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

2002 ◽  
Vol 13 (3) ◽  
pp. 866-879 ◽  
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.

scholarly journals CAP1 binds and activates adenylyl cyclase in mammalian cells

2021 ◽  
Vol 118 (24) ◽  
pp. e2024576118
Author(s):  
Xuefeng Zhang ◽  
Alejandro Pizzoni ◽  
Kyoungja Hong ◽  
Nyla Naim ◽  
Chao Qi ◽  
...  
Keyword(s):  
Adenylyl Cyclase ◽  
Mammalian Cells ◽  
Feedback Loop ◽  
Critical Role ◽  
Small Gtpase ◽  
Actin Dynamics ◽  
Live Cells ◽  
Dependent Manner ◽  
Downstream Effector

CAP1 (Cyclase-Associated Protein 1) is highly conserved in evolution. Originally identified in yeast as a bifunctional protein involved in Ras-adenylyl cyclase and F-actin dynamics regulation, the adenylyl cyclase component seems to be lost in mammalian cells. Prompted by our recent identification of the Ras-like small GTPase Rap1 as a GTP-independent but geranylgeranyl-specific partner for CAP1, we hypothesized that CAP1-Rap1, similar to CAP-Ras-cyclase in yeast, might play a critical role in cAMP dynamics in mammalian cells. In this study, we report that CAP1 binds and activates mammalian adenylyl cyclase in vitro, modulates cAMP in live cells in a Rap1-dependent manner, and affects cAMP-dependent proliferation. Utilizing deletion and mutagenesis approaches, we mapped the interaction of CAP1-cyclase with CAP’s N-terminal domain involving critical leucine residues in the conserved RLE motifs and adenylyl cyclase’s conserved catalytic loops (e.g., C1a and/or C2a). When combined with a FRET-based cAMP sensor, CAP1 overexpression–knockdown strategies, and the use of constitutively active and negative regulators of Rap1, our studies highlight a critical role for CAP1-Rap1 in adenylyl cyclase regulation in live cells. Similarly, we show that CAP1 modulation significantly affected cAMP-mediated proliferation in an RLE motif–dependent manner. The combined study indicates that CAP1-cyclase-Rap1 represents a regulatory unit in cAMP dynamics and biology. Since Rap1 is an established downstream effector of cAMP, we advance the hypothesis that CAP1-cyclase-Rap1 represents a positive feedback loop that might be involved in cAMP microdomain establishment and localized signaling.

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

1997 ◽  
Vol 137 (2) ◽  
pp. 319-333 ◽  
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.

scholarly journals Prohibitin: A Novel Molecular Player in KDEL Receptor Signalling

2015 ◽  
Vol 2015 ◽  
pp. 1-13 ◽  
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.

scholarly journals Pathogenic Effects of Impaired Retrieval between the Endoplasmic Reticulum and Golgi Complex

2019 ◽  
Vol 20 (22) ◽  
pp. 5614 ◽  
Author(s):  
Hiroshi Kokubun ◽  
Hisayo Jin ◽  
Tomohiko Aoe
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Receptor Activation ◽  
Toxic Substances ◽  
Bidirectional Transport ◽  
The Unfolded Protein Response ◽  
Kdel Receptor ◽  
Kdel Sequence

Cellular activities, such as growth and secretion, are dependent on correct protein folding and intracellular protein transport. Injury, like ischemia, malnutrition, and invasion of toxic substances, affect the folding environment in the endoplasmic reticulum (ER). The ER senses this information, following which cells adapt their response to varied situations through the unfolded protein response. Activation of the KDEL receptor, resulting from the secretion from the ER of chaperones containing the KDEL sequence, plays an important role in this adaptation. The KDEL receptor was initially shown to be necessary for the retention of KDEL sequence-containing proteins in the ER. However, it has become clear that the activated KDEL receptor also regulates bidirectional transport between the ER and the Golgi complex, as well as from the Golgi to the secretory pathway. In addition, it has been suggested that the signal for KDEL receptor activation may also affect several other cellular activities. In this review, we discuss KDEL receptor-mediated bidirectional transport and signaling and describe disease models and human diseases related to KDEL receptor dysfunction.

