Actin Microfilaments Facilitate the Retrograde Transport from the Golgi Complex to the Endoplasmic Reticulum in Mammalian Cells

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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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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Influenza virus hemagglutinin trimers and monomers maintain distinct biochemical modifications and intracellular distribution in brefeldin A-treated cells.

Cell Regulation ◽

10.1091/mbc.2.7.549 ◽

1991 ◽

Vol 2 (7) ◽

pp. 549-563 ◽

Cited By ~ 16

Author(s):

G Russ ◽

J R Bennink ◽

T Bächi ◽

J W Yewdell

Keyword(s):

Endoplasmic Reticulum ◽

Influenza Virus ◽

Monoclonal Antibodies ◽

Golgi Complex ◽

Brefeldin A ◽

Retrograde Transport ◽

Cell Fractionation ◽

Influenza Virus Hemagglutinin ◽

Infected Cells ◽

Vesicular Compartment

Brefeldin A (BFA) induces the retrograde transport of proteins from the Golgi complex (GC) to the endoplasmic reticulum (ER). It is uncertain, however, whether the drug completely merges the ER with post-ER compartments, or whether some of their elements remain physically and functionally distinct. We investigated this question by the use of monoclonal antibodies specific for monomers and trimers of the influenza virus hemagglutinin (HA). In untreated influenza virus-infected cells, monomers and trimers almost exclusively partition into the ER and GC, respectively. In BFA-treated cells, both monomers and trimers are detected in the ER by immunofluorescence. Cell fractionation experiments indicate, however, that whereas HA monomers synthesized in the presence of BFA reside predominantly in vesicles with a characteristic density of the ER, HA trimers are primarily located in lighter vesicles characteristic of post-ER compartments. Biochemical experiments confirm that in BFA-treated cells, trimers are more extensively modified than monomers by GC-associated enzymes. Additional immunofluorescence experiments reveal that in BFA-treated cells, HA monomers can exist in an ER subcompartment less accessible to trimers and, conversely, that trimers are present in a vesicular compartment less accessible to monomers. These findings favor the existence of a post-ER compartment for which communication with the ER is maintained in the presence of BFA and suggest that trimers cycle between this compartment and the ER, but have access to only a portion of the ER.

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Requirement for Neo1p in Retrograde Transport from the Golgi Complex to the Endoplasmic Reticulum

Molecular Biology of the Cell ◽

10.1091/mbc.e03-07-0463 ◽

2003 ◽

Vol 14 (12) ◽

pp. 4971-4983 ◽

Cited By ~ 65

Author(s):

Zhaolin Hua ◽

Todd R. Graham

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Complex ◽

Secretory Pathway ◽

Protein Transport ◽

Retrograde Transport ◽

Transport Pathway ◽

Transport Defect ◽

Copii Vesicles ◽

P Type ◽

Adp Ribosylation Factor

Neo1p from Saccharomyces cerevisiae is an essential P-type ATPase and potential aminophospholipid translocase (flippase) in the Drs2p family. We have previously implicated Drs2p in protein transport steps in the late secretory pathway requiring ADP-ribosylation factor (ARF) and clathrin. Here, we present evidence that epitope-tagged Neo1p localizes to the endoplasmic reticulum (ER) and Golgi complex and is required for a retrograde transport pathway between these organelles. Using conditional alleles of NEO1, we find that loss of Neo1p function causes cargo-specific defects in anterograde protein transport early in the secretory pathway and perturbs glycosylation in the Golgi complex. Rer1-GFP, a protein that cycles between the ER and Golgi complex in COPI and COPII vesicles, is mislocalized to the vacuole in neo1-ts at the nonpermissive temperature. These phenotypes suggest that the anterograde protein transport defect is a secondary consequence of a defect in a COPI-dependent retrograde pathway. We propose that loss of lipid asymmetry in the cis Golgi perturbs retrograde protein transport to the ER.

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Faculty Opinions recommendation of Association of tapasin and COPI provides a mechanism for the retrograde transport of major histocompatibility complex (MHC) class I molecules from the Golgi complex to the endoplasmic reticulum.

