scholarly journals Identification of Rab6 as an N-ethylmaleimide-sensitive fusion protein-binding protein

2000 ◽  
Vol 352 (1) ◽  
pp. 165-173 ◽  
Author(s):  
Sang Yeul HAN ◽  
Dong Yoon PARK ◽  
Sang Dai PARK ◽  
Seung Hwan HONG
Keyword(s):  
Fusion Protein ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Polyclonal Antibodies ◽  
Binding Protein ◽  
Vesicular Transport ◽  
Brain Extract ◽  
Terminal Domain ◽  
Protein Receptor ◽  
And Function

In this study we show the interaction of N-ethylmaleimide-sensitive fusion protein (NSF) with a small GTP-binding protein, Rab6. NSF is an ATPase involved in the vesicular transport within eukaryotic cells. Using the yeast two-hybrid system, we have isolated new NSF-binding proteins from the rat lung cDNA library. One of them was Rab6, which is involved in the vesicular transport within the Golgi and trans-Golgi network as a Ras-like GTPase. We demonstrated that the N-terminal domain of NSF interacted with the C-terminal domain of Rab6, and these proteins were co-immunoprecipitated from the rat brain extract. This interaction was maintained preferentially in the presence of hydrolysable ATP. Recombinant NSF-His6 can also bind to C-terminal Rab6–glutathione S-transferase under the conditions to allow the ATP hydrolysis. Surprisingly, Rab6 stimulates the ATPase activity of NSF by approx. 2-fold as does α-soluble NSF attachment protein receptor. Anti-Rab6 polyclonal antibodies significantly inhibited the Rab6-stimulated ATPase activity of NSF. Furthermore, we found that Rab3 and Rab4 can also associate with NSF and stimulate its ATPase activity. Taken together, we propose a model in which Rab can form an ATP hydrolysis-regulated complex with NSF, and function as a signalling molecule to deliver the signal of vesicle fusion through the interaction with NSF.

scholarly journals Ordering the Final Events in Yeast Exocytosis

2000 ◽  
Vol 151 (2) ◽  
pp. 439-452 ◽  
Author(s):  
Eric Grote ◽  
Chavela M. Carr ◽  
Peter J. Novick
Keyword(s):  
Plasma Membrane ◽  
Fusion Protein ◽  
Late Stage ◽  
Binding Protein ◽  
Secretory Vesicle ◽  
Snare Complex ◽  
Wild Type ◽  
Attachment Protein ◽  
Complex Assembly ◽  
Protein Receptor

In yeast, assembly of exocytic soluble N-ethylmaleimide–sensitive fusion protein (NSF) attachment protein receptor (SNARE) complexes between the secretory vesicle SNARE Sncp and the plasma membrane SNAREs Ssop and Sec9p occurs at a late stage of the exocytic reaction. Mutations that block either secretory vesicle delivery or tethering prevent SNARE complex assembly and the localization of Sec1p, a SNARE complex binding protein, to sites of secretion. By contrast, wild-type levels of SNARE complexes persist in the sec1-1 mutant after a secretory block is imposed, suggesting a role for Sec1p after SNARE complex assembly. In the sec18-1 mutant, cis-SNARE complexes containing surface-accessible Sncp accumulate in the plasma membrane. Thus, one function of Sec18p is to disassemble SNARE complexes on the postfusion membrane.

scholarly journals RAVE and Rabconnectin-3 Complexes as Signal Dependent Regulators of Organelle Acidification

2021 ◽  
Vol 9 ◽  
Author(s):  
Michael C. Jaskolka ◽  
Samuel R. Winkley ◽  
Patricia M. Kane
Keyword(s):  
Atpase Activity ◽  
Proton Transport ◽  
Atp Hydrolysis ◽  
Glucose Deprivation ◽  
Vacuolar Membrane ◽  
Proton Pumps ◽  
Endosomal Acidification ◽  
Endosomal Membranes ◽  
Higher Eukaryotes ◽  
And Function

The yeast RAVE (Regulator of H+-ATPase of Vacuolar and Endosomal membranes) complex and Rabconnectin-3 complexes of higher eukaryotes regulate acidification of organelles such as lysosomes and endosomes by catalyzing V-ATPase assembly. V-ATPases are highly conserved proton pumps consisting of a peripheral V1 subcomplex that contains the sites of ATP hydrolysis, attached to an integral membrane Vo subcomplex that forms the transmembrane proton pore. Reversible disassembly of the V-ATPase is a conserved regulatory mechanism that occurs in response to multiple signals, serving to tune ATPase activity and compartment acidification to changing extracellular conditions. Signals such as glucose deprivation can induce release of V1 from Vo, which inhibits both ATPase activity and proton transport. Reassembly of V1 with Vo restores ATP-driven proton transport, but requires assistance of the RAVE or Rabconnectin-3 complexes. Glucose deprivation triggers V-ATPase disassembly in yeast and is accompanied by binding of RAVE to V1 subcomplexes. Upon glucose readdition, RAVE catalyzes both recruitment of V1 to the vacuolar membrane and its reassembly with Vo. The RAVE complex can be recruited to the vacuolar membrane by glucose in the absence of V1 subunits, indicating that the interaction between RAVE and the Vo membrane domain is glucose-sensitive. Yeast RAVE complexes also distinguish between organelle-specific isoforms of the Vo a-subunit and thus regulate distinct V-ATPase subpopulations. Rabconnectin-3 complexes in higher eukaryotes appear to be functionally equivalent to yeast RAVE. Originally isolated as a two-subunit complex from rat brain, the Rabconnectin-3 complex has regions of homology with yeast RAVE and was shown to interact with V-ATPase subunits and promote endosomal acidification. Current understanding of the structure and function of RAVE and Rabconnectin-3 complexes, their interactions with the V-ATPase, their role in signal-dependent modulation of organelle acidification, and their impact on downstream pathways will be discussed.

Regulation of SNAP-25 trafficking and function by palmitoylation

2010 ◽  
Vol 38 (1) ◽  
pp. 163-166 ◽  
Author(s):  
Jennifer Greaves ◽  
Gerald R. Prescott ◽  
Oforiwa A. Gorleku ◽  
Luke H. Chamberlain
Keyword(s):  
Fusion Protein ◽  
Intracellular Trafficking ◽  
Neuroendocrine Cells ◽  
Snare Proteins ◽  
Receptor Protein ◽  
Attachment Protein ◽  
Rich Region ◽  
Protein Receptor ◽  
And Function ◽  
Protein Attachment

The SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) protein SNAP-25 (25 kDa synaptosome-associated protein) is essential for regulated exocytosis in neuronal and neuroendocrine cells. Whereas the majority of SNARE proteins contain transmembrane domains, SNAP-25 is instead anchored to membranes by the palmitoylation of a central cysteine-rich region. In this review, we discuss the mechanisms of SNAP-25 palmitoylation and how this modification regulates the intracellular trafficking and exocytotic function of this essential protein.

scholarly journals In Vivo Function of Hsp90 Is Dependent on ATP Binding and ATP Hydrolysis

1998 ◽  
Vol 143 (4) ◽  
pp. 901-910 ◽  
Author(s):  
Wolfgang M.J. Obermann ◽  
Holger Sondermann ◽  
Alicia A. Russo ◽  
Nikola P. Pavletich ◽  
F. Ulrich Hartl
Keyword(s):  
Crystal Structure ◽  
Atpase Activity ◽  
Binding Site ◽  
Atp Hydrolysis ◽  
Specific Inhibitor ◽  
Nucleotide Binding ◽  
Basic Mechanism ◽  
Atp Binding ◽  
Terminal Domain

Heat shock protein 90 (Hsp90), an abundant molecular chaperone in the eukaryotic cytosol, is involved in the folding of a set of cell regulatory proteins and in the re-folding of stress-denatured polypeptides. The basic mechanism of action of Hsp90 is not yet understood. In particular, it has been debated whether Hsp90 function is ATP dependent. A recent crystal structure of the NH2-terminal domain of yeast Hsp90 established the presence of a conserved nucleotide binding site that is identical with the binding site of geldanamycin, a specific inhibitor of Hsp90. The functional significance of nucleotide binding by Hsp90 has remained unclear. Here we present evidence for a slow but clearly detectable ATPase activity in purified Hsp90. Based on a new crystal structure of the NH2-terminal domain of human Hsp90 with bound ADP-Mg and on the structural homology of this domain with the ATPase domain of Escherichia coli DNA gyrase, the residues of Hsp90 critical in ATP binding (D93) and ATP hydrolysis (E47) were identified. The corresponding mutations were made in the yeast Hsp90 homologue, Hsp82, and tested for their ability to functionally replace wild-type Hsp82. Our results show that both ATP binding and hydrolysis are required for Hsp82 function in vivo. The mutant Hsp90 proteins tested are defective in the binding and ATP hydrolysis–dependent cycling of the co-chaperone p23, which is thought to regulate the binding and release of substrate polypeptide from Hsp90. Remarkably, the complete Hsp90 protein is required for ATPase activity and for the interaction with p23, suggesting an intricate allosteric communication between the domains of the Hsp90 dimer. Our results establish Hsp90 as an ATP-dependent chaperone.

scholarly journals Preparation of Specific Polyclonal Antibody Against the Recombinant Mutacin Produced by sfGFP Fusion Protein Technology

2015 ◽  
Vol 9 (1) ◽  
pp. 70-80 ◽  
Author(s):  
Lamis Al-Homsi ◽  
Souad Al-Okla ◽  
Abdul Q. Abbady
Keyword(s):  
Fusion Protein ◽  
Large Scale ◽  
Polyclonal Antibodies ◽  
Gel Filtration ◽  
High Yield ◽  
Additional Advantage ◽  
Short Peptide ◽  
Tev Protease ◽  
Green Florescent Protein ◽  
And Function

Mutacin I, a bacteriocin produced bystreptococcus mutans, displays an antimicrobial activity against many gram positive and some gram negative bacteria. Because of its medical importance, production of this short peptide in large scale for future applications is a significant challenge. This work described the improvement of a novel system to produce the recombinant mutacin using fusion protein technology.The short peptide was expressed directly as a fusion protein with a superfolder form of the green florescent protein (sfGFP), resulting in a high yield expression of solublesfGFP-mutacin fusion protein (30 kDa) in the cytoplasm of E. coli. Mutacin was released from the fusion by enzymatic cleavage at the tobacco etch virus (TEV) protease recognition site and separated from the carriersfGFP by nickel affinity and gel filtration chromatography. An additional advantage of this fusion system was tested in the generation of mutacin-specific polyclonal antibodies. Specific anti-mutacin IgGs were affinity purified, and were able to recognize the mutacin-sfGFP fusion protein or the cleaved forms of mutacin.Even though it was efficiently produced (25 mg/L) by this method, pure mutacin was devoid of antibiotic activity. Fourier transform infrared spectroscopy (FTIR) analysis revealed the absence of thioether bonds in the purified mutacin, which are critical for final structure and function of this antibiotic. Determining whether the activity of pure mutacin could be recovered by the reformation of such structures by chemical reaction needs more investigations. The development of this system will provide large quantities of mutacin for future studies and applications as broad spectrum antibacterial peptide.

scholarly journals Vanadate-Induced Trapping of Nucleotides by Purified Maltose Transport Complex Requires ATP Hydrolysis

2000 ◽  
Vol 182 (23) ◽  
pp. 6570-6576 ◽  
Author(s):  
Susan Sharma ◽  
Amy L. Davidson
Keyword(s):  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Binding Protein ◽  
Nucleotide Binding ◽  
Maltose Binding Protein ◽  
High Energy ◽  
Maltose Transport ◽  
Binding Domains ◽  
Mechanism Of Inhibition ◽  
Transport Complex

ABSTRACT The maltose transport system in Escherichia coli is a member of the ATP-binding cassette superfamily of transporters that is defined by the presence of two nucleotide-binding domains or subunits and two transmembrane regions. The bacterial import systems are unique in that they require a periplasmic substrate-binding protein to stimulate the ATPase activity of the transport complex and initiate the transport process. Upon stimulation by maltose-binding protein, the intact MalFGK2 transport complex hydrolyzes ATP with positive cooperativity, suggesting that the two nucleotide-binding MalK subunits interact to couple ATP hydrolysis to transport. The ATPase activity of the intact transport complex is inhibited by vanadate. In this study, we investigated the mechanism of inhibition by vanadate and found that incubation of the transport complex with MgATP and vanadate results in the formation of a stably inhibited species containing tightly bound ADP that persists after free vanadate and nucleotide are removed from the solution. The inhibited species does not form in the absence of MgCl2 or of maltose-binding protein, and ADP or another nonhydrolyzable analogue does not substitute for ATP. Taken together, these data conclusively show that ATP hydrolysis must precede the formation of the vanadate-inhibited species in this system and implicate a role for a high-energy, ADP-bound intermediate in the transport cycle. Transport complexes containing a mutation in a single MalK subunit are still inhibited by vanadate during steady-state hydrolysis; however, a stably inhibited species does not form. ATP hydrolysis is therefore necessary, but not sufficient, for vanadate-induced nucleotide trapping.

Characteristics and Function of the Chitin Binding Protein from Xenorhabdus nematophila

2019 ◽  
Vol 26 (6) ◽  
pp. 414-422
Author(s):  
Jia Liu ◽  
Ping Song ◽  
Jie Zhang ◽  
Ziyan Nangong ◽  
Xiaobei Liu ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Growth Inhibition ◽  
Insecticidal Activity ◽  
Spore Germination ◽  
Binding Protein ◽  
Colloidal Chitin ◽  
Inhibition Rate ◽  
Xenorhabdus Nematophila ◽  
Chitin Binding Protein ◽  
And Function

Background: Genome sequence analysis (GenBank access No.: FN667742.1) shows that Xenorhabdus nematophila ATCC19061 contains one gene (Xn-cbp) encoding chitin binding protein (Xn-CBP). Objective: The present work aims to clarify the characteristics and function of Xn-CBP from X. nematophila HB310. Methods: In this study, the Xn-cbp gene was cloned and expressed in Escherichia coli BL21 (DE3). Substrate binding assays were performed to explain the ability of Xn-CBP combined with the polysaccharide. The insecticidal toxicity of Xn-CBP against the second-instar larvae of Helicoverpa armigera was determined by feeding method. Besides, the antifungal activity of Xn-CBP against Coniothyrium diplodiella, Verticillium dahlia, and Fusarium oxysporum was tested by spore germination assay and hyphal extension assay. Results: Xn-CBP encoded 199 amino acids with a calculated mass of 28 kDa, which contained a signal peptide and a chitin binding domain. The Bmax and Kd values of Xn-CBP to colloidal chitin were 2.46 and 4.08, respectively. Xn-CBP had insecticidal activity against the H. armigera with a growth inhibition rate of 84.08%. Xn-CBP had the highest spore germination inhibitory effect on C. diplodiella with the inhibition rate of 83.11%. The hyphal growth inhibition rate of Xn-CBP to F. oxysporum, 41.52%, was higher than the other two fungi. Conclusion: The Xn-CBP had the highest binding ability to colloidal chitin and it showed insecticidal activity and antifungal activity. The present study laid a foundation for further exploitation and utilization of X. nematophila.

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