scholarly journals The Retrieval Function of the KDEL Receptor Requires PKA Phosphorylation of Its C-Terminus

2003 ◽  
Vol 14 (10) ◽  
pp. 4114-4125 ◽  
Author(s):  
Margarita Cabrera ◽  
Manuel Muñiz ◽  
Josefina Hidalgo ◽  
Lucia Vega ◽  
María Esther Martín ◽  
...  
Keyword(s):  
Aspartic Acid ◽  
Retrograde Transport ◽  
Integral Membrane Protein ◽  
Truncated Form ◽  
Permeabilized Cells ◽  
C Terminus ◽  
Intermediate Compartment ◽  
Peptide Binding Assay ◽  
Kdel Receptor

The KDEL receptor is a Golgi/intermediate compartment-located integral membrane protein that carries out the retrieval of escaped ER proteins bearing a C-terminal KDEL sequence. This occurs throughout retrograde traffic mediated by COPI-coated transport carriers. The role of the C-terminal cytoplasmic domain of the KDEL receptor in this process has been investigated. Deletion of this domain did not affect receptor subcellular localization although cells expressing this truncated form of the receptor failed to retain KDEL ligands intracellularly. Permeabilized cells incubated with ATP and GTP exhibited tubular processes-mediated redistribution from the Golgi area to the ER of the wild-type receptor, whereas the truncated form lacking the C-terminal domain remained concentrated in the Golgi. As revealed with a peptide-binding assay, this domain did not interact with both coatomer and ARF-GAP unless serine 209 was mutated to aspartic acid. In contrast, alanine replacement of serine 209 inhibited coatomer/ARF-GAP recruitment, receptor redistribution into the ER, and intracellular retention of KDEL ligands. Serine 209 was phosphorylated by both cytosolic and recombinant protein kinase A (PKA) catalytic subunit. Inhibition of endogenous PKA activity with H89 blocked Golgi-ER transport of the native receptor but did not affect redistribution to the ER of a mutated form bearing aspartic acid at position 209. We conclude that PKA phosphorylation of serine 209 is required for the retrograde transport of the KDEL receptor from the Golgi complex to the ER from which the retrieval of proteins bearing the KDEL signal depends.

scholarly journals Retrograde Transport from the Pre-Golgi Intermediate Compartment and the Golgi Complex Is Affected by the Vacuolar H+-ATPase Inhibitor Bafilomycin A1

1998 ◽  
Vol 9 (12) ◽  
pp. 3561-3578 ◽  
Author(s):  
Harri Palokangas ◽  
Ming Ying ◽  
Kalervo Väänänen ◽  
Jaakko Saraste
Keyword(s):  
Brefeldin A ◽  
Retrograde Transport ◽  
Small Gtpase ◽  
Bafilomycin A1 ◽  
Atpase Inhibitor ◽  
Marker Proteins ◽  
Golgi Region ◽  
Intermediate Compartment ◽  
Acidic Compartments

The effect of the vacuolar H+-ATPase inhibitor bafilomycin A1 (Baf A1) on the localization of pre-Golgi intermediate compartment (IC) and Golgi marker proteins was used to study the role of acidification in the function of early secretory compartments. Baf A1 inhibited both brefeldin A- and nocodazole-induced retrograde transport of Golgi proteins to the endoplasmic reticulum (ER), whereas anterograde ER-to-Golgi transport remained largely unaffected. Furthermore, p58/ERGIC-53, which normally cycles between the ER, IC, and cis-Golgi, was arrested in pre-Golgi tubules and vacuoles, and the number of p58-positive ∼80-nm Golgi (coatomer protein I) vesicles was reduced, suggesting that the drug inhibits the retrieval of the protein from post-ER compartments. In parallel, redistribution of β-coatomer protein from the Golgi to peripheral pre-Golgi structures took place. The small GTPase rab1p was detected in short pre-Golgi tubules in control cells and was efficiently recruited to the tubules accumulating in the presence of Baf A1. In contrast, these tubules showed no enrichment of newly synthesized, anterogradely transported proteins, indicating that they participate in retrograde transport. These results suggest that the pre-Golgi structures contain an active H+-ATPase that regulates retrograde transport at the ER–Golgi boundary. Interestingly, although Baf A1 had distinct effects on peripheral pre-Golgi structures, only more central, p58-containing elements accumulated detectable amounts of 3-(2,4-dinitroanilino)-3′-amino-N-methyldipropylamine (DAMP), a marker for acidic compartments, raising the possibility that the lumenal pH of the pre-Golgi structures gradually changes in parallel with their translocation to the Golgi region.

scholarly journals Localization of the Lys, Asp, Glu, Leu tetrapeptide receptor to the Golgi complex and the intermediate compartment in mammalian cells.