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

10.3410/f.1004885.183280 ◽

2003 ◽

Author(s):

Lutz Walter

Keyword(s):

Endoplasmic Reticulum ◽

Major Histocompatibility Complex ◽

Mhc Class I ◽

Golgi Complex ◽

Retrograde Transport ◽

Class I ◽

Major Histocompatibility ◽

Histocompatibility Complex ◽

Mhc Class I Molecules

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Retrograde transport of toxins across the endoplasmic reticulum membrane

Biochemical Society Transactions ◽

10.1042/bst0311260 ◽

2003 ◽

Vol 31 (6) ◽

pp. 1260-1262 ◽

Cited By ~ 42

Author(s):

J.M. Lord ◽

E. Deeks ◽

C.J. Marsden ◽

K. Moore ◽

C. Pateman ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Mammalian Cells ◽

Retrograde Transport ◽

Proteasomal Degradation ◽

Degradation Pathway ◽

Endoplasmic Reticulum Membrane ◽

Cellular Processes ◽

A Chain ◽

Toxin Degradation ◽

Cellular Components

Several protein toxins, including the A chain of the plant protein ricin (RTA), enter mammalian cells by endocytosis and catalytically modify cellular components to disrupt essential cellular processes. In the case of ricin, the process inhibited is protein synthesis. In order to reach their cytosolic substrates, several toxins undergo retrograde transport to the ER (endoplasmic reticulum) before translocating across the ER membrane. To achieve this export, these toxins exploit the ERAD (ER-associated protein degradation) pathway but must escape, at least in part, the normal degradative fate of ERAD substrates in order to intoxicate the cell. Toxins that translocate from the ER have an unusually low lysine content that reduces the likelihood of ubiquitination and ubiquitin-mediated proteasomal degradation. We have changed the two lysyl residues normally present in RTA to arginyl residues. Their replacement in RTA did not have a significant stabilizing effect on the protein, suggesting that the endogenous lysyl residues are not sites for ubiquitin attachment. However, when four additional lysyl residues were introduced into RTA in a way that did not compromise the activity, structure or stability of the toxin, degradation was significantly enhanced. Enhanced degradation resulted from ubiquitination that predisposed the toxin to proteasomal degradation. Treatment with the proteasomal inhibitor lactacystin increased the cytotoxicity of the lysine-enriched RTA to a level approaching that of wild-type RTA.

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The Biogenesis of the Golgi Ribbon: The Roles of Membrane Input from the ER and of GM130

Molecular Biology of the Cell ◽

10.1091/mbc.e06-10-0886 ◽

2007 ◽

Vol 18 (5) ◽

pp. 1595-1608 ◽

Cited By ~ 119

Author(s):

Pierfrancesco Marra ◽

Lorena Salvatore ◽

Alexander Mironov ◽

Antonella Di Campli ◽

Giuseppe Di Tullio ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Complex ◽

Mammalian Cells ◽

Matrix Protein ◽

Golgi Ribbon ◽

Continuous Input ◽

Golgi Matrix ◽

Flat Cisternae

The Golgi complex in mammalian cells forms a continuous ribbon of interconnected stacks of flat cisternae. We show here that this distinctive architecture reflects and requires the continuous input of membranes from the endoplasmic reticulum (ER), in the form of pleiomorphic ER-to-Golgi carriers (EGCs). An important step in the biogenesis of the Golgi ribbon is the complete incorporation of the EGCs into the stacks. This requires the Golgi-matrix protein GM130, which continuously cycles between the cis-Golgi compartments and the EGCs. On acquiring GM130, the EGCs undergo homotypic tethering and fusion, maturing into larger and more homogeneous membrane units that appear primed for incorporation into the Golgi stacks. In the absence of GM130, this process is impaired and the EGCs remain as distinct entities. This induces the accumulation of tubulovesicular membranes, the shortening of the cisternae, and the breakdown of the Golgi ribbon. Under these conditions, however, secretory cargo can still be delivered to the Golgi complex, although this occurs less efficiently, and apparently through transient and/or limited continuities between the EGCs and the Golgi cisternae.

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OSBP-Related Protein Family in Lipid Transport over Membrane Contact Sites

Lipid Insights ◽

10.4137/lpi.s31726 ◽

2015 ◽

Vol 8s1 ◽

pp. LPI.S31726 ◽

Cited By ~ 15

Author(s):

Vesa M. Olkkonen

Keyword(s):

Endoplasmic Reticulum ◽

Mammalian Cells ◽

Binding Protein ◽

High Capacity ◽

Retrograde Transport ◽

Protein Family ◽

Oxysterol Binding Protein ◽

Membrane Contact Sites ◽

Contact Sites ◽

Membrane Contact

Increasing evidence suggests that oxysterol-binding protein-related proteins (ORPs) localize at membrane contact sites, which are high-capacity platforms for inter-organelle exchange of small molecules and information. ORPs can simultaneously associate with the two apposed membranes and transfer lipids across the interbilayer gap. Oxysterol-binding protein moves cholesterol from the endoplasmic reticulum to trans-Golgi, driven by the retrograde transport of phosphatidylinositol-4-phosphate (PI4P). Analogously, yeast Osh6p mediates the transport of phosphatidylserine from the endoplasmic reticulum to the plasma membrane in exchange for PI4P, and ORP5 and -8 are suggested to execute similar functions in mammalian cells. ORPs may share the capacity to bind PI4P within their ligand-binding domain, prompting the hypothesis that bidirectional transport of a phosphoinositide and another lipid may be a common theme among the protein family. This model, however, needs more experimental support and does not exclude a function of ORPs in lipid signaling.

