Caspase-resistant Golgin-160 Disrupts Apoptosis Induced by Secretory Pathway Stress and Ligation of Death Receptors

Golgin-160 is a coiled-coil protein on the cytoplasmic face of the Golgi complex that is cleaved by caspases during apoptosis. We assessed the sensitivity of cell lines stably expressing wild-type or caspase-resistant golgin-160 to several proapoptotic stimuli. Cells expressing a caspase-resistant mutant of golgin-160 were strikingly resistant to apoptosis induced by ligation of death receptors and by drugs that induce endoplasmic reticulum (ER) stress, including brefeldin-A, dithiothreitol, and thapsigargin. However, both cell lines responded similarly to other proapoptotic stimuli, including staurosporine, anisomycin, and etoposide. The caspase-resistant golgin-160 dominantly prevented cleavage of endogenous golgin-160 after ligation of death receptors or induction of ER stress, which could be explained by a failure of initiator caspase activation. The block in apoptosis in cells expressing caspase-resistant golgin-160 could not be bypassed by expression of potential caspase cleavage fragments of golgin-160, or by drug-induced disassembly of the Golgi complex. Our results suggest that some apoptotic signals (including those initiated by death receptors and ER stress) are sensed and integrated at Golgi membranes and that golgin-160 plays an important role in transduction of these signals.

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Death Receptors DR4 and DR5 Undergo Spontaneous and Ligand-Mediated Endocytosis and Recycling Regardless of the Sensitivity of Cancer Cells to TRAIL

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.733688 ◽

2021 ◽

Vol 9 ◽

Author(s):

Artem A. Artykov ◽

Anne V. Yagolovich ◽

Dmitry A. Dolgikh ◽

Mikhail P. Kirpichnikov ◽

Daria B. Trushina ◽

...

Keyword(s):

Cell Surface ◽

Cell Lines ◽

Death Receptor ◽

Brefeldin A ◽

Death Receptors ◽

Resistant Cell ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Receptor Endocytosis ◽

Trail Signaling

Tumor necrosis factor-associated ligand inducing apoptosis (TRAIL) induces apoptosis through the death receptors (DRs) 4 and 5 expressed on the cell surface. Upon ligand stimulation, death receptors are rapidly internalized through clathrin-dependent and -independent mechanisms. However, there have been conflicting data on the role of death receptor endocytosis in apoptotic TRAIL signaling and possible cell type-specific differences in TRAIL signaling have been proposed. Here we have compared the kinetics of TRAIL-mediated internalization and subsequent recycling of DR4 and DR5 in resistant (HT-29 and A549) and sensitive (HCT116 and Jurkat) tumor cell lines of various origin. TRAIL stimulated the internalization of both receptors in a concentration-dependent manner with similar kinetics in sensitive and resistant cell lines without affecting the steady-state expression of DR4 and DR5 in cell lysates. Using the receptor-selective TRAIL variant DR5-B, we have shown that DR5 is internalized independently of DR4 receptor. After internalization and elimination of TRAIL from culture medium, the receptors slowly return to the plasma membrane. Within 4 h in resistant or 6 h in sensitive cells, the surface expression of receptors was completely restored. Recovery of receptors occurred both from newly synthesized molecules or from trans-Golgi network, as cycloheximide and brefeldin A inhibited this process. These agents also suppressed the expression of cell surface receptors in a time- and concentration-dependent manner, indicating that DRs undergo constitutive endocytosis. Inhibition of receptor endocytosis by sucrose led to sensitization of resistant cells to TRAIL and to an increase in its cytotoxic activity against sensitive cells. Our results confirm the universal nature of TRAIL-induced death receptor endocytosis, thus cell sensitivity to TRAIL can be associated with post-endocytic events.

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Characterization of Class I and II ADP-Ribosylation Factors (Arfs) in Live Cells: GDP-bound Class II Arfs Associate with the ER-Golgi Intermediate Compartment Independently of GBF1

Molecular Biology of the Cell ◽

10.1091/mbc.e08-04-0373 ◽

2008 ◽

Vol 19 (8) ◽

pp. 3488-3500 ◽

Cited By ~ 55

Author(s):

Justin Chun ◽

Zoya Shapovalova ◽

Selma Y. Dejgaard ◽

John F. Presley ◽

Paul Melançon

Keyword(s):

Golgi Complex ◽

Binding Sites ◽

Secretory Pathway ◽

Brefeldin A ◽

Live Cells ◽

Adp Ribosylation ◽

Rapid Release ◽

Intermediate Compartment ◽

Adp Ribosylation Factor

Despite extensive work on ADP-ribosylation factor (Arf) 1 at the Golgi complex, the functions of Arf2–5 in the secretory pathway, or for that of any Arf at the ER-Golgi intermediate compartment (ERGIC) remain uncharacterized. Here, we examined the recruitment of fluorescently tagged Arf1, -3, -4, and -5 onto peripheral ERGIC. Live cell imaging detected Arfs on peripheral puncta that also contained Golgi-specific brefeldin A (BFA) resistance factor (GBF) 1 and the ERGIC marker p58. Unexpectedly, BFA did not promote corecruitment of Arfs with GBF1 either at the Golgi complex or the ERGIC, but it uncovered striking differences between Arf1,3 and Arf4,5. Although Arf1,3 quickly dissociated from all endomembranes after BFA addition, Arf4,5 persisted on ERGIC structures, even after redistribution of GBF1 to separate compartments. The GDP-arrested Arf4(T31N) mutant localized to the ERGIC, even with BFA and Exo1 present. In addtion, loss of Arf · GTP after treatment with Exo1 caused rapid release of all Arfs from the Golgi complex and led to GBF1 accumulation on both Golgi and ERGIC membranes. Our results demonstrate that GDP-bound Arf4,5 associate with ERGIC membranes through binding sites distinct from those responsible for GBF1 recruitment. Furthermore, they provide the first evidence that GBF1 accumulation on membranes may be caused by loss of Arf · GTP, rather than the formation of an Arf · GDP · BFA · GBF1 complex.

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Golgi-Disturbing Agents Lead to the Elimination of Intracellular Toxoplasma gondii

The Open Biology Journal ◽

10.2174/1874196700902010010 ◽

2009 ◽

Vol 2 (1) ◽

pp. 10-19 ◽

Cited By ~ 1

Author(s):

C. S. Carvalho ◽

G. R. Figueiredo ◽

E. J. T. de Melo

Keyword(s):

Retinoic Acid ◽

Toxoplasma Gondii ◽

Golgi Apparatus ◽

Okadaic Acid ◽

Golgi Complex ◽

Secretory Pathway ◽

Brefeldin A ◽

Host Cells ◽

Parasite Development ◽

Intracellular Medium

The Golgi apparatus is responsible for the genesis of secretory organelles of Toxoplasma gondii and lipid traffic to the vacuole. This study used anti-Golgi agents to demonstrate the importance of Golgi in Toxoplasma development. Monensin, Brefeldin A, Retinoic Acid and Okadaic Acid reduced the infection, leading to parasite elimination. Mon, BFA and RA affected secretory organelles and the Golgi Complex of the parasites, with faster parasite elimination in the presence of Monensin; in addition, the vesicular transit of host cell C6-NBD-ceramide metabolites was interrupted, but the GC of host cells was preserved. Our results suggest that several targets in the secretory pathway are affected in the intracellular Toxoplasma rather than in the host cells, resulting in interruption of parasite development and its elimination from the intracellular medium.

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Involvement of a Golgi-resident GPI-anchored Protein in Maintenance of the Golgi Structure

Molecular Biology of the Cell ◽

10.1091/mbc.e06-03-0236 ◽

2007 ◽

Vol 18 (4) ◽

pp. 1261-1271 ◽

Cited By ~ 11

Author(s):

Xueyi Li ◽

Dora Kaloyanova ◽

Martin van Eijk ◽

Ruud Eerland ◽

Gisou van der Goot ◽

...

Keyword(s):

Secretory Pathway ◽

Protein Transport ◽

Coiled Coil ◽

Brefeldin A ◽

Repeat Sequence ◽

Strong Interactions ◽

Golgi Localization ◽

Associated Proteins ◽

Golgi Structure ◽

And Function

The Golgi apparatus consists of a series of flattened cisternal membranes that are aligned in parallel to form stacks. Cytosolic-oriented Golgi-associated proteins have been identified that may coordinate or maintain the Golgi architecture. Here, we describe a novel GPI-anchored protein, Golgi-resident GPI-anchored protein (GREG) that has a brefeldin A-sensitive Golgi localization. GREG resides in the Golgi lumen as a cis-oriented homodimer, due to strong interactions between coiled-coil regions in the C termini. Dimerization of GREG as well as its Golgi localization depends on a unique tandem repeat sequence within the coiled-coil region. RNA-mediated interference of GREG expression or expression of GREG mutants reveals an essential role for GREG in maintenance of the Golgi integrity. Under these conditions, secretion of the vesicular stomatitis virus glycoprotein protein as a marker for protein transport along the secretory pathway is inhibited, suggesting a loss of Golgi function as well. These results imply the involvement of a luminal protein in Golgi structure and function.

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The Golgi-Associated Hook3 Protein Is a Member of a Novel Family of Microtubule-Binding Proteins

The Journal of Cell Biology ◽

10.1083/jcb.152.5.923 ◽

2001 ◽

Vol 152 (5) ◽

pp. 923-934 ◽

Cited By ~ 138

Author(s):

Jason H. Walenta ◽

Aaron J. Didier ◽

Xinran Liu ◽

Helmut Krämer

Keyword(s):

Golgi Complex ◽

Membrane Trafficking ◽

Spatial Organization ◽

Large Fraction ◽

Coiled Coil ◽

Brefeldin A ◽

Associated Proteins ◽

Terminal Domains

Microtubules are central to the spatial organization of diverse membrane-trafficking systems. Here, we report that Hook proteins constitute a novel family of cytosolic coiled coil proteins that bind to organelles and to microtubules. The conserved NH2-terminal domains of Hook proteins mediate attachment to microtubules, whereas the more divergent COOH-terminal domains mediate the binding to organelles. Human Hook3 bound to Golgi membranes in vitro and was enriched in the cis-Golgi in vivo. Unlike other cis-Golgi–associated proteins, however, a large fraction of Hook3 maintained its juxtanuclear localization after Brefeldin A treatment, indicating a Golgi-independent mechanism for Hook3 localization. Because overexpression of Hook3 caused fragmentation of the Golgi complex, we propose that Hook3 participates in defining the architecture and localization of the mammalian Golgi complex.

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Cerulenin inhibits the cytotoxicity of ricin, modeccin, Pseudomonas toxin, and diphtheria toxin in brefeldin A-resistant cell lines

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)31430-3 ◽

1993 ◽

Vol 268 (17) ◽

pp. 12596-12602

Author(s):

T. Oda ◽

H.C. Wu

Keyword(s):

Cell Lines ◽

Diphtheria Toxin ◽

Brefeldin A ◽

Resistant Cell

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Hijacking Components of the Cellular Secretory Pathway for Replication of Poliovirus RNA

Journal of Virology ◽

10.1128/jvi.01820-06 ◽

2006 ◽

Vol 81 (2) ◽

pp. 558-567 ◽

Cited By ~ 126

Author(s):

George A. Belov ◽

Nihal Altan-Bonnet ◽

Gennadiy Kovtunovych ◽

Catherine L. Jackson ◽

Jennifer Lippincott-Schwartz ◽

...

Keyword(s):

Secretory Pathway ◽

Brefeldin A ◽

Rna Replication ◽

Bound State ◽

Small Gtpases ◽

Nucleotide Exchange ◽

Virus Growth ◽

Guanine Nucleotide Exchange ◽

Membrane Reorganization

ABSTRACT Infection of cells with poliovirus induces a massive intracellular membrane reorganization to form vesicle-like structures where viral RNA replication occurs. The mechanism of membrane remodeling remains unknown, although some observations have implicated components of the cellular secretory and/or autophagy pathways. Recently, we showed that some members of the Arf family of small GTPases, which control secretory trafficking, became membrane-bound after the synthesis of poliovirus proteins in vitro and associated with newly formed membranous RNA replication complexes in infected cells. The recruitment of Arfs to specific target membranes is mediated by a group of guanine nucleotide exchange factors (GEFs) that recycle Arf from its inactive, GDP-bound state to an active GTP-bound form. Here we show that two different viral proteins independently recruit different Arf GEFs (GBF1 and BIG1/2) to the new structures that support virus replication. Intracellular Arf-GTP levels increase ∼4-fold during poliovirus infection. The requirement for these GEFs explains the sensitivity of virus growth to brefeldin A, which can be rescued by the overexpression of GBF1. The recruitment of Arf to membranes via specific GEFs by poliovirus proteins provides an important clue toward identifying cellular pathways utilized by the virus to form its membranous replication complex.

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Yeast Surface Two-hybrid for Quantitative in Vivo Detection of Protein-Protein Interactions via the Secretory Pathway

Journal of Biological Chemistry ◽

10.1074/jbc.m109.001743 ◽

2009 ◽

Vol 284 (24) ◽

pp. 16369-16376 ◽

Cited By ~ 23

Author(s):

Xuebo Hu ◽

Sungkwon Kang ◽

Xiaoyue Chen ◽

Charles B. Shoemaker ◽

Moonsoo M. Jin

Keyword(s):

Protein Interactions ◽

Secretory Pathway ◽

Coiled Coil ◽

Affinity Maturation ◽

Antibody Binding ◽

Soluble Form ◽

Protein Protein Interactions ◽

Yeast Surface ◽

Two Hybrid

A quantitative in vivo method for detecting protein-protein interactions will enhance our understanding of protein interaction networks and facilitate affinity maturation as well as designing new interaction pairs. We have developed a novel platform, dubbed “yeast surface two-hybrid (YS2H),” to enable a quantitative measurement of pairwise protein interactions via the secretory pathway by expressing one protein (bait) anchored to the cell wall and the other (prey) in soluble form. In YS2H, the prey is released either outside of the cells or remains on the cell surface by virtue of its binding to the bait. The strength of their interaction is measured by antibody binding to the epitope tag appended to the prey or direct readout of split green fluorescence protein (GFP) complementation. When two α-helices forming coiled coils were expressed as a pair of prey and bait, the amount of the prey in complex with the bait progressively decreased as the affinity changes from 100 pm to 10 μm. With GFP complementation assay, we were able to discriminate a 6-log difference in binding affinities in the range of 100 pm to 100 μm. The affinity estimated from the level of antibody binding to fusion tags was in good agreement with that measured in solution using a surface plasmon resonance technique. In contrast, the level of GFP complementation linearly increased with the on-rate of coiled coil interactions, likely because of the irreversible nature of GFP reconstitution. Furthermore, we demonstrate the use of YS2H in exploring the nature of antigen recognition by antibodies and activation allostery in integrins and in isolating heavy chain-only antibodies against botulinum neurotoxin.

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