scholarly journals Thylakoid-integrated recombinant Hcf106 participates in the chloroplast Twin Arginine Transport (cpTat) system

2018 ◽  
Author(s):  
Qianqian Ma ◽  
Kristen Fite ◽  
Christopher Paul New ◽  
Carole Dabney-Smith
Keyword(s):  
Protein Transport ◽  
Transmembrane Domain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sole Source ◽  
Receptor Complex ◽  
Amphipathic Helix ◽  
Transport Pathway ◽  
Independent Manner ◽  
Arginine Transport

AbstractThe chloroplast Twin arginine transport (cpTat) system distinguishes itself as a protein transport pathway by translocating fully-folded proteins, using the proton-motive force (PMF) as the sole source of energy. The cpTat pathway is evolutionarily conserved with the Tat pathway found in the plasma membrane of many prokaryotes. The cpTat (E. coli) system uses three proteins, Tha4 (TatA), Hcf106 (TatB), and cpTatC (TatC), to form a transient translocase allowing the passage of precursor proteins. Briefly, cpTatC and Hcf106, with Tha4, form the initial receptor complex responsible for precursor protein recognition and binding in an energy-independent manner, while a separate pool of Tha4 assembles with the precursor-bound receptor complex in the presence the PMF. Analysis by blue-native polyacrylamide gel electrophoresis (BN-PAGE) shows that the receptor complex, in the absence of precursor, migrates near 700 kDa and contains cpTatC and Hcf106 with little Tha4 remaining after detergent solubilization. To investigate the role that Hcf106 may play in receptor complex oligomerization and/or stability, systematic cysteine substitutions were made in positions from the N-terminal transmembrane domain to the end of the predicted amphipathic helix of the protein. BN-PAGE analysis allowed us to identify the locations of amino acids in Hcf106 that were critical for interacting with cpTatC. Oxidative cross-linking allowed us to map interactions of the transmembrane domain and amphipathic helix region of Hcf106. In addition, we showed that in vitro expressed, integrated Hcf106 can interact with the precursor signal peptide domain and imported cpTatC, strongly suggesting that a subpopulation of the integrated Hcf106 is participating in competent cpTat complexes.

Syntaxin 11 is associated with SNAP-23 on late endosomes and the trans-Golgi network

1999 ◽  
Vol 112 (6) ◽  
pp. 845-854 ◽  
Author(s):  
A.C. Valdez ◽  
J.P. Cabaniols ◽  
M.J. Brown ◽  
P.A. Roche
Keyword(s):  
Mammalian Cells ◽  
Protein Transport ◽  
Transmembrane Domain ◽  
Family Members ◽  
Snare Proteins ◽  
Trans Golgi Network ◽  
Late Endosomes ◽  
Syntaxin 11 ◽  
Golgi Network

SNARE proteins are known to play a role in regulating intracellular protein transport between donor and target membranes. This docking and fusion process involves the interaction of specific vesicle-SNAREs (e.g. VAMP) with specific cognate target-SNAREs (e.g. syntaxin and SNAP-23). Using human SNAP-23 as the bait in a yeast two-hybrid screen of a human B-lymphocyte cDNA library, we have identified the 287-amino-acid SNARE protein syntaxin 11. Like other syntaxin family members, syntaxin 11 binds to the SNARE proteins VAMP and SNAP-23 in vitro and also exists in a complex with SNAP-23 in transfected HeLa cells and in native human B lymphocytes. Unlike other syntaxin family members, no obvious transmembrane domain is present in syntaxin 11. Nevertheless, syntaxin 11 is predominantly membrane-associated and colocalizes with the mannose 6-phosphate receptor on late endosomes and the trans-Golgi network. These data suggest that syntaxin 11 is a SNARE that acts to regulate protein transport between late endosomes and the trans-Golgi network in mammalian cells.

scholarly journals Clustering of C-Terminal Stromal Domains of Tha4 Homo-oligomers during Translocation by the Tat Protein Transport System

