scholarly journals Coatomer and dimeric ADP ribosylation factor 1 promote distinct steps in membrane scission

2011 ◽  
Vol 194 (5) ◽  
pp. 765-777 ◽  
Author(s):  
Rainer Beck ◽  
Simone Prinz ◽  
Petra Diestelkötter-Bachert ◽  
Simone Röhling ◽  
Frank Adolf ◽  
...  
Keyword(s):  
Membrane Curvature ◽  
Coated Vesicles ◽  
Cross Linking ◽  
Vesicle Release ◽  
Reconstituted System ◽  
Copi Vesicle ◽  
Membrane Scission ◽  
Adp Ribosylation Factor ◽  
Chemical Cross Linking

Formation of coated vesicles requires two striking manipulations of the lipid bilayer. First, membrane curvature is induced to drive bud formation. Second, a scission reaction at the bud neck releases the vesicle. Using a reconstituted system for COPI vesicle formation from purified components, we find that a dimerization-deficient Arf1 mutant, which does not display the ability to modulate membrane curvature in vitro or to drive formation of coated vesicles, is able to recruit coatomer to allow formation of COPI-coated buds but does not support scission. Chemical cross-linking of this Arf1 mutant restores vesicle release. These experiments show that initial curvature of the bud is defined primarily by coatomer, whereas the membrane curvature modulating activity of dimeric Arf1 is required for membrane scission.

Membrane curvature and the control of GTP hydrolysis in Arf1 during COPI vesicle formation

2005 ◽  
Vol 33 (4) ◽  
pp. 619-622 ◽  
Author(s):  
B. Antonny ◽  
J. Bigay ◽  
J.-F. Casella ◽  
G. Drin ◽  
B. Mesmin ◽  
...  
Keyword(s):  
Lipid Membranes ◽  
Membrane Curvature ◽  
Gtp Hydrolysis ◽  
Membrane Fission ◽  
Transport Vesicles ◽  
Highly Sensitive ◽  
Copi Vesicle ◽  
Membrane Budding ◽  
Gtpase Cycle ◽  
Adp Ribosylation Factor

The GTP switch of the small G-protein Arf1 (ADP-ribosylation factor 1) on lipid membranes promotes the polymerization of the COPI (coat protein complex I) coat, which acts as a membrane deforming shell to form transport vesicles. Real-time measurements for coat assembly on liposomes gives insights into how the GTPase cycle of Arf1 is coupled in time with the polymerization of the COPI coat and the resulting membrane deformation. One key parameter seems to be the membrane curvature. Arf-GAP1 (where GAP stands for GTPase-activating protein), which promotes GTP hydrolysis in the Arf1–COPI complex is highly sensitive to lipid packing. Its activity on Arf1-GTP increases by two orders of magnitude as the diameter of the liposomes approaches that of authentic transport vesicles (60 nm). This suggests that during membrane budding, Arf1-GTP molecules are progressively eliminated from the coated area where the membrane curvature is positive, but are protected from Arf-GAP1 at the bud neck due to the negative curvature of this region. As a result, the coat should be stable as long as the bud remains attached and should disassemble as soon as membrane fission occurs.

scholarly journals Mapping the Contact Sites of the Escherichia coli Division-Initiating Proteins FtsZ and ZapA by BAMG Cross-Linking and Site-Directed Mutagenesis

2018 ◽  
Vol 19 (10) ◽  
pp. 2928 ◽  
Author(s):  
Winfried Roseboom ◽  
Madhvi Nazir ◽  
Nils Meiresonne ◽  
Tamimount Mohammadi ◽  
Jolanda Verheul ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Cross Linking ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Binding Interface ◽  
Tetrameric Structure ◽  
Z Ring ◽  
Cross Links ◽  
Chemical Cross Linking

Cell division in bacteria is initiated by the polymerization of FtsZ at midcell in a ring-like structure called the Z-ring. ZapA and other proteins assist Z-ring formation and ZapA binds ZapB, which senses the presence of the nucleoids. The FtsZ–ZapA binding interface was analyzed by chemical cross-linking mass spectrometry (CXMS) under in vitro FtsZ-polymerizing conditions in the presence of GTP. Amino acids residue K42 from ZapA was cross-linked to amino acid residues K51 and K66 from FtsZ, close to the interphase between FtsZ molecules in protofilaments. Five different cross-links confirmed the tetrameric structure of ZapA. A number of FtsZ cross-links suggests that its C-terminal domain of 55 residues, thought to be largely disordered, has a limited freedom to move in space. Site-directed mutagenesis of ZapA reveals an interaction site in the globular head of the protein close to K42. Using the information on the cross-links and the mutants that lost the ability to interact with FtsZ, a model of the FtsZ protofilament–ZapA tetramer complex was obtained by information-driven docking with the HADDOCK2.2 webserver.

scholarly journals Discrete Determinants in ArfGAP2/3 Conferring Golgi Localization and Regulation by the COPI Coat

2009 ◽  
Vol 20 (3) ◽  
pp. 859-869 ◽  
Author(s):  
Lena Kliouchnikov ◽  
Joëlle Bigay ◽  
Bruno Mesmin ◽  
Anna Parnis ◽  
Moran Rawet ◽  
...  
Keyword(s):  
Lipid Membranes ◽  
Membrane Curvature ◽  
In Vitro Assays ◽  
Minor Role ◽  
Gtp Hydrolysis ◽  
Gtpase Activating Proteins ◽  
Golgi Localization ◽  
Adp Ribosylation Factor ◽  
Fusion Approach

From yeast to mammals, two types of GTPase-activating proteins, ArfGAP1 and ArfGAP2/3, control guanosine triphosphate (GTP) hydrolysis on the small G protein ADP-ribosylation factor (Arf) 1 at the Golgi apparatus. Although functionally interchangeable, they display little similarity outside the catalytic GTPase-activating protein (GAP) domain, suggesting differential regulation. ArfGAP1 is controlled by membrane curvature through its amphipathic lipid packing sensor motifs, whereas Golgi targeting of ArfGAP2 depends on coatomer, the building block of the COPI coat. Using a reporter fusion approach and in vitro assays, we identified several functional elements in ArfGAP2/3. We show that the Golgi localization of ArfGAP3 depends on both a central basic stretch and a carboxy-amphipathic motif. The basic stretch interacts directly with coatomer, which we found essential for the catalytic activity of ArfGAP3 on Arf1-GTP, whereas the carboxy-amphipathic motif interacts directly with lipid membranes but has minor role in the regulation of ArfGAP3 activity. Our findings indicate that the two types of ArfGAP proteins that reside at the Golgi use a different combination of protein–protein and protein–lipid interactions to promote GTP hydrolysis in Arf1-GTP.

scholarly journals Localization of Calmodulin and Dynein Light Chain Lc8 in Flagellar Radial Spokes

2001 ◽  
Vol 153 (6) ◽  
pp. 1315-1326 ◽  
Author(s):  
Pinfen Yang ◽  
Dennis R. Diener ◽  
Joel L. Rosenbaum ◽  
Winfield S. Sale
Keyword(s):  
Light Chain ◽  
Crucial Role ◽  
Cross Linking ◽  
Dynein Light Chain ◽  
Flagellar Motility ◽  
Radial Spoke ◽  
Size And Shape ◽  
Radial Spokes ◽  
Chemical Cross Linking

Genetic and in vitro analyses have revealed that radial spokes play a crucial role in regulation of ciliary and flagellar motility, including control of waveform. However, the mechanisms of regulation are not understood. Here, we developed a novel procedure to isolate intact radial spokes as a step toward understanding the mechanism by which these complexes regulate dynein activity. The isolated radial spokes sediment as 20S complexes that are the size and shape of radial spokes. Extracted radial spokes rescue radial spoke structure when reconstituted with isolated axonemes derived from the radial spoke mutant pf14. Isolated radial spokes are composed of the 17 previously defined spoke proteins as well as at least five additional proteins including calmodulin and the ubiquitous dynein light chain LC8. Analyses of flagellar mutants and chemical cross-linking studies demonstrated calmodulin and LC8 form a complex located in the radial spoke stalk. We postulate that calmodulin, located in the radial spoke stalk, plays a role in calcium control of flagellar bending.

Preparation of Carboxymethyl Chitosan Microspheres and Their Application in Hemostasis

2015 ◽  
Vol 11 (6) ◽  
pp. 660-667 ◽  
Author(s):  
Lu Liu ◽  
Qi Lv ◽  
Qingyun Zhang ◽  
Hui Zhu ◽  
Wei Liu ◽  
...  
Keyword(s):  
Hepatic Injury ◽  
Carboxymethyl Chitosan ◽  
Water Soluble ◽  
Cross Linking ◽  
Clotting Time ◽  
Hemostatic Agents ◽  
Hemostatic Effect ◽  
Injury Model ◽  
Chemical Cross Linking

AbstractObjectiveChitosan (CS) is currently used as a hemostatic agent in emergencies and in military settings. However, its application is limited owing to its poor hydrophilia at neutral pH. Carboxymethyl chitosan (CMCS) is an important, water-soluble derivative of CS. In this study, we prepared CS and CMCS microspheres (CSMs and CMCSMs, respectively) and evaluated their hemostatic effect.MethodsTo prepare the microspheres of various sizes, we used the emulsion cross-linking technique. CMCSMs were also loaded with etamsylate (DIC). Clotting time in vitro and in a hepatic injury model was examined to evaluate the hemostatic effect.ResultsCMCSMs swelled more and clotted faster than did CSMs. CMCSMs loaded with DIC had no effect on hemostasis.ConclusionsBoth increasing material hydrophilicity and expanding the contact area promoted clotting, whereas chemical cross-linking hampered it because of decreased swelling. CMCSMs are promising candidates for the production of effective hemostatic agents. (Disaster Med Public Health Preparedness. 2017;11:660–667)

scholarly journals P1 ParB Domain Structure Includes Two Independent Multimerization Domains

1999 ◽  
Vol 181 (19) ◽  
pp. 5898-5908 ◽  
Author(s):  
Jennifer A. Surtees ◽  
Barbara E. Funnell
Keyword(s):  
Amino Acids ◽  
Domain Structure ◽  
Cross Linking ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
C Terminus ◽  
Self Association ◽  
Two Hybrid ◽  
Chemical Cross Linking

ABSTRACT ParB is one of two P1-encoded proteins that are required for active partition of the P1 prophage in Escherichia coli. To probe the native domain structure of ParB, we performed limited proteolytic digestions of full-length ParB, as well as of several N-terminal and C-terminal deletion fragments of ParB. The C-terminal 140 amino acids of ParB form a very trypsin-resistant domain. In contrast, the N terminus is more susceptible to proteolysis, suggesting that it forms a less stably folded domain or domains. Because native ParB is a dimer in solution, we analyzed the ability of ParB fragments to dimerize, using both the yeast two-hybrid system and in vitro chemical cross-linking of purified proteins. These studies revealed that the C-terminal 59 amino acids of ParB, a region within the protease-resistant domain, are sufficient for dimerization. Cross-linking and yeast two-hybrid experiments also revealed the presence of a second self-association domain within the N-terminal half of ParB. The cross-linking data also suggest that the C terminus is inhibitory to multimerization through the N-terminal domain in vitro. We propose that the two multimerization domains play distinct roles in partition complex formation.

scholarly journals The Assembly of AP-3 Adaptor Complex-containing Clathrin-coated Vesicles on Synthetic Liposomes

2000 ◽  
Vol 11 (11) ◽  
pp. 3723-3736 ◽  
Author(s):  
Matthew T. Drake ◽  
Yunxiang Zhu ◽  
Stuart Kornfeld
Keyword(s):  
Protein Transport ◽  
Adaptor Protein ◽  
Coated Vesicles ◽  
Thin Section Electron Microscopy ◽  
Section Electron ◽  
Dependent Protein ◽  
Lysosomal System ◽  
Adp Ribosylation Factor ◽  
Membrane Concentration

The heterotetrameric adaptor protein complex AP-3 has been shown to function in the sorting of proteins to the endosomal/lysosomal system. However, the mechanism of AP-3 recruitment onto membranes is poorly understood, and it is still uncertain whether AP-3 nucleates clathrin-coated vesicles. Using purified components, we show that AP-3 and clathrin are recruited onto protein-free liposomes and Golgi-enriched membranes by a process that requires ADP-ribosylation factor (ARF) and GTP but no other proteins or nucleotides. The efficiency of recruitment onto the two sources of membranes is comparable and independent of the composition of the liposomes. Clathrin binding occurred in a cooperative manner as a function of the membrane concentration of AP-3. Thin-section electron microscopy of liposomes and Golgi-enriched membranes that had been incubated with AP-3, clathrin, and ARF·GTP showed the presence of clathrin-coated buds and vesicles. These results establish that AP-3–containing clathrin-coated vesicles form in vitro and are consistent with AP-3–dependent protein transport being mediated by clathrin-coated vesicles.

scholarly journals Mammalian GGAs act together to sort mannose 6-phosphate receptors

2003 ◽  
Vol 163 (4) ◽  
pp. 755-766 ◽  
Author(s):  
Pradipta Ghosh ◽  
Janice Griffith ◽  
Hans J. Geuze ◽  
Stuart Kornfeld
Keyword(s):  
Protein Trafficking ◽  
Immunogold Electron Microscopy ◽  
Partial Redistribution ◽  
Golgi Membranes ◽  
Vitro Binding ◽  
Mannose 6 Phosphate ◽  
Golgi Network ◽  
Adp Ribosylation Factor ◽  
Chemical Cross Linking

The GGAs (Golgi-localized, γ ear–containing, ADP ribosylation factor–binding proteins) are multidomain proteins implicated in protein trafficking between the Golgi and endosomes. We examined whether the three mammalian GGAs act independently or together to mediate their functions. Using cryo-immunogold electron microscopy, the three GGAs were shown to colocalize within coated buds and vesicles at the trans-Golgi network (TGN) of HeLa cells. In vitro binding experiments revealed multidomain interactions between the GGAs, and chemical cross-linking experiments demonstrated that GGAs 1 and 2 form a complex on Golgi membranes. RNA interference of each GGA resulted in decreased levels of the other GGAs and their redistribution from the TGN to cytosol. This was associated with impaired incorporation of the cation-independent mannose 6-phosphate receptor into clathrin-coated vesicles at the TGN, partial redistribution of the receptor to endosomes, and missorting of cathepsin D. The morphology of the TGN was also altered. These findings indicate that the three mammalian GGAs cooperate to sort cargo and are required for maintenance of TGN structure.

scholarly journals FORMULATION AND EVALUATION OF RACECADOTRIL MUCOADHESIVE MICROSPHERES

2019 ◽  
pp. 55-61
Author(s):  
KANCHAN JAMKAR ◽  
SWATI MUTHA ◽  
SHARWAREE HARDIKAR ◽  
NIKHIL KUMBHAR
Keyword(s):  
Drug Release ◽  
Sustained Release ◽  
Xanthan Gum ◽  
Entrapment Efficiency ◽  
Cross Linking ◽  
Natural Polymers ◽  
In Vitro Drug Release ◽  
Mucoadhesive Microsphere ◽  
Chemical Cross Linking

Objective: To develop and evaluate the mucoadhesive microsphere using combinations of natural polymers chitosan and xanthan gum for sustained release. Methods: In the present work mucoadhesive microspheres were prepared by using natural polymers like chitosan and xanthan gum by using the emulsion chemical cross-linking method. Chemical cross-linking was done by using glutaraldehyde. The 22 factorial design was employed to show the effect of cross-linking agent and processing factor-like stirring and speed. Prepared microspheres were evaluated for their particle size, surface morphology, drug entrapment efficiency, in vitro drug release, swelling index, and mucoadhesive strength. Results: The size of microspheres of factorial batches were in the range of 26-46 µm. The swelling index was showed in the range of 1.51-1.66 percentage. The equation of multiple regression revealed that there was significant interaction among factors. The glutaraldehyde concentration had a positive effect on % entrapment efficiency, % cumulative drug release and % mucoadhesion. Stirring speed showed a negative impact on % entrapment efficiency, % cumulative drug release and % mucoadhesion. The interactive effect of glutaraldehyde concentration and the stirring speed was found to be positive for % entrapment efficiency and % cumulative drug release. In vitro drug release study of optimized formulation F2 show 96 % of drug release with 6 h indicating sustained release behavior with diffusion mechanism. The SEM image of the optimized batch was spherical with a porous surface. Conclusion: The results obtained in this research work indicated that a promising potential of chitosan and xanthan gum combination for the preparation of the mucoadhesive microsphere of Racecadotril.

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