1994 ◽  
Vol 127 (6) ◽  
pp. 1557-1574 ◽  
Author(s):  
G Griffiths ◽  
M Ericsson ◽  
J Krijnse-Locker ◽  
T Nilsson ◽  
B Goud ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Hepatitis Virus ◽  
Significant Shift ◽  
Related Sequence ◽  
Golgi Stack ◽  
Permeabilized Cells ◽  
Intermediate Compartment ◽  
Carboxyl Terminal ◽  
Golgi Network ◽  
Kdel Receptor

The carboxyl-terminal Lys-Asp-Glu-Leu (KDEL), or a closely-related sequence, is important for ER localization of both lumenal as well as type II membrane proteins. This sequence functions as a retrieval signal at post-ER compartment(s), but the exact compartment(s) where the retrieval occurs remains unresolved. With an affinity-purified antibody against the carboxyl-terminal sequence of the mammalian KDEL receptor, we have investigated its subcellular localization using immunogold labeling on thawed cryosections of different tissues, such as mouse spermatids and rat pancreas, as well as HeLa, Vero, NRK, and mouse L cells. We show that rab1 is an excellent marker of the intermediate compartment, and we use this marker, as well as budding profiles of the mouse hepatitis virus (MHV) in cells infected with this virus, to identify this compartment. Our results demonstrate that the KDEL receptor is concentrated in the intermediate compartment, as well as in the Golgi stack. Lower but significant labeling was detected in the rough ER. In general, only small amounts of the receptor were detected on the trans side of the Golgi stack, including the trans-Golgi network (TGN) of normal cells and tissues. However, some stress conditions, such as infection with vaccinia virus or vesicular stomatitis virus, as well as 20 degrees C or 43 degrees C treatment, resulted in a significant shift of the distribution towards the trans-TGN side of the Golgi stack. This shift could be quantified in HeLa cells stably expressing a TGN marker. No significant labeling was detected in structures distal to the TGN under all conditions tested. After GTP gamma S treatment of permeabilized cells, the receptor was detected in the beta-COP-containing buds/vesicles that accumulate after this treatment, suggesting that these vesicles may transport the receptor between compartments. We propose that retrieval of KDEL-containing proteins occurs at multiple post-ER compartments up to the TGN along the exocytotic pathway, and that within this pathway, the amounts of the receptor in different compartments varies according to physiological conditions.

scholarly journals Light-dependent THRUMIN1 phosphorylation regulates its association with actin filaments and 14-3-3 proteins

2021 ◽  
Author(s):  
Matthew E Dwyer ◽  
Roger P. Hangarter
Keyword(s):  
Aspartic Acid ◽  
Actin Filaments ◽  
Chloroplast Movement ◽  
Cell Surfaces ◽  
Intrinsically Disordered ◽  
C Terminus ◽  
Intrinsically Disordered Region ◽  
Chloroplast Movements ◽  
Cell Layers

Light-dependent chloroplast movements in leaf cells contribute to the optimization of photosynthesis. Low light conditions induce chloroplast accumulation along periclinal cell surfaces, providing greater access to the available light, whereas high light induces movement of chloroplasts to anticlinal cell surfaces providing photodamage protection and allowing more light to reach underlying cell layers. The THRUMIN1 protein is required for normal chloroplast movements in Arabidopsis thaliana and has been shown to localize at the plasma membrane and to undergo rapid light-dependent interactions with actin filaments through the N-terminal intrinsically disordered region. A predicted WASP-Homology 2 (WH2) domain was found in the intrinsically disordered region but mutations in this domain did not disrupt localization of THRUMIN1:YFP to actin filaments. A series of other protein truncations and site-directed mutations of known and putative phosphorylation sites indicated that a phosphomimetic mutation (serine to aspartic acid) at position 170 disrupted localization of THRUMIN1 with actin filaments. However, the phosphomimetic mutant rescued the thrumin1-2 mutant phenotype for chloroplast movement and raises questions about the role of THRUMIN1's interaction with actin. Mutation of serine 146 to aspartic acid also resulted in cytoplasmic localization of THRUMIN1:YFP in Nicotiana benthamiana. Mutations to a group of putative zinc-binding cysteine clusters implicates the C-terminus of THRUMIN1 in chloroplast movement. Phosphorylation-dependent association of THRUMIN1 with 14-3-3 KAPPA and OMEGA were also identified. Together, these studies provide new insights into the mechanistic role of THRUMIN1 in light-dependent chloroplast movements.

scholarly journals KDEL Receptor (Erd2p)-mediated Retrograde Transport of the Cholera Toxin A Subunit from the Golgi Involves COPI, p23, and the COOH Terminus of Erd2p