2009 ◽  
Vol 20 (7) ◽  
pp. 2060-2069 ◽  
Author(s):  
Carole Dabney-Smith ◽  
Kenneth Cline
Keyword(s):  
Protein Transport ◽  
Transmembrane Domain ◽  
Protein Translocation ◽  
Proton Gradient ◽  
Receptor Complex ◽  
Tat Protein ◽  
Twin Arginine Translocation ◽  
Conducting Component ◽  
Cross Links ◽  
Folded Proteins

The chloroplast Twin arginine translocation (Tat) pathway uses three membrane proteins and the proton gradient to transport folded proteins across sealed membranes. Precursor proteins bind to the cpTatC-Hcf106 receptor complex, triggering Tha4 assembly and protein translocation. Tha4 is required only for the translocation step and is thought to be the protein-conducting component. The organization of Tha4 oligomers was examined by substituting pairs of cysteine residues into Tha4 and inducing disulfide cross-links under varying stages of protein translocation. Tha4 formed tetramers via its transmembrane domain in unstimulated membranes and octamers in membranes stimulated by precursor and the proton gradient. Tha4 formed larger oligomers of at least 16 protomers via its carboxy tail, but such C-tail clustering only occurred in stimulated membranes. Mutational studies showed that transmembrane domain directed octamers as well as C-tail clusters require Tha4's transmembrane glutamate residue and its amphipathic helix, both of which are necessary for Tha4 function. A novel double cross-linking strategy demonstrated that both transmembrane domain directed- and C-tail directed oligomerization occur in the translocase. These results support a model in which Tha4 oligomers dock with a precursor–receptor complex and undergo a conformational switch that results in activation for protein transport. This possibly involves accretion of additional Tha4 into a larger transport-active homo-oligomer.

scholarly journals Construction and Characterization ofMoraxella catarrhalis Mutants Defective in Expression of Transferrin Receptors

1999 ◽  
Vol 67 (11) ◽  
pp. 5815-5819 ◽  
Author(s):  
Nicole R. Luke ◽  
Anthony A. Campagnari
Keyword(s):  
Solid Phase ◽  
Iron Acquisition ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sole Source ◽  
Pcr Analysis ◽  
Binding Studies ◽  
Binding Characteristics ◽  
Isogenic Mutants

ABSTRACT We have previously reported the construction of an isogenic mutant defective in expression of OmpB1, the TbpB homologue, inMoraxella catarrhalis 7169. In this report, we have extended these studies by constructing and characterizing two new isogenic mutants in this clinical isolate. One mutant is defective in expression of TbpA, and the other mutant is defective in expression of both TbpA and TbpB. These isogenic mutants were confirmed by using PCR analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and sequencing. In vitro growth studies, comparing all three mutants, demonstrated that the tbpA mutant and the tbpABmutant were severely limited in their ability to grow with human holotransferrin as the sole source of iron. In contrast, theompB1 (tbpB) mutant was capable of utilizing iron from human transferrin, although not to the extent of the parental strain. While affinity chromatography with human holotransferrin showed that each Tbp was capable of binding independently to transferrin, solid-phase transferrin binding studies using whole cells demonstrated that the tbpA mutant exhibited binding characteristics similar to those seen with the wild-type bacteria. However, theompB1 (tbpB) mutant exhibited a diminished capacity for binding transferrin, and no binding was detected with the double mutant. These data suggest that the M. catarrhalisTbpA is necessary for the acquisition of iron from transferrin. In contrast, TbpB is not essential but may serve as a facilitory protein that functions to optimize this process. Together these mutants are essential to provide a more thorough understanding of iron acquisition mechanisms utilized by M. catarrhalis.

scholarly journals Co-operation between different targeting pathways during integration of a membrane protein

2012 ◽  
Vol 199 (2) ◽  
pp. 303-315 ◽  
Author(s):  
Rebecca Keller ◽  
Jeanine de Keyzer ◽  
Arnold J.M. Driessen ◽  
Tracy Palmer
Keyword(s):  
Membrane Protein ◽  
Protein Transport ◽  
Transmembrane Domain ◽  
Energy Coupling ◽  
Integral Membrane Protein ◽  
Transmembrane Domains ◽  
Dependent Manner ◽  
Rieske Protein ◽  
Transport Pathway ◽  
Iron Sulfur