scholarly journals The Pseudomonas aeruginosa product pyochelin interferes with Trypanosoma cruzi infection and multiplication in vitro

2020 ◽  
Vol 114 (7) ◽  
pp. 492-498 ◽  
Author(s):  
Gabriele Sass ◽  
Laura C Miller Conrad ◽  
Terrence-Thang H Nguyen ◽  
David A Stevens
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Trypanosoma Cruzi ◽  
Mammalian Cells ◽  
Iron Acquisition ◽  
Vero Cells ◽  
Dependent Manner ◽  
Wild Type ◽  
Current Standard ◽  
Cruzi Infection

Abstract Background Bacteria are sources of numerous molecules used in treatment of infectious diseases. We investigated effects of molecules produced by 26 Pseudomonas aeruginosa strains against infection of mammalian cell cultures with Trypanosoma cruzi, the aetiological agent of Chagas disease. Methods Vero cells were infected with T. cruzi in the presence of wild-type P. aeruginosa supernatants or supernatants of mutants with defects in the production of various virulence, quorum sensing and iron acquisition factors. Quantification of T. cruzi infection (percentage of infected cells) and multiplication (number of amastigotes per infected cell) was performed and cell viability was determined. Results Wild-type P. aeruginosa products negatively affected T. cruzi infection and multiplication in a dose-dependent manner, without evident toxicity for mammalian cells. PvdD/pchE mutation (loss of the P. aeruginosa siderophores pyoverdine and pyochelin) had the greatest impact on anti–T. cruzi activity. Negative effects on T. cruzi infection by pure pyochelin, but not pyoverdine, or other P. aeruginosa exoproducts studied, were quantitatively similar to the effects of benznidazole, the current standard therapy against T. cruzi. Conclusions The P. aeruginosa product pyochelin showed promising activity against T. cruzi and might become a new lead molecule for therapy development.

scholarly journals Inhibition of Cyclin-dependent Kinase 1 Induces Cytokinesis without Chromosome Segregation in an ECT2 and MgcRacGAP-dependent Manner

2005 ◽  
Vol 280 (43) ◽  
pp. 36502-36509 ◽  
Author(s):  
Fumihiko Niiya ◽  
Xiaozhen Xie ◽  
Kyung S. Lee ◽  
Hiroki Inoue ◽  
Toru Miki
Keyword(s):  
Rna Interference ◽  
Mammalian Cells ◽  
Specific Inhibitor ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Cleavage Furrow ◽  
Dependent Manner ◽  
Cyclin Dependent Kinase ◽  
Mitotic Spindles ◽  
Initiation Mechanism

Cleavage furrow formation marks the onset of cell division during early anaphase. The small GTPase RhoA and its regulators ECT2 and MgcRacGAP have been implicated in furrow ingression in mammalian cells, but the signaling upstream of these molecules remains unclear. We now show that the inhibition of cyclin-dependent kinase (Cdk)1 is sufficient to initiate cytokinesis. When mitotically synchronized cells were treated with the Cdk-specific inhibitor BMI-1026, the initiation of cytokinesis was induced precociously before chromosomal separation. Cytokinesis was also induced by the Cdk1-specific inhibitor purvalanol A but not by Cdk2/Cdk5- or Cdk4-specific inhibitors. Consistent with initiation of precocious cytokinesis by Cdk1 inhibition, introduction of anti-Cdk1 monoclonal antibody resulted in cells with aberrant nuclei. Depolymerization of mitotic spindles by nocodazole inhibited BMI-1026-induced precocious cytokinesis. However, in the presence of a low concentration of nocodazole, BMI-1026 induced excessive membrane blebbing, which appeared to be caused by formation of ectopic cleavage furrows. Depletion of ECT2 or MgcRacGAP by RNA interference abolished both of the phenotypes (precocious furrowing after nocodazole release and excessive blebbing in the presence of nocodazole). RNA interference of RhoA or expression of dominant-negative RhoA efficiently reduced both phenotypes. RhoA was localized at the cleavage furrow or at the necks of blebs. We propose that Cdk1 inactivation is sufficient to activate a signaling pathway leading to cytokinesis, which emanates from mitotic spindles and is regulated by ECT2, MgcRacGAP, and RhoA. Chemical induction of cytokinesis will be a valuable tool to study the initiation mechanism of cytokinesis.