1998 ◽  
Vol 143 (3) ◽  
pp. 601-612 ◽  
Author(s):  
Irina Majoul ◽  
Kai Sohn ◽  
Felix Theodor Wieland ◽  
Rainer Pepperkok ◽  
Mariagrazia Pizza ◽  
...  
Keyword(s):  
Coat Protein ◽  
Cholera Toxin ◽  
Retrograde Transport ◽  
Vero Cells ◽  
Toxin A ◽  
P24 Protein ◽  
A Subunit ◽  
Intermediate Compartment ◽  
Camp Levels ◽  
Kdel Receptor

A cholera toxin mutant (CTX–K63) unable to raise cAMP levels was used to study in Vero cells the retrograde transport of the toxin A subunit (CTX-A–K63), which possesses a COOH-terminal KDEL retrieval signal. Microinjected GTP-γ-S inhibits the internalization as well as Golgi–ER transport of CTX-A–K63. The appearance of CTX-A–K63 in the Golgi induces a marked dispersion of Erd2p and p53 but not of the Golgi marker giantin. Erd2p is translocated under these conditions most likely to the intermediate compartment as indicated by an increased colocalization of Erd2p with mSEC13, a member of the mammalian coat protein II complex. IgGs as well as Fab fragments directed against Erd2p, β-COP, or p23, a new member of the p24 protein family, inhibit or block retrograde transport of CTX-A–K63 from the Golgi without affecting its internalization or its transport to the Golgi. Anti-Erd2p antibodies do not affect the binding of CTX-A to Erd2p, but inhibit the CTX-K63–induced translocation of Erd2p and p53.

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

1999 ◽  
Vol 112 (4) ◽  
pp. 467-475 ◽  
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.

Factor Xa Enhances the Binding of Tissue Factor Pathway Inhibitor to Acidic Phospholipids

1996 ◽  
Vol 75 (05) ◽  
pp. 796-800 ◽  
Author(s):  
Sanne Valentin ◽  
Inger Schousboe
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Xa ◽  
Microtitre Plate ◽  
C Terminus ◽  
Microtitre Plate Assay ◽  
Kunitz Domain ◽  
Tissue Factor Pathway ◽  
Acidic Phospholipids

SummaryIn the present study, the interaction between tissue factor pathway inhibitor (TFPI) and phospholipids has been characterized using a microtitre plate assay. TFPI was shown to bind calcium-independently to an acidic phospholipid surface composed of phosphatidylserine, but not a surface composed of the neutral phosphatidylcholine. The interaction was demonstrated to be dependent on the presence of the TFPI C-terminus. The presence of heparin (1 U/ml, unfractionated) was able to significantly reduce the binding of TFPI to phospholipid. The interaction of TFPI with phosphatidylserine was significantly decreased in the presence of calcium, but this was counteracted, and even enhanced, following complex formation of TFPI with factor Xa prior to incubation with the phospholipid surface. Moreover, a TFPI variant, not containing the third Kunitz domain and the C-terminus, was unable to bind to phospholipid. However, following the formation of a TFPI/factor Xa-complex this TFPI variant was capable of interacting with the phospholipid surface. This indicates that the role of factor Xa as a TFPI cofactor, at least in part, is to mediate the binding of TFPI to the phospholipid surface.

scholarly journals Homeostatic regulation of STING by retrograde membrane traffic to the ER

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Kojiro Mukai ◽  
Emari Ogawa ◽  
Rei Uematsu ◽  
Yoshihiko Kuchitsu ◽  
Fumika Kiku ◽  
...  
Keyword(s):  
Resting State ◽  
Complex I ◽  
Membrane Traffic ◽  
Retrograde Transport ◽  
Homeostatic Regulation ◽  
Gene Encoding ◽  
Downstream Signaling ◽  
Intermediate Compartment ◽  
Downstream Signaling Pathway ◽  
Copa Syndrome

AbstractCoat protein complex I (COP-I) mediates the retrograde transport from the Golgi apparatus to the endoplasmic reticulum (ER). Mutation of the COPA gene, encoding one of the COP-I subunits (α-COP), causes an immune dysregulatory disease known as COPA syndrome. The molecular mechanism by which the impaired retrograde transport results in autoinflammation remains poorly understood. Here we report that STING, an innate immunity protein, is a cargo of the retrograde membrane transport. In the presence of the disease-causative α-COP variants, STING cannot be retrieved back to the ER from the Golgi. The forced Golgi residency of STING results in the cGAS-independent and palmitoylation-dependent activation of the STING downstream signaling pathway. Surf4, a protein that circulates between the ER/ ER-Golgi intermediate compartment/ Golgi, binds STING and α-COP, and mediates the retrograde transport of STING to the ER. The STING/Surf4/α-COP complex is disrupted in the presence of the disease-causative α-COP variant. We also find that the STING ligand cGAMP impairs the formation of the STING/Surf4/α-COP complex. Our results suggest a homeostatic regulation of STING at the resting state by retrograde membrane traffic and provide insights into the pathogenesis of COPA syndrome.