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Regulation of Protein Transport from the Golgi Complex to the Endoplasmic Reticulum by CDC42 and N-WASP

Molecular Biology of the Cell ◽

10.1091/mbc.01-12-0579 ◽

2002 ◽

Vol 13 (3) ◽

pp. 866-879 ◽

Cited By ~ 115

Author(s):

Ana Luna ◽

Olga B. Matas ◽

José Angel Martı́nez-Menárguez ◽

Eugenia Mato ◽

Juan M. Durán ◽

...

Keyword(s):

Golgi Complex ◽

Mammalian Cells ◽

Protein Transport ◽

Retrograde Transport ◽

Small Gtpase ◽

Actin Dynamics ◽

Mutant Form ◽

Dependent Manner ◽

Wild Type ◽

Kdel Receptor

Actin is involved in the organization of the Golgi complex and Golgi-to-ER protein transport in mammalian cells. Little, however, is known about the regulation of the Golgi-associated actin cytoskeleton. We provide evidence that Cdc42, a small GTPase that regulates actin dynamics, controls Golgi-to-ER protein transport. We located GFP-Cdc42 in the lateral portions of Golgi cisternae and in COPI-coated and noncoated Golgi-associated transport intermediates. Overexpression of Cdc42 and its activated form Cdc42V12 inhibited the retrograde transport of Shiga toxin from the Golgi complex to the ER, the redistribution of the KDEL receptor, and the ER accumulation of Golgi-resident proteins induced by the active GTP-bound mutant of Sar1 (Sar1[H79G]). Coexpression of wild-type or activated Cdc42 and N-WASP also inhibited Golgi-to-ER transport, but this was not the case in cells expressing Cdc42V12 and N-WASP(ΔWA), a mutant form of N-WASP that lacks Arp2/3 binding. Furthermore, Cdc42V12 recruited GFP-N-WASP to the Golgi complex. We therefore conclude that Cdc42 regulates Golgi-to-ER protein transport in an N-WASP–dependent manner.

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Mycoplasma pneumoniae CARDS toxin exploits host cell endosomal acidic pH and vacuolar ATPase proton pump to execute its biological activities

Scientific Reports ◽

10.1038/s41598-021-90948-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Kumaraguruparan Ramasamy ◽

Sowmya Balasubramanian ◽

Alejandra Kirkpatrick ◽

Daniel Szabo ◽

Lavanya Pandranki ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Mycoplasma Pneumoniae ◽

Mammalian Cells ◽

Biological Activities ◽

Electrical Potential ◽

Retrograde Transport ◽

The United States ◽

Community Acquired Pneumonia ◽

Amino Terminal ◽

Cards Toxin

AbstractMycoplasma pneumoniae is the leading cause of bacterial community-acquired pneumonia among hospitalized children in the United States. It is also responsible for a spectrum of other respiratory tract disorders and extrapulmonary manifestations in children and adults. The main virulence factor of M. pneumoniae is a 591 amino acid multifunctional protein called Community Acquired Respiratory Distress Syndrome (CARDS) toxin. The amino terminal region of CARDS toxin (N-CARDS) retains ADP-ribosylating activity and the carboxy region (C-CARDS) contains the receptor binding and vacuolating activities. After internalization, CARDS toxin is transported in a retrograde manner from endosome through the Golgi complex into the endoplasmic reticulum. However, the mechanisms and criteria by which internalized CARDS toxin is transported and activated to execute its cytotoxic effects remain unknown. In this study, we used full-length CARDS toxin and its mutant and truncated derivatives to analyze how pharmacological drugs that alter pH of intracellular vesicles and electrical potential across vesicular membranes affect translocation of CARDS toxin in mammalian cells. Our results indicate that an acidic environment is essential for CARDS toxin retrograde transport to endoplasmic reticulum. Moreover, retrograde transport facilitates toxin clipping and is required to induce vacuole formation. Additionally, toxin-mediated cell vacuolation is strictly dependent on the function of vacuolar type-ATPase.

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