scholarly journals CP-31398 Restores DNA-binding Activity to Mutant p53in Vitrobut Does Not Affect p53 Homologs p63 and p73

2004 ◽  
Vol 279 (44) ◽  
pp. 45887-45896 ◽  
Author(s):  
Mark J. Demma ◽  
Serena Wong ◽  
Eugene Maxwell ◽  
Bimalendu Dasmahapatra
Keyword(s):  
Dna Binding ◽  
Mammalian Cells ◽  
P53 Protein ◽  
Binding Activity ◽  
Mutant P53 ◽  
Dependent Manner ◽  
Wild Type ◽  
Dna Binding Activity

The p53 protein plays a major role in the maintenance of genome stability in mammalian cells. Mutations of p53 occur in over 50% of all cancers and are indicative of highly aggressive cancers that are hard to treat. Recently, there has been a high degree of interest in therapeutic approaches to restore growth suppression functions to mutant p53. Several compounds have been reported to restore wild type function to mutant p53. One such compound, CP-31398, has been shown effectivein vivo, but questions have arisen to whether it actually affects p53. Here we show that mutant p53, isolated from cells treated with CP-31398, is capable of binding to p53 response elementsin vitro. We also show the compound restores DNA-binding activity to mutant p53 in cells as determined by a chromatin immunoprecipitation assay. In addition, using purified p53 core domain from two different hotspot mutants (R273H and R249S), we show that CP-31398 can restore DNA-binding activity in a dose-dependent manner. Using a quantitative DNA binding assay, we also show that CP-31398 increases significantly the amount of mutant p53 that binds to cognate DNA (Bmax) and its affinity (Kd) for DNA. The compound, however, does not affect the affinity (Kdvalue) of wild type p53 for DNA and only increasesBmaxslightly. In a similar assay PRIMA1 does not have any effect on p53 core DNA-binding activity. We also show that CP-31398 had no effect on the DNA-binding activity of p53 homologs p63 and p73.

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

2003 ◽  
Vol 14 (12) ◽  
pp. 4971-4983 ◽  
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.

Lactadherin and Clearance of Platelet-Derived Microvesicles.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1840-1840
Author(s):  
Swapan Kumar Dasgupta ◽  
Hanan Abdel-Monem ◽  
Polly Niravath ◽  
Anhquyen Le ◽  
Ricardo Bellera ◽  
...  
Keyword(s):  
Milk Fat ◽  
Mammalian Cells ◽  
Procoagulant Activity ◽  
In Vivo Model ◽  
Maximal Effect ◽  
Dependent Manner ◽  
Wild Type ◽  
Membrane Bilayer ◽  
Anionic Phospholipids ◽  
Deficient Mice

