽

Vol 10 (12) ◽

pp. 4021-4032 ◽

Cited By ~ 39

Author(s):

Daniel Drecktrah ◽

William J. Brown

Keyword(s):

Endoplasmic Reticulum ◽

G Protein ◽

Immunoelectron Microscopy ◽

Vesicular Stomatitis Virus ◽

Brefeldin A ◽

Rat Hepatocytes ◽

Retrograde Transport ◽

Golgi Transport ◽

Vesicular Stomatitis ◽

In Cells

Evidence has been presented both for and against obligate retrograde movement of resident Golgi proteins through the endoplasmic reticulum (ER) during nocodazole-induced Golgi ministack formation. Here, we studied the nocodazole-induced formation of ministacks using phospholipase A2(PLA2) antagonists, which have been shown previously to inhibit brefeldin A–stimulated Golgi-to-ER retrograde transport. Examination of clone 9 rat hepatocytes by immunofluorescence and immunoelectron microscopy revealed that a subset of PLA2antagonists prevented nocodazole-induced ministack formation by inhibiting two different trafficking pathways for resident Golgi enzymes; at 25 μM, retrograde Golgi-to-ER transport was inhibited, whereas at 5 μM, Golgi-to-ER trafficking was permitted, but resident Golgi enzymes accumulated in the ER. Moreover, resident Golgi enzymes gradually redistributed from the juxtanuclear Golgi or Golgi ministacks to the ER in cells treated with these PLA2antagonists alone. Not only was ER-to-Golgi transport of resident Golgi enzymes inhibited in cells treated with these PLA2antagonists, but transport of the vesicular stomatitis virus G protein out of the ER was also prevented. These results support a model of obligate retrograde recycling of Golgi resident enzymes during nocodazole-induced ministack formation and provide additional evidence that resident Golgi enzymes slowly and constitutively cycle between the Golgi and ER.

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The KDEL retrieval system is exploited by Pseudomonas exotoxin A, but not by Shiga-like toxin-1, during retrograde transport from the Golgi complex to the endoplasmic reticulum

Journal of Cell Science ◽

10.1242/jcs.112.4.467 ◽

1999 ◽

Vol 112 (4) ◽

pp. 467-475 ◽

Cited By ~ 1

Author(s):

M.E. Jackson ◽

J.C. Simpson ◽

A. Girod ◽

R. Pepperkok ◽

L.M. Roberts ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Complex ◽

Retrograde Transport ◽

Vero Cells ◽

Exotoxin A ◽

Pseudomonas Exotoxin ◽

Pseudomonas Exotoxin A ◽

Protein Toxins ◽

Kdel Receptor

To investigate the role of the KDEL receptor in the retrieval of protein toxins to the mammalian cell endoplasmic reticulum (ER), lysozyme variants containing AARL or KDEL C-terminal tags, or the human KDEL receptor, have been expressed in toxin-treated COS 7 and HeLa cells. Expression of the lysozyme variants and the KDEL receptor was confirmed by immunofluorescence. When such cells were challenged with diphtheria toxin (DT) or Escherichia coli Shiga-like toxin 1 (SLT-1), there was no observable difference in their sensitivities as compared to cells which did not express these exogenous proteins. By contrast, the cytotoxicity of Pseudomonas exotoxin A (PE) is reduced by expressing lysozyme-KDEL, which causes a redistribution of the KDEL receptor from the Golgi complex to the ER, and cells are sensitised to this toxin when they express additional KDEL receptors. These data suggest that, in contrast to SLT-1, PE can exploit the KDEL receptor in order to reach the ER lumen where it is believed that membrane transfer to the cytosol occurs. This contention was confirmed by microinjecting into Vero cells antibodies raised against the cytoplasmically exposed tail of the KDEL receptor. Immunofluorescence confirmed that these antibodies prevented the retrograde transport of the KDEL receptor from the Golgi complex to the ER, and this in turn reduced the cytotoxicity of PE, but not that of SLT-1, to these cells.

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