Membrane protein assembly is a fundamental process in all cells. The membrane-bound Rieske iron-sulfur protein is an essential component of the cytochrome bc1 and cytochrome b6f complexes, and it is exported across the energy-coupling membranes of bacteria and plants in a folded conformation by the twin arginine protein transport pathway (Tat) transport pathway. Although the Rieske protein in most organisms is a monotopic membrane protein, in actinobacteria, it is a polytopic protein with three transmembrane domains. In this work, we show that the Rieske protein of Streptomyces coelicolor requires both the Sec and the Tat pathways for its assembly. Genetic and biochemical approaches revealed that the initial two transmembrane domains were integrated into the membrane in a Sec-dependent manner, whereas integration of the third transmembrane domain, and thus the correct orientation of the iron-sulfur domain, required the activity of the Tat translocase. This work reveals an unprecedented co-operation between the mechanistically distinct Sec and Tat systems in the assembly of a single integral membrane protein.

scholarly journals Functional reconstitution of TatB into thylakoidal Tat translocase

2019 ◽  
Author(s):  
Sarah Zinecker ◽  
Mario Jakob ◽  
Ralf Bernd Klösgen
Keyword(s):  
Functional Analysis ◽  
Heterologous Expression ◽  
Protein Transport ◽  
Transmembrane Helix ◽  
Experimental System ◽  
Receptor Complex ◽  
Transport Activity ◽  
E Coli ◽  
Purified Antigen

AbstractWe have established an experimental system for the functional analysis of thylakoidal TatB, a component of the membrane-integral TatBC receptor complex of the thylakoidal Twin-arginine protein transport (Tat1) machinery. For this purpose, the intrinsic TatB activity of isolated pea thylakoids was inhibited by affinity-purified antibodies and substituted by supplementing the assays with TatB protein either obtained by in vitro translation or purified after heterologous expression in E. coli. Tat transport activity of such reconstituted thylakoids, which was analyzed with the authentic Tat substrate pOEC16, reached routinely 20 - 25% of the activity of mock-treated thylakoid vesicles analysed in parallel. In contrast, supplementation of the assays with the purified antigen comprising all but the N-terminal transmembrane helix of thylakoidal TatB did not result in Tat transport reconstitution which confirms that transport relies strictly on the activity of the TatB protein added and is not due to restoration of the intrinsic TatB activity by antibody release. Unexpectedly, even a mutant TatB protein (TatB,E10C) assumed to be incapable of assembling into the TatBC receptor complex showed low but considerable transport reconstitution underlining the sensitivity of the approach and its suitability for further functional mutant analyses. Finally, quantification of TatB demand suggests that TatA and TatB are required in approximately equimolar amounts to achieve Tat-dependent thylakoid transport.

scholarly journals Precursor–Receptor Interactions in the Twin Arginine Protein Transport Pathway Probed with a New Receptor Complex Preparation

Biochemistry ◽  
2018 ◽  
Vol 57 (10) ◽  
pp. 1663-1671 ◽  
Author(s):  
Marta Wojnowska ◽  
Joseph Gault ◽  
Shee Chien Yong ◽  
Carol V. Robinson ◽  
Ben C. Berks
Keyword(s):  
Protein Transport ◽  
Receptor Complex ◽  
Transport Pathway ◽  
Receptor Interactions ◽  
Complex Preparation

Calpain Functions in a Caspase-Independent Manner to Promote Apoptosis-Like Events During Platelet Activation

Blood ◽  
1999 ◽  
Vol 94 (5) ◽  
pp. 1683-1692 ◽  
Author(s):  
Beni B. Wolf ◽  
Joshua C. Goldstein ◽  
Henning R. Stennicke ◽  
Helen Beere ◽  
Gustavo P. Amarante-Mendes ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Platelet Activation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Calcium Ionophore ◽  
Phosphatidyl Serine ◽  
Human Platelets ◽  
Independent Manner ◽  
Calcium Dependent