Abstract Objective— In platelets, as in most mammalian cells, the anionic phospholipids such as phosphatidylserine are present only in the inner leaflet of the membrane bilayer. During platelet activation, phosphatidylserine moves from the inner to the outer leaflet of the membrane bilayer. The transbilayer movement of phosphatidylserine is responsible for platelet procoagulant activity as the exposed phosphatidylserine provides high affinity binding sites for the assembly of the prothrombinase and tenase complex. Externalization of anionic phospholipids in platelet is accompanied by the release of phosphatidylserine-rich microvesicles. These microvesicles account for the procoagulant activity of plasma by providing an efficient catalytic surface. Lactadherin, also known milk fat globule-EGF 8, is a 45 kDa glycoprotein secreted by macrophages. Lactadherin contains EGF-like domains at the amino terminus and two C-domains at the carboxy terminus that share homology to the phosphatidylserinebinding domains of blood coagulation factors V and VIII. Lactadherin binds to apoptotic lymphocytes and phosphatidylserine-expressing red blood cells via the C-domains and anchors them to macrophage integrins via its RGD sequence in the EGF domain. We have examined the role of lactadherin in clearance of phosphatidylserine-rich platelet-derived microvesicles. Methods and Results—Platelet-derived microvesicles were labeled with the fluorophore BODIPY-maleimide and incubated with THP-1 cell derived macrophages. The extent of phagocytosis was quantified by measuring the intracellular fluorescence by flow cytometry. Lactadherin promoted phagocytosis in a concentration-dependent manner with a half-maximal effect at ~ 5 ng/ml. A monoclonal antibody to lactadherin and a carboxy terminal fragment of lactadherin inhibited lactadherin-dependent phagocytosis. Lactadherin-deficient mice had increased number of microvesicles in their plasma and generated more thrombin compared to their wild type littermates. In addition, splenic macrophages from lactadherin-deficient mice showed decreased capacity to phagocytose platelet microvesicles. Finally, in a in vivo model of light/dye-induced endothelial injury/ thrombosis model, lactadherin-deficient mice, showed enhanced thrombus formation (5.93 ± 0.43 min) compared to their wild-type littermates ( 9.80 ± 1.14 min; P=0.01, n=9 in each group) in the cremastric venules. Conclusion— Our studies show that lactadherin mediates the clearance of PS expressing platelet-derived microvesicles from the circulation and that a defective clearance can induce a hypercoagulable state.

scholarly journals The Ca2+-binding Protein ALG-2 Is Recruited to Endoplasmic Reticulum Exit Sites by Sec31A and Stabilizes the Localization of Sec31A

2006 ◽  
Vol 17 (11) ◽  
pp. 4876-4887 ◽  
Author(s):  
Akinori Yamasaki ◽  
Katsuko Tani ◽  
Akitsugu Yamamoto ◽  
Naomi Kitamura ◽  
Masayuki Komada
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Binding Protein ◽  
Small Gtpase ◽  
Dependent Manner ◽  
Rich Region ◽  
Linked Gene ◽  
Transport Vesicles ◽  
Er Exit Sites ◽  
A Cell

The formation of transport vesicles that bud from endoplasmic reticulum (ER) exit sites is dependent on the COPII coat made up of three components: the small GTPase Sar1, the Sec23/24 complex, and the Sec13/31 complex. Here, we provide evidence that apoptosis-linked gene 2 (ALG-2), a Ca2+-binding protein of unknown function, regulates the COPII function at ER exit sites in mammalian cells. ALG-2 bound to the Pro-rich region of Sec31A, a ubiquitously expressed mammalian orthologue of yeast Sec31, in a Ca2+-dependent manner and colocalized with Sec31A at ER exit sites. A Ca2+binding-deficient ALG-2 mutant, which did not bind Sec31A, lost the ability to localize to ER exit sites. Overexpression of the Pro-rich region of Sec31A or RNA interference-mediated Sec31A depletion also abolished the ALG-2 localization at these sites. In contrast, depletion of ALG-2 substantially reduced the level of Sec31A associated with the membrane at ER exit sites. Finally, treatment with a cell-permeable Ca2+chelator caused the mislocalization of ALG-2, which was accompanied by a reduced level of Sec31A at ER exit sites. We conclude that ALG-2 is recruited to ER exit sites via Ca2+-dependent interaction with Sec31A and in turn stabilizes the localization of Sec31A at these sites.

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