scholarly journals A CACNA1A variant associated with trigeminal neuralgia alters the gating of Cav2.1 channels

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Eder Gambeta ◽  
Maria A. Gandini ◽  
Ivana A. Souza ◽  
Laurent Ferron ◽  
Gerald W. Zamponi
Keyword(s):  
Trigeminal Neuralgia ◽  
Missense Mutation ◽  
Neurotransmitter Release ◽  
Trigeminal System ◽  
Wild Type ◽  
Calcium Dependent ◽  
Whole Cell Patch Clamp ◽  
C Terminus ◽  
Dependent Inactivation

AbstractA novel missense mutation in the CACNA1A gene that encodes the pore forming α1 subunit of the CaV2.1 voltage-gated calcium channel was identified in a patient with trigeminal neuralgia. This mutation leads to a substitution of proline 2455 by histidine (P2455H) in the distal C-terminus region of the channel. Due to the well characterized role of this channel in neurotransmitter release, our aim was to characterize the biophysical properties of the P2455H variant in heterologously expressed CaV2.1 channels. Whole-cell patch clamp recordings of wild type and mutant CaV2.1 channels expressed in tsA-201 cells reveal that the mutation mediates a depolarizing shift in the voltage-dependence of activation and inactivation. Moreover, the P2455H mutant strongly reduced calcium-dependent inactivation of the channel that is consistent with an overall gain of function. Hence, the P2455H CaV2.1 missense mutation alters the gating properties of the channel, suggesting that associated changes in CaV2.1-dependent synaptic communication in the trigeminal system may contribute to the development of trigeminal neuralgia.

scholarly journals Estrogen- and Progesterone (P4)-Mediated Epigenetic Modifications of Endometrial Stromal Cells (EnSCs) and/or Mesenchymal Stem/Stromal Cells (MSCs) in the Etiopathogenesis of Endometriosis

2021 ◽  
Author(s):  
Dariusz Szukiewicz ◽  
Aleksandra Stangret ◽  
Carmen Ruiz-Ruiz ◽  
Enrique G. Olivares ◽  
Olga Soriţău ◽  
...  
Keyword(s):  
Stromal Cells ◽  
Uterine Cavity ◽  
Retrograde Transport ◽  
Surrounding Tissue ◽  
Pelvic Cavity ◽  
Endometrial Stromal Cells ◽  
Endometrial Cells ◽  
Estrogen Exposure ◽  
Chronic Inflammatory Condition

AbstractEndometriosis is a common chronic inflammatory condition in which endometrial tissue appears outside the uterine cavity. Because ectopic endometriosis cells express both estrogen and progesterone (P4) receptors, they grow and undergo cyclic proliferation and breakdown similar to the endometrium. This debilitating gynecological disease affects up to 15% of reproductive aged women. Despite many years of research, the etiopathogenesis of endometrial lesions remains unclear. Retrograde transport of the viable menstrual endometrial cells with retained ability for attachment within the pelvic cavity, proliferation, differentiation and subsequent invasion into the surrounding tissue constitutes the rationale for widely accepted implantation theory. Accordingly, the most abundant cells in the endometrium are endometrial stromal cells (EnSCs). These cells constitute a particular population with clonogenic activity that resembles properties of mesenchymal stem/stromal cells (MSCs). Thus, a significant role of stem cell-based dysfunction in formation of the initial endometrial lesions is suspected. There is increasing evidence that the role of epigenetic mechanisms and processes in endometriosis have been underestimated. The importance of excess estrogen exposure and P4 resistance in epigenetic homeostasis failure in the endometrial/endometriotic tissue are crucial. Epigenetic alterations regarding transcription factors of estrogen and P4 signaling pathways in MSCs are robust in endometriotic tissue. Thus, perspectives for the future may include MSCs and EnSCs as the targets of epigenetic therapies in the prevention and treatment of endometriosis. Here, we reviewed the current known changes in the epigenetic background of EnSCs and MSCs due to estrogen/P4 imbalances in the context of etiopathogenesis of endometriosis.

scholarly journals Studies on the role of actin's aspartic acid 3 and aspartic acid 11 using oligodeoxynucleotide-directed site-specific mutagenesis.

1988 ◽  
Vol 263 (36) ◽  
pp. 19662-19669
Author(s):  
T L Solomon ◽  
L R Solomon ◽  
L S Gay ◽  
P A Rubenstein
Keyword(s):  
Aspartic Acid ◽  
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