Apoptosis and platelet activation share common morphological and biochemical features. Because caspases are essential mediators of apoptosis, we examined whether platelets contain these proteinases and use them during platelet activation. Human platelets contained caspase-9, caspase-3, and the caspase activators APAF-1 and cytochrome c as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Upon treatment with cytochrome c and dATP, platelet cytoplasmic extracts recapitulated apoptotic events, including sequential activation of procaspase-9 and procaspase-3 and subsequent proteolysis of caspase substrates. Calcium ionophore-stimulated platelets also recapitulated apoptotic events, including cell shrinkage, plasma membrane microvesiculation, phosphatidyl serine externalization, and proteolysis of procaspase-9, procaspase-3, gelsolin, and protein kinase C-δ. Strikingly, however, these events occurred without caspase activation or release of mitochondrial cytochrome c, suggesting a role for a noncaspase proteinase. Supporting this, inhibition of the calcium-dependent proteinase, calpain, prevented caspase proteolysis, ‘apoptotic’ substrate cleavage, and platelet microvesiculation. In vitro, purified calpain cleaved recombinant procaspase-9 and procaspase-3 without activating either caspase, confirming the inhibitor studies. These data implicate calpain as a potential regulator of caspases and suggest that calpain, not caspases, promotes apoptosis-like events during platelet activation.

scholarly journals Betacellulin-δ4, a Novel Differentiation Factor for Pancreatic β-Cells, Ameliorates Glucose Intolerance in Streptozotocin-Treated Rats

Endocrinology ◽  
2005 ◽  
Vol 146 (11) ◽  
pp. 4673-4681 ◽  
Author(s):  
Takeki Ogata ◽  
Andrew J. Dunbar ◽  
Yoritsuna Yamamoto ◽  
Yuji Tanaka ◽  
Masaharu Seno ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Cell Mass ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Independent Manner ◽  
Ductal Cells ◽  
Alternatively Spliced ◽  
Epidermal Growth

We previously described a novel alternatively spliced mRNA transcript of the betacellulin (BTC) gene. This splice isoform, termed BTC-δ4, lacks the C-loop of the epidermal growth factor motif and the transmembrane domain as a result of exon 4 ‘skipping’. In this study, we expressed BTC-δ4 recombinantly to explore its biological function. When BTC-δ4 was expressed in COS-7 cells, it was secreted largely into the culture medium, in contrast to BTC. Unlike BTC, highly purified recombinant BTC-δ4 produced in Escherichia coli failed to bind or induce tyrosine phosphorylation of either ErbB1 or ErbB4, nor did it antagonize the binding of BTC to these receptors. Consistent with this, BTC-δ4 failed to stimulate DNA synthesis in Balb/c 3T3 and INS-1 cells. However, BTC-δ4 induced differentiation of pancreatic β-cells; BTC-δ4 converted AR42J cells to insulin-producing cells. When recombinant BTC-δ4 was administered to streptozotocin-treated neonatal rats, it reduced the plasma glucose concentration and improved glucose tolerance. Importantly, BTC-δ4 significantly increased the insulin content, the β-cell mass, and the numbers of islet-like cell clusters and PDX-1-positive ductal cells. Thus, BTC-δ4 is a secreted protein that stimulates differentiation of β-cells in vitro and in vivo in an apparent ErbB1- and ErbB4-independent manner. The mechanism by which BTC-δ4 exerts this action on β-cells remains to be defined but presumably involves an, as yet, unidentified unique receptor.

scholarly journals The Cell Adhesion Molecule DdCAD-1 in Dictyostelium Is Targeted to the Cell Surface by a Nonclassical Transport Pathway Involving Contractile Vacuoles

1997 ◽  
Vol 138 (4) ◽  
pp. 939-951 ◽  
Author(s):  
Hiromi Sesaki ◽  
Estella F.S. Wong ◽  
Chi-Hung Siu
Keyword(s):  
Cell Adhesion ◽  
Cell Surface ◽  
Adhesion Molecule ◽  
Transport Mechanism ◽  
Cell Adhesion Molecule ◽  
Transmembrane Domain ◽  
Specific Inhibitor ◽  
Transport Pathway ◽  
Contractile Vacuoles

DdCAD-1 is a 24-kD Ca2+-dependent cell– cell adhesion molecule that is expressed soon after the initiation of development in Dictyostelium cells. DdCAD-1 is present on the cell surface as well as in the cytosol. However, the deduced amino acid sequence of DdCAD-1 lacks a hydrophobic signal peptide or any predicted transmembrane domain, suggesting that it may be presented on the cell surface via a nonclassical transport mechanism. Here we report that DdCAD-1 is transported to the cell surface via contractile vacuoles, which are normally involved in osmoregulation. Immunofluorescence microscopy and subcellular fractionation revealed a preferential association of DdCAD-1 with contractile vacuoles. Proteolytic treatment of isolated contractile vacuoles degraded vacuole-associated calmodulin but not DdCAD-1, demonstrating that DdCAD-1 was present in the lumen. The use of hyperosmotic conditions that suppress contractile vacuole activity led to a dramatic decrease in DdCAD-1 accumulation on the cell surface and the absence of cell cohesiveness. Shifting cells back to a hypotonic condition after hypertonic treatments induced a rapid increase in DdCAD-1–positive contractile vacuoles, followed by the accumulation of DdCAD-1 on the cell membrane. 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, a specific inhibitor of vacuolar-type H+-ATPase and thus of the activity of contractile vacuoles, also inhibited the accumulation of DdCAD-1 on the cell surface. Furthermore, an in vitro reconstitution system was established, and isolated contractile vacuoles were shown to import soluble DdCAD-1 into their lumen in an ATP-stimulated manner. Taken together, these data provide the first evidence for a nonclassical protein transport mechanism that uses contractile vacuoles to target a soluble cytosolic protein to the cell surface.

Calpain Functions in a Caspase-Independent Manner to Promote Apoptosis-Like Events During Platelet Activation

Blood ◽  
1999 ◽  
Vol 94 (5) ◽  
pp. 1683-1692 ◽  
Author(s):  
Beni B. Wolf ◽  
Joshua C. Goldstein ◽  
Henning R. Stennicke ◽  
Helen Beere ◽  
Gustavo P. Amarante-Mendes ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Platelet Activation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Calcium Ionophore ◽  
Phosphatidyl Serine ◽  
Human Platelets ◽  
Independent Manner ◽  
Calcium Dependent

Abstract Apoptosis and platelet activation share common morphological and biochemical features. Because caspases are essential mediators of apoptosis, we examined whether platelets contain these proteinases and use them during platelet activation. Human platelets contained caspase-9, caspase-3, and the caspase activators APAF-1 and cytochrome c as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Upon treatment with cytochrome c and dATP, platelet cytoplasmic extracts recapitulated apoptotic events, including sequential activation of procaspase-9 and procaspase-3 and subsequent proteolysis of caspase substrates. Calcium ionophore-stimulated platelets also recapitulated apoptotic events, including cell shrinkage, plasma membrane microvesiculation, phosphatidyl serine externalization, and proteolysis of procaspase-9, procaspase-3, gelsolin, and protein kinase C-δ. Strikingly, however, these events occurred without caspase activation or release of mitochondrial cytochrome c, suggesting a role for a noncaspase proteinase. Supporting this, inhibition of the calcium-dependent proteinase, calpain, prevented caspase proteolysis, ‘apoptotic’ substrate cleavage, and platelet microvesiculation. In vitro, purified calpain cleaved recombinant procaspase-9 and procaspase-3 without activating either caspase, confirming the inhibitor studies. These data implicate calpain as a potential regulator of caspases and suggest that calpain, not caspases, promotes apoptosis-like events during platelet